**Expression Profile of Galectins (Gal-1, Gal-9, Gal-11 and Gal-13) in Human Bone Marrow Derived Mesenchymal Stem Cells in Different Culture Mediums**

Faouzi Jenhani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55383

#### **1. Introduction**

Mesenchymal stem cells (MCSs) are refined as undifferencitaed cells that are capable of self renewal and differentiation into several cell types such chondrocyte, adipocyte osteocyte, myocyte and neuron-like cells. MSC can be isolated from bone marrow umbilical cord blood adipose tissue placenta. Although bone marrow(BM) has been regarded as a major source of MSC umbilical cord blood has been regarded as an alternative source for isolation of MSC. Human umbilical cord blood derived mesenchymal stem cell (hUCB-MSCs) have a capacity similar to that of BM-MCSs for multi-lineage differentiation

Researchers are interested in these cells because of their ability of differentiating into multiple mesenchymal and non mesenchymal lineages. Furthermore, MSCs evoke only minimal immunoreactivity and they display trophic, anti-inflammatory, and immunomodulatory capacities, through secretion of bioactive soluble factors with anti-inflammatory and immu‐ nomodulatory effects in vivo. These properties were confirmed by both experimental and clinical studies which demonstrated that the MSCs support hematopoiesis and enhance the engraftment of hematopoietic stem cells (HSC) after cotransplantation, which may contribute to a reduced incidence of graft versus host disease (GVHD). [1,2,3,4,5,6,7]

The galectins are a family of soluble lectins characterized by their affinity for b-galactoside residues. These proteins have recently attracted increasing attention because of theirinvolve‐ ment in various physiological and pathological processes. In addition, these proteins have recently attracted increasing attention of cancer biologists because of their essential functions including development, differentiation, cell–cell adhesion, cell–matrix interaction, growth

© 2013 Jenhani; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

regulation, apoptosis, RNA splicing, and tumor metastasis. Also, it has been shown that galectins' levels are altered in various cancers. [8,9,10]

*2.2.1. Osteogenic induction (O)for analysis galectins expression*

*2.2.2. Adipogenic induction (A) for analysis galectins expression*

with Nile Red (Sigma) and observed by fluorescent microscopy.

*2.2.3. Vascular smooth muscle induction (V) for analysis galectins expression*

days. The VSM differentiation was evaluated after 3 weeks of induction.

**2.3. Immunophenotyping of mesenchymal stem cells by flow cytometry**

**2.4. Detection of galectins (Gal-1, Gal-9 Gal-11 and Gal-13), by flow cytometry**

recombinant galectin (Gal-1, Gal-9, Gal-11 and Gal-13). Thus we incubated 1,5 106

software (Becton–Dickinson, BD Biosciences San Jose, CA).

the presence of calcium deposition into osteocytes.

Jose CA).

primary antibody.

At 50% confluence, the cells were cultured for 14 days in DMEM-HG (High Glucose 4. 5 g/L) containing 2% FBS, 0. 1 µM dexamethasone (Sigma), 2 mM β-glycerolphosphate (Sigma), and 100 µM ascorbate-2-phosphate (Sigma) with medium changes every 3 days. After 2 weeks of induction, the cells were stained according to Von Kossa's and Alizarin Red methods to detect

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35

The adipogenic induction medium consisted of DMEM-LG (Low Glucose 1g/L) supplemented with 20% FBS, 1 µM/L dexamethasone, 0. 5 mmol/L isobutylmethylxanthine (IBMX; Invitro‐ gen), and 60 µmol/L indomethacin (Sigma). Adipogenic differentiation was evaluated after 2 weeks of induction, by the cellular accumulation of neutral lipid vacuoles that were stained

Vascular smooth muscle (VSM) differentiation was obtained in a Mc Coy'5A medium supplemented with 12. 5% FBS, 12. 5% HS (Horse Serum, Invitrogen), 20 µM L-glutamine, 0,8 mM L-serine, 0,15 mM L-asparagine, 1 Mm sodium pyruvate, 5 mM sodium bicarbonate, 1 µM hydrocortisone and amphotericin B and antibodies. The medium was changed every 4

MSCs were immunophenotypically characterized by flow cytometry using the following fluorochrome (FITC PE, PerCP) marked monoclonal antibodies anti : CD45, CD105, CD106, CD90,CD49, CD34, et CD73 and CD14 (Becton Dickinson and Company BD Biosciences San

To reveal the expression of the galactin we used biotynaled antibodies against human

with the antibody which is specific to the focused galectin, for 30 minutes, at 4° C. We incubated also the MSCs with a control antibody which doesn't recognize any protein, but has the same isotype to evaluate a background noise which correspond to non-specific fixation of the

The MSCs were washed 2 times with PBS 1X solution, then incubated for 30 minutes with Streptavidin coupled with a phycoerythrin (PE) fluorochrome phycoerythrin (PE). Flow cytometry analysis was performed on a FACS calibur, and data were analyzed using CellQuest

MSCs cells

Galectins-1,3,9 and 13 were among the best characterized members of this family. These galactins possesses several functions and were expressed in many tissues.

Given the importance of galectins, we investigated in this work, their expression by BM MSCs in different culture mediums

### **2. Materiel and methods**

#### **2.1. Preparation of hBM-MSC**

MSCs were extracted and isolated from two sources adult the bone marrow. H- *BM cells* was harvested from the sternum or the iliac crest of healthy donors [20] after obtaining an informed consent was obtained in the Bone Marrow Graft National Center of Tunisia. The mean age of donors was 30 ± 2 years (range, 15-40 years).

MSCs were isolated using the classical plastic adhesion method. Mononuclear cells (MNCs) were isolated from hBM by density gradient (Ficoll Hypaque solution d=1. 077) centrifugation. After centrifugation at 800 g for 20 min at room temperature. MNC layer was removed from the interphase and washed twice with Hank's buffered salt solution and seeded into uncoated T25 or T75 flasks (Becton Dickinson, Bedford, MA, USA) at a cell concentration of 1×105 cells/ cm2 for BM cells and cultured in three condition mediums:


#### **2.2. Induction of the differentiation directions**

We induced the differentiation of the MSCs when they reached the confluence. We used three differentiation mediums according to the focused differentiation line:

#### *2.2.1. Osteogenic induction (O)for analysis galectins expression*

regulation, apoptosis, RNA splicing, and tumor metastasis. Also, it has been shown that

Galectins-1,3,9 and 13 were among the best characterized members of this family. These

Given the importance of galectins, we investigated in this work, their expression by BM MSCs

MSCs were extracted and isolated from two sources adult the bone marrow. H- *BM cells* was harvested from the sternum or the iliac crest of healthy donors [20] after obtaining an informed consent was obtained in the Bone Marrow Graft National Center of Tunisia. The mean age of

MSCs were isolated using the classical plastic adhesion method. Mononuclear cells (MNCs) were isolated from hBM by density gradient (Ficoll Hypaque solution d=1. 077) centrifugation. After centrifugation at 800 g for 20 min at room temperature. MNC layer was removed from the interphase and washed twice with Hank's buffered salt solution and seeded into uncoated T25 or T75 flasks (Becton Dickinson, Bedford, MA, USA) at a cell concentration of 1×105

**i.** *(M1) : basic growth* medium consisting of alpha-Minimum Essential Mediums (alpha

**ii.** (M2): basic growth medium with fetal bovine serum and FGF2 replaced by 5 % (v/v)

**iii.** (M3): basic growth medium with fetal bovine serum and FGF2 replaced by 10 %

We induced the differentiation of the MSCs when they reached the confluence. We used three

differentiation mediums according to the focused differentiation line:

(v/v) h (HPL). Cell cultures were incubated at 37°C in a 5% CO2 humidified atmos‐ phere. The medium was changed twice weekly thereafter. When reaching 60% -80% confluence, the adherent cells were detached after treatment with 0. 05% (v/v) trypsin/ 1 mM EDTA solution (Gibco BRL) and expanded by replating at a lower density at

. All the studies were performed after 2 passages (P2).


cells/

galactins possesses several functions and were expressed in many tissues.

galectins' levels are altered in various cancers. [8,9,10]

in different culture mediums

34 Stem Cell Biology in Normal Life and Diseases

**2. Materiel and methods**

**2.1. Preparation of hBM-MSC**

France)

103

cells per cm2

cm2

donors was 30 ± 2 years (range, 15-40 years).

human patelet lysed (HPL)

**2.2. Induction of the differentiation directions**

for BM cells and cultured in three condition mediums:

At 50% confluence, the cells were cultured for 14 days in DMEM-HG (High Glucose 4. 5 g/L) containing 2% FBS, 0. 1 µM dexamethasone (Sigma), 2 mM β-glycerolphosphate (Sigma), and 100 µM ascorbate-2-phosphate (Sigma) with medium changes every 3 days. After 2 weeks of induction, the cells were stained according to Von Kossa's and Alizarin Red methods to detect the presence of calcium deposition into osteocytes.

#### *2.2.2. Adipogenic induction (A) for analysis galectins expression*

The adipogenic induction medium consisted of DMEM-LG (Low Glucose 1g/L) supplemented with 20% FBS, 1 µM/L dexamethasone, 0. 5 mmol/L isobutylmethylxanthine (IBMX; Invitro‐ gen), and 60 µmol/L indomethacin (Sigma). Adipogenic differentiation was evaluated after 2 weeks of induction, by the cellular accumulation of neutral lipid vacuoles that were stained with Nile Red (Sigma) and observed by fluorescent microscopy.

#### *2.2.3. Vascular smooth muscle induction (V) for analysis galectins expression*

Vascular smooth muscle (VSM) differentiation was obtained in a Mc Coy'5A medium supplemented with 12. 5% FBS, 12. 5% HS (Horse Serum, Invitrogen), 20 µM L-glutamine, 0,8 mM L-serine, 0,15 mM L-asparagine, 1 Mm sodium pyruvate, 5 mM sodium bicarbonate, 1 µM hydrocortisone and amphotericin B and antibodies. The medium was changed every 4 days. The VSM differentiation was evaluated after 3 weeks of induction.

#### **2.3. Immunophenotyping of mesenchymal stem cells by flow cytometry**

MSCs were immunophenotypically characterized by flow cytometry using the following fluorochrome (FITC PE, PerCP) marked monoclonal antibodies anti : CD45, CD105, CD106, CD90,CD49, CD34, et CD73 and CD14 (Becton Dickinson and Company BD Biosciences San Jose CA).

#### **2.4. Detection of galectins (Gal-1, Gal-9 Gal-11 and Gal-13), by flow cytometry**

To reveal the expression of the galactin we used biotynaled antibodies against human recombinant galectin (Gal-1, Gal-9, Gal-11 and Gal-13). Thus we incubated 1,5 106 MSCs cells with the antibody which is specific to the focused galectin, for 30 minutes, at 4° C. We incubated also the MSCs with a control antibody which doesn't recognize any protein, but has the same isotype to evaluate a background noise which correspond to non-specific fixation of the primary antibody.

The MSCs were washed 2 times with PBS 1X solution, then incubated for 30 minutes with Streptavidin coupled with a phycoerythrin (PE) fluorochrome phycoerythrin (PE). Flow cytometry analysis was performed on a FACS calibur, and data were analyzed using CellQuest software (Becton–Dickinson, BD Biosciences San Jose, CA).

#### **2.5. Galectins immunofluorescence detection**

MSCs were fixed using 4% Methanol for 30 minutes at +4°C and washed with PBS. The primary antibodies against many anti-antibodies: Gal-1-Streptavidine, Gal-3-Streptavidine, Gal-9- Streptavidin and Gal-13 Streptavidin were diluted in 10% BSA and 0. 2% Tween 20 (1:100) and were incubated at 4°C for 12h followed by washings 3 times with PBS. For immunofluorescence non-specific binding sites were blocked with 10% BSA-PBS. For secondary immunofluorecence Biotine –fluoroscein were diluted in 10% BSA-PBS and incubated for 60 min at +4°C in the dark. Images were taken with camera and with inverse fluorescence microscopy

*2.6.3. Reverse transcription – Polymerase chain reaction before and after induction for*

min, 42°C for 60 min and 95°C for 5 min. Primers used are shown in Table 2.

**Construct Sequence forward Sequence reverse**

After denaturation at 65°C for 5 min, amplification was carried out by 30 cycles at 30°C for 10

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GAPDH 5'AATCCCATCACCATCTTCCAGG3' 5'AGAGGCAGGGATGATGTTCTGG3' Gal 1 5'ATGGCTTGTGGTCTGGTC3' 5'TCAGTCAAAGGCCACACA3' Gal 9 5'ATGGCCTTCAGCGGTTCC3' 5'CTATGTCTGCACATGGGTCAG3' Gal 11 5'ATGAGTCAGCCCAGTGGG3' 5'TCAGGAGTGGACACAGTAGAG3' Gal 13 5'ATGTCCCTGACCCACAG3' 5'TCAATCGCTGATAAGCACT3'

Thermocycling was performed with a gradient thermocycler (Takara, Japan and Applied Biosystems). The analyse of the PCR products was carried out in an electrophoresis gel 1%

Data are expressed as mean ± standard error of the mean. Statistical comparisons were performed using the t-test student or Mann Whitney test with the program Graphpad, version. 5. A one-way analysis of variance (ANOVA) was done for paired samples; p < 0. 05 was

**3.1. Morphologic analysis of MSCs derived from bone marrow in terms of culture mediums**

To prepare MSCs cultures, we isolated mononuclear cells (MNCs) from bone marrow (hBM).

Adherent cell populations from the MNC fraction of hBM samples were generated by expansion culture using 3 different media: (M1) Alpha MEM with 10% FBS and 1 ng/mL FGF2, (M2) Alpha MEM with 5% HPL, (M3) Alpha with 10% HPL. After two weeks of culture, an adherent and stable cell layer was obtained from BM derived MNC with all medium Mean time for the primary culture to reach subconfluence was 15 days. After one passage(P1), adherent cells displayed a fibroblast-like morphology in culture plate (n=20). The figure 1

The MNCs derived from bone marrow were put in a density of 1. 10⁶ cells / cm².

*Galectin1,Gal-9,Gal-11 and Gal-13 expression*

**Table 2.** Sequences of the primers used in RT-PCR study

considered to be statistically significant.

showed a particular morphology of cultured MSC.

(Sigma).

**3. Results**

**2.7. Statistical analysis**

#### **2.6. RNA isolation and reverse transcription – Polymerase chain reaction before and after induction**

#### *2.6.1. RNA isolation*

For RT-PCR, Total RNA was extracted from cell culture in total confluence [90% ) using TRIZOL reagent (Gibco BRL) according to the manufacturer's instructions. Reverse transcrip‐ tion was carried out using PrimeScript™RTase (Takara: Japan and Applied Biosystems) and the cDNA fragments were amplified using RNase Inhibitor (Takara: Japan and Applied Biosystems): For each reaction, we mixed 5 µl of ARN or 5µl of distilled water (for the control), 6 µl desoxyribonucleotides (dNTP)(TaKaRa Japan and Applied Biosystems), 1µl primers specific of the gene sequence (table 1) et 0. 5 µl of enzyme la Taq polymerase enzyme (Takara: Japan and Applied Biosystems).

#### *2.6.2. RT-PCR before and after induction for the confirmation of the three directions of differentiation*

After denaturation at 65°C for 5 min, amplification was carried out by 30 cycles at 30°C for 10 min, 42°C for 60 min and 95°C for 5 min. Primers used are shown in Table 1. The expression of the following genes were analyzed before (D0) and after differentiation (D14) into three directions of differentiation: osteogenesis (O), adipogenesis (A) and vascular smooth muscle (V). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was used as control. The sequences of the oligonucleotides were reported in Table 1. Thermocycling was performed with a gradient thermocycler (Takara, Japan and Applied Biosystems). The analyse of the PCR products was carried out in an electrophoresis gel 1% (Sigma).


**Table 1.** Sequences of the primers used in RT-PCR study

*2.6.3. Reverse transcription – Polymerase chain reaction before and after induction for Galectin1,Gal-9,Gal-11 and Gal-13 expression*

After denaturation at 65°C for 5 min, amplification was carried out by 30 cycles at 30°C for 10 min, 42°C for 60 min and 95°C for 5 min. Primers used are shown in Table 2.


**Table 2.** Sequences of the primers used in RT-PCR study

Thermocycling was performed with a gradient thermocycler (Takara, Japan and Applied Biosystems). The analyse of the PCR products was carried out in an electrophoresis gel 1% (Sigma).

#### **2.7. Statistical analysis**

Data are expressed as mean ± standard error of the mean. Statistical comparisons were performed using the t-test student or Mann Whitney test with the program Graphpad, version. 5. A one-way analysis of variance (ANOVA) was done for paired samples; p < 0. 05 was considered to be statistically significant.

#### **3. Results**

**2.5. Galectins immunofluorescence detection**

36 Stem Cell Biology in Normal Life and Diseases

**induction**

*2.6.1. RNA isolation*

Japan and Applied Biosystems).

**Table 1.** Sequences of the primers used in RT-PCR study

MSCs were fixed using 4% Methanol for 30 minutes at +4°C and washed with PBS. The primary antibodies against many anti-antibodies: Gal-1-Streptavidine, Gal-3-Streptavidine, Gal-9- Streptavidin and Gal-13 Streptavidin were diluted in 10% BSA and 0. 2% Tween 20 (1:100) and were incubated at 4°C for 12h followed by washings 3 times with PBS. For immunofluorescence non-specific binding sites were blocked with 10% BSA-PBS. For secondary immunofluorecence Biotine –fluoroscein were diluted in 10% BSA-PBS and incubated for 60 min at +4°C in the

**2.6. RNA isolation and reverse transcription – Polymerase chain reaction before and after**

For RT-PCR, Total RNA was extracted from cell culture in total confluence [90% ) using TRIZOL reagent (Gibco BRL) according to the manufacturer's instructions. Reverse transcrip‐ tion was carried out using PrimeScript™RTase (Takara: Japan and Applied Biosystems) and the cDNA fragments were amplified using RNase Inhibitor (Takara: Japan and Applied Biosystems): For each reaction, we mixed 5 µl of ARN or 5µl of distilled water (for the control), 6 µl desoxyribonucleotides (dNTP)(TaKaRa Japan and Applied Biosystems), 1µl primers specific of the gene sequence (table 1) et 0. 5 µl of enzyme la Taq polymerase enzyme (Takara:

*2.6.2. RT-PCR before and after induction for the confirmation of the three directions of differentiation*

After denaturation at 65°C for 5 min, amplification was carried out by 30 cycles at 30°C for 10 min, 42°C for 60 min and 95°C for 5 min. Primers used are shown in Table 1. The expression of the following genes were analyzed before (D0) and after differentiation (D14) into three directions of differentiation: osteogenesis (O), adipogenesis (A) and vascular smooth muscle (V). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was used as control. The sequences of the oligonucleotides were reported in Table 1. Thermocycling was performed with a gradient thermocycler (Takara, Japan and Applied Biosystems). The analyse

of the PCR products was carried out in an electrophoresis gel 1% (Sigma).

**Construct Sequence forward Sequence reverse**

GAPDH 5'- AATCCCATCACCATCTTCCAGG-3' 5'-AGAGGCAGGGATGATGTTCTGG-3' PAL 5'-CTGGACCTCGTTGACACCTG-3' 5'-GACATTCTCTCGTTCACCGC-3' Runx2 5'-AACTTCCTGTGCTCGGTGCTG-3' 5'-GGGGAGGATTTGTGAAGACGG-3' LPL 5'-AAAGCCCTGCTCGTGCTGAC-3' 5'-TAAACCGGGCCACATCCTGT-3' PPRγ 5'-GGAGAAGCTGTTGGCGGAGA-3' 5'-TCAAGGAGGCCAGCATTGTG-3' ASMA 5'-TCATGATGCTGTTGTAGGTGGT-3' 5'-CTGTTCCAGCCATCCTTCAT-3'

dark. Images were taken with camera and with inverse fluorescence microscopy

#### **3.1. Morphologic analysis of MSCs derived from bone marrow in terms of culture mediums**

To prepare MSCs cultures, we isolated mononuclear cells (MNCs) from bone marrow (hBM). The MNCs derived from bone marrow were put in a density of 1. 10⁶ cells / cm².

Adherent cell populations from the MNC fraction of hBM samples were generated by expansion culture using 3 different media: (M1) Alpha MEM with 10% FBS and 1 ng/mL FGF2, (M2) Alpha MEM with 5% HPL, (M3) Alpha with 10% HPL. After two weeks of culture, an adherent and stable cell layer was obtained from BM derived MNC with all medium Mean time for the primary culture to reach subconfluence was 15 days. After one passage(P1), adherent cells displayed a fibroblast-like morphology in culture plate (n=20). The figure 1 showed a particular morphology of cultured MSC.

Isotype

CD90

CD105

CD106

CD73

CD49

CD34

CD14

CD45

Control

αMEM+FGF2 + 10 % FBS

**Figure 2.** Representative flow cytometry analysis of BM-MSCs.Comparison of membrane antigen expression of BM-

MSCs cultured at P2 in different media: M1 :(10% FBS+FGF2) M2: (5% HPL), M3: (10% HPL)

αMEM+ 5%HPL

αMEM+ 10%HPL

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39

**Figure 1.** Morphological characterization of hMSC-BM cultured in different media in the P2 (passage): M1: (10% FBS +FGF2), M2:(5% HPL), M3:(10% HPL).

**hM**

**MSC-BM**

**M1** 

38 Stem Cell Biology in Normal Life and Diseases

**M2** 

**M3** 

+FGF2), M2:(5% HPL), M3:(10% HPL).

**Figure 1.** Morphological characterization of hMSC-BM cultured in different media in the P2 (passage): M1: (10% FBS

**Figure 2.** Representative flow cytometry analysis of BM-MSCs.Comparison of membrane antigen expression of BM-MSCs cultured at P2 in different media: M1 :(10% FBS+FGF2) M2: (5% HPL), M3: (10% HPL)

     

Also MSCs were immunophenotypically characterized by flow cytometry. This analysis revealed that MSCs were uniformly positive for CD73 CD90 CD105 CD106 but negative for CD14 CD34 and CD45 (figure 2).

The influence of HPL or FBS on the osteogenic and adipogenic differentiation potential of MSCs was investigated after appropriate induction at the second passage (P2). Cells grown in HPL or FBS deposited an extensive mineralized matrix when cultured for 2 weeks in an osteogenic medium [2% FBS), as demonstrated by strong alizarin red staining. These cells also efficiently differentiated into the adipogenic lineage, as indicated by Oil Red O staining of lipid droplets in the cytoplasm following culture in an adipogenic medium.

All populations of BM-MSCs cultured in different conditions with the HPL showed osteogenic, Vascular smooth muscle and chondrogenic differentiation capacity. The difference appears in adipogenic differentiation with 10% HPL (Fig 3 ).

ogenic, adipog

Osteo

 differentia ation capacity assessed by s taining of BM M-derived MS Cs

scular smooth

muscular

To confirm their differentiation potential, after P1 MSCs were plated in specific induction

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41

To select the differentiated MSCs, we used the RT-PCR to search markers specific for the three differentiation directions. These markers are the alkalin phosphatase (PAL) as osteoblast specific marker the lipoprotein lipase (LPL)as specific of the adipocyte line and the actin-ASMA specific of vascular smooth muscles line. The positive control is the GAPDH. These markers were not expressed before the induction of the differentiation and presented an overexpression after the 14th day from the induction of specific differentiation in the three expansion mediums (M1, M2,

mediums to generate for adipocytes, osteoblasts, or chondrocytes.

et M3) demonstrating the multipotent nature of the MSCs (figure 4).

 **A B**

 **BM (D0) BM (D14)**  M1 M2 M3 M1 M2 M3

**ASMA** 

**PPRγ** 

**Runx 2** 

**PAL** 

**GAPDH** 

**Figure 4.** RT-PCR analyses of vascular smooth muscle (ASMA), adipogenic (PPRγ), and osteogenic (Runx2, PAL) mark‐ ers prior to (D0) (**A**) and after 14 days of differentiation induction (D14) (**B**) of BM-derived MSCs previously cultured in

**3.2. Detection of galectins in MSCs in terms of the culture mediums by flow cytometry**

MSCs from different BM samples were characterized by flow cytometry with a panel of biotynaled antibodies against human recombinant galectin (Gal-1, Gal-9, Gal-11 and Gal-13) at P2 after culture in the three mediums (M1, M2, et M3). Flow cytometry analysis revealed that MSC derived-hBM constitutively expressed galectins (Gal-1, Gal-9, Gal-11, Gal-13) at

expansion media (M1, M2, M3)

genic, and vas

**Figure 3.** Morphological characterization of hBM- MSCs derived cultured in different media M1, M2 and M3 at P2 and Osteogenic, chondrogenic, adipogenic, and vascular smooth muscular differentiation capacity assessed by staining

To confirm their differentiation potential, after P1 MSCs were plated in specific induction mediums to generate for adipocytes, osteoblasts, or chondrocytes.

Also MSCs were immunophenotypically characterized by flow cytometry. This analysis revealed that MSCs were uniformly positive for CD73 CD90 CD105 CD106 but negative for

The influence of HPL or FBS on the osteogenic and adipogenic differentiation potential of MSCs was investigated after appropriate induction at the second passage (P2). Cells grown in HPL or FBS deposited an extensive mineralized matrix when cultured for 2 weeks in an osteogenic medium [2% FBS), as demonstrated by strong alizarin red staining. These cells also efficiently differentiated into the adipogenic lineage, as indicated by Oil Red O staining of lipid

All populations of BM-MSCs cultured in different conditions with the HPL showed osteogenic, Vascular smooth muscle and chondrogenic differentiation capacity. The difference appears in

> ogenic, adipog ation capacity **\_\_\_\_\_\_\_\_\_\_ arin Red**

genic, and vas assessed by s **\_\_\_\_\_\_\_\_\_\_ Nil R (A)**

scular smooth taining of BM **\_\_\_\_\_\_\_\_\_\_**

muscular M-derived MS **\_\_\_\_\_\_\_\_\_\_ ASMA (VSM)** 

Cs **\_\_**

**Red )** 

**Figure 3.** Morphological characterization of hBM- MSCs derived cultured in different media M1, M2 and M3 at P2 and Osteogenic, chondrogenic, adipogenic, and vascular smooth muscular differentiation capacity assessed by staining

droplets in the cytoplasm following culture in an adipogenic medium.

Osteo differentia  **\_\_\_\_\_\_ Aliza (0)** 

adipogenic differentiation with 10% HPL (Fig 3 ).

 **gical Cs** 

CD14 CD34 and CD45 (figure 2).

40 Stem Cell Biology in Normal Life and Diseases

 **M h**

**Morpholog hBM MSC**

**M1**

 **M2**

**M2**

To select the differentiated MSCs, we used the RT-PCR to search markers specific for the three differentiation directions. These markers are the alkalin phosphatase (PAL) as osteoblast specific marker the lipoprotein lipase (LPL)as specific of the adipocyte line and the actin-ASMA specific of vascular smooth muscles line. The positive control is the GAPDH. These markers were not expressed before the induction of the differentiation and presented an overexpression after the 14th day from the induction of specific differentiation in the three expansion mediums (M1, M2, et M3) demonstrating the multipotent nature of the MSCs (figure 4).

**Figure 4.** RT-PCR analyses of vascular smooth muscle (ASMA), adipogenic (PPRγ), and osteogenic (Runx2, PAL) mark‐ ers prior to (D0) (**A**) and after 14 days of differentiation induction (D14) (**B**) of BM-derived MSCs previously cultured in expansion media (M1, M2, M3)

#### **3.2. Detection of galectins in MSCs in terms of the culture mediums by flow cytometry**

MSCs from different BM samples were characterized by flow cytometry with a panel of biotynaled antibodies against human recombinant galectin (Gal-1, Gal-9, Gal-11 and Gal-13) at P2 after culture in the three mediums (M1, M2, et M3). Flow cytometry analysis revealed that MSC derived-hBM constitutively expressed galectins (Gal-1, Gal-9, Gal-11, Gal-13) at different mediums but with unequal percentages. We noticed that galectins 9 and 11 are more expressed by cells derived from hBM with an average of [88. 1 ± 1. 8 % ) in M1 Meduim in comparison to M2 (16,8 ± 3. 9 % ) and M3 (11,6 ± 3. 9% ) with p < 0. 01.

We noticed also that galectin 1 is expressed with in average of (91. 2 ± 1. 4 % ) at P2 after culture in the three mediums (M1,M2,M3). In the contrary no expression of galactin 13 have been noted in any medium. (figure 5)

Gal 1

 st (a

tandard med alpha MEM+

dium (M1) +FGF)

Medium HPL 5% (M2) Medium HPL 10% (M3)

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**Figure 6.** Immunofluorescence Galectin-1, Galectin-9, Galectin -11 and Galactin-13 expression by cultured at P2 in dif‐

ferent media: M1 (10% FBS+FGF2) M2 (5% HPL), M3 (10% HPL)

Gal 9

Gal 11

Gal 13

**Figure 5.** Galectins Gal-1, Gal-9,Gal-11 and Gal-13 antigen expression of BM-MSCs cultured at P2 in different media: M1 (10% FBS+FGF2) M2(5% HPL), M3 (10% HPL) by flow cytometry

#### *3.2.1. Expression of galectins of MSC-BM by microscopy immunofluorecence*

Immunofluorecsence of galectin-1 in MSC of normal adult bone marrow was showed expres‐ sion in three mediums M1,M2,M3. Following this technique, we are also identified galectins gal-9 and gal-11 expression but only in standard medium M1. No expression of gal- 13 have been reveled by hBM-CSM cultured in all media M1,M2 and M3. we are determined the localization of galectin mostly in nucleus cells. (Fig6).

different mediums but with unequal percentages. We noticed that galectins 9 and 11 are more expressed by cells derived from hBM with an average of [88. 1 ± 1. 8 % ) in M1 Meduim in

We noticed also that galectin 1 is expressed with in average of (91. 2 ± 1. 4 % ) at P2 after culture in the three mediums (M1,M2,M3). In the contrary no expression of galactin 13 have been noted

**Figure 5.** Galectins Gal-1, Gal-9,Gal-11 and Gal-13 antigen expression of BM-MSCs cultured at P2 in different media:

Immunofluorecsence of galectin-1 in MSC of normal adult bone marrow was showed expres‐ sion in three mediums M1,M2,M3. Following this technique, we are also identified galectins gal-9 and gal-11 expression but only in standard medium M1. No expression of gal- 13 have been reveled by hBM-CSM cultured in all media M1,M2 and M3. we are determined the

M1 (10% FBS+FGF2) M2(5% HPL), M3 (10% HPL) by flow cytometry

localization of galectin mostly in nucleus cells. (Fig6).

*3.2.1. Expression of galectins of MSC-BM by microscopy immunofluorecence*

comparison to M2 (16,8 ± 3. 9 % ) and M3 (11,6 ± 3. 9% ) with p < 0. 01.

in any medium. (figure 5)

42 Stem Cell Biology in Normal Life and Diseases

**Figure 6.** Immunofluorescence Galectin-1, Galectin-9, Galectin -11 and Galactin-13 expression by cultured at P2 in dif‐ ferent media: M1 (10% FBS+FGF2) M2 (5% HPL), M3 (10% HPL)

MSCs cultivated in M2, M3, and M4. And After the MSCs differentiation Gal-11 was no more expressed in the standard culture's medium (M1 : MEM+ FGF2), while MSCs culti‐ vated in mediums containing the HPL (M2, M3) expressed the Gal- 11 in adipocytes (A) and vascular smooth muscle cells (VML). As to Gal-13, we reported that this galectin wasn't expressed by the undiferenciated MSCs neither in the standard medium nor in the mediums containing the HPL (M1, M2, and M3). Whereas, after the MSCs differentiation, Gal-13 was

Human Platelet Lysate for MSC Expression Galectins

http://dx.doi.org/10.5772/55383

45

Multipotent mesenchymal stromal cells (MSCs) have generated a great debate as a potential source of cells for cell-based therapeutic strategies primarily owing to their intrinsic ability to selfrenewanddifferentiate intofunctional celltypes that constitutethe tissueinwhichtheyexist and also because MSCs express a large number of molecules including the galectins. [11,12]

The galectins are a family of soluble lectins characterized by their affinity for β-galactoside residues. In recent years, galectins have become a major focus of investigation because of their

Given that galectins have been shown to have important effects [13,14] we decided to evaluate

In this study we analyzed MSCs cultured and expanded in three conditions mediums: *basic growth* medium consisting of alpha-Minimum Essential Medium in which HPL replaced FBS in order to investigate their morphology, plasticity, proliferation differentiation to osteoblasts, adipocytes, and vascular smooth muscles and their capacity to express galectins in terms of

In our laboratory MSCs were characterized and defined according to the International Society for Cellular Therapy minimal criteria. [17,18]. So first, we demonstrated that BM-MSC adhered to plastic. Second as measured by flow cytometry all of MSC derived from BM-MSC were nearly 100% positive for the makers CD90 CD105, and CD73 (≥95% ), while they lack the expression of CD45 CD34, and CD14 (≤ 2% ). Third, BM-MSC differentiated to osteoblasts

First, we investigated the effect of culture mediums on the adherence and the proliferation of MSCs. Then we demonstrated that the three mediums allow the adherence of MSCs to plastic and their proliferation, but the percentage of confluence and the times it takes to get it, change in terms of the composition of the culture medium. So, the culture mediums containing 5% HPL induced the best MSCs confluence. Thus the most important confluence of the bone marrow's MSCs was reached at the first passage (P1) in the culture's medium 5% HPL after 30 days from their implantation. So, we confirmed that HPL-expanded MSCs show a significantly higher proliferation rate, in comparison with MSCs expanded in the standard medium. So, we can use the HPL as a substitute of FBS in mediums specific to the culture of MSCs destined for clinical

applications, to avoid a possible xenogenic contamination caused by the FBS. [19]

involvement in various physiological and pathological processes. [13,14]

the expression of galectins (Gal-1, Gal-3, Gal-9 and Gal-13) by MSCs.

only expressed by adipocytes. (figure 5)

the mediums' compositions. [15,16]

adipocytes and vascular smooth cells.

**4. Discussion**

**Figure 7.** RT –PCR Galectin-1, Galectin-9, Galectin -11 and Galactin-13 expression by cultured at P2 in different media: M1 (10% FBS+FGF2) M2 (5% HPL), M3 (10% HPL) and after differentiation process ( O: osteogenic induction, A: Adipo‐ genic induction,V: Vascular Smooth Muscle induction, GAPDH : Glyceraldheyde 3-phospate dehydrogenase, PM: Weight molecular )

#### **3.3. RT –PCR galectin-1,Gal-9,Gal-11 and Gal-13 expression by hBM-MSCs**

By RT-PCR, we evaluated the messenger RNA expression of Galectin-1,Galectin-9, Galectin-11 and Galectin -13 in hBM-MSCs before and after differentiation to the three direction : osteo‐ blastic [0), adipocyte (A) and to vascular smooth muscle (VSM).

We had a positive results for hBM-MSCs both before their differentiation and after the induction of the three differentiation directions:


MSCs cultivated in M2, M3, and M4. And After the MSCs differentiation Gal-11 was no more expressed in the standard culture's medium (M1 : MEM+ FGF2), while MSCs culti‐ vated in mediums containing the HPL (M2, M3) expressed the Gal- 11 in adipocytes (A) and vascular smooth muscle cells (VML). As to Gal-13, we reported that this galectin wasn't expressed by the undiferenciated MSCs neither in the standard medium nor in the mediums containing the HPL (M1, M2, and M3). Whereas, after the MSCs differentiation, Gal-13 was only expressed by adipocytes. (figure 5)

### **4. Discussion**

Gal 1

standar (alpha

44 Stem Cell Biology in Normal Life and Diseases

rd medium ( MEM+FGF)

M1) Medium HPL 5% (M2) Medium HPL 10% (M3)

**Figure 7.** RT –PCR Galectin-1, Galectin-9, Galectin -11 and Galactin-13 expression by cultured at P2 in different media: M1 (10% FBS+FGF2) M2 (5% HPL), M3 (10% HPL) and after differentiation process ( O: osteogenic induction, A: Adipo‐ genic induction,V: Vascular Smooth Muscle induction, GAPDH : Glyceraldheyde 3-phospate dehydrogenase, PM:

By RT-PCR, we evaluated the messenger RNA expression of Galectin-1,Galectin-9, Galectin-11 and Galectin -13 in hBM-MSCs before and after differentiation to the three direction : osteo‐

We had a positive results for hBM-MSCs both before their differentiation and after the

**•** before the induction of the differentiation process :Gal-1 was expressed by hBM- MSCs in the standard medium M1 (MEM+ FGF2) and also in the three mediums (M2, M3, et M4) which contain the human platelet lysat (HPL). While, after the induction of the MSCs differentiation, Gal-1 was only expressed in MSCs cultivated in mediums containing the

**•** Concerning the expression of Gal-9 by BM-MSCs: we observed that Gal-9 was expressed by undifferentiated BM-MSCs cultivated in the standard medium (M1: MEM+ FGF2), but absent in MSCs cultivated in mediums containing the HPL (M2, M3). However, after inducing the differentiation process, this galectin was absent in MSCs of the standard

**•** Regarding the expression of Gal-11 by BM-MSCs, we found that Gal- 11 was expressed by undifferenciated BM-MSCs, in the standard medium M1 (MEM+ FGF2) but absent in BM-

medium and had a very low expression in mediums containing the HPL (M2, M3).

**3.3. RT –PCR galectin-1,Gal-9,Gal-11 and Gal-13 expression by hBM-MSCs**

blastic [0), adipocyte (A) and to vascular smooth muscle (VSM).

HPL (M2, M3), in the three differentiation directions (O, A, VML)

induction of the three differentiation directions:

Gal 9

Gal 11

Gal 13

Weight molecular )

Multipotent mesenchymal stromal cells (MSCs) have generated a great debate as a potential source of cells for cell-based therapeutic strategies primarily owing to their intrinsic ability to selfrenewanddifferentiate intofunctional celltypes that constitutethe tissueinwhichtheyexist and also because MSCs express a large number of molecules including the galectins. [11,12]

The galectins are a family of soluble lectins characterized by their affinity for β-galactoside residues. In recent years, galectins have become a major focus of investigation because of their involvement in various physiological and pathological processes. [13,14]

Given that galectins have been shown to have important effects [13,14] we decided to evaluate the expression of galectins (Gal-1, Gal-3, Gal-9 and Gal-13) by MSCs.

In this study we analyzed MSCs cultured and expanded in three conditions mediums: *basic growth* medium consisting of alpha-Minimum Essential Medium in which HPL replaced FBS in order to investigate their morphology, plasticity, proliferation differentiation to osteoblasts, adipocytes, and vascular smooth muscles and their capacity to express galectins in terms of the mediums' compositions. [15,16]

In our laboratory MSCs were characterized and defined according to the International Society for Cellular Therapy minimal criteria. [17,18]. So first, we demonstrated that BM-MSC adhered to plastic. Second as measured by flow cytometry all of MSC derived from BM-MSC were nearly 100% positive for the makers CD90 CD105, and CD73 (≥95% ), while they lack the expression of CD45 CD34, and CD14 (≤ 2% ). Third, BM-MSC differentiated to osteoblasts adipocytes and vascular smooth cells.

First, we investigated the effect of culture mediums on the adherence and the proliferation of MSCs. Then we demonstrated that the three mediums allow the adherence of MSCs to plastic and their proliferation, but the percentage of confluence and the times it takes to get it, change in terms of the composition of the culture medium. So, the culture mediums containing 5% HPL induced the best MSCs confluence. Thus the most important confluence of the bone marrow's MSCs was reached at the first passage (P1) in the culture's medium 5% HPL after 30 days from their implantation. So, we confirmed that HPL-expanded MSCs show a significantly higher proliferation rate, in comparison with MSCs expanded in the standard medium. So, we can use the HPL as a substitute of FBS in mediums specific to the culture of MSCs destined for clinical applications, to avoid a possible xenogenic contamination caused by the FBS. [19]

The expansion-promoting effect of the HPL is likely to result from the high concentration of natural growth factors that HPLcontains.In factplatelet granules containmany growth factors, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor (TGFb), platelet factor 4 (PF-4), and plateletderivedepidermalgrowthfactor(PDEGF)[20,21].Thesegrowthfactors,whicharereleasedfrom platelet lysate, have been shown to enhance MSC expansion in vitro. [20, 21,22,23,24]

We noted that MSCs expressed the galectins constitutively, but this expression was modulated differently depending on the type of the galectin the culture's stage and the composition of

Human Platelet Lysate for MSC Expression Galectins

http://dx.doi.org/10.5772/55383

47

These findings are in agreement with previous reports. Thus, Friederike Gieseke and al. [27] claim that m RNAs of Gal-1, Gal-3, -8 and -9 were detected in MSCs by the RT-PCR and when performing a quantitative real time –PCR the m RNAs of Gal-3, -8 and -9 were detected to a lesser extent than Gal-1 which is similar to the results we obtained when performing the flow

Najar M and al. [14] confirmed the constitutive expression of galectin-1 m RNA by BM-MSCs. Regarding Gal-3 Ju-Yeon Kim and al. [28] demonstrated that Gal-3 is secreted by UCB-MSC under pathological conditions whereas low level of GAL-3 was detected by Western blot analysis in conditioned medium of naïve UCB-MSC. This report is in contrast with our results since we confirmed the absence of galectins' in both membranes and RNAs of UC-MSCs.

The present study provides new insights concerning the expression of the galectins by the MSCs. According to our observations and the reports mentioned above the galectins are located both in nuclear and membrane levels. This suggests that the galectins are released by MSCs. Because of their their multilineage differentiation potential, immunomodulatory and anti-inflammatory properties, MSCs have become increasingly attractive as a therapeutic approach. Careful characterization of MSC physiology and the effects of culture mediums context on their proliferation differentiation and molecules' secretion will increase the safety

Address all correspondence to: faouzi.jenhani@yahoo.tn, tunisiacelltherapy@ymail.com

2 Unit Immunology Research, Faculty of pharmacy, Monastir, Tunisia

transplantation. Transplantation,2003, 75, 389–397.

1 Cell Immunology and Cytometry and Cell Therapy Laboratories, Blood National Center,

[1] Tse, W. T. , Pendleton, J. D. , Beyer, W. M. , Egalka, M. C. , Guinan, E. C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in

the culture medium.

and efficiency of MSCs in clinical settings.

**Author details**

Faouzi Jenhani1,2\*

Tunisia

**References**

cytometry.

Second, we analyzed the morphology of MSC, and observed fibroblast-like cells in BM. Thus the MSCs formed a monolayer of homogenous spindle-shaped cells with a whirlpool-like array. This result is in accordance with the literature. [25]. However, two cell phenotypes were observed among fibroblast-like BM-MSC colonies: thin spindle shaped MSC and star-shaped MSC. These BM-MSC two phenotypes were described by Wolgang et Wagnera and al [25]

To detect the expression of galectins Gal-1, Gal-9, Gal-11 and Gal-13 by MSCs, we used two techniques which are the flow cytometry (FC) and the RT-PCR. We searched the expression of galectins in MSCs cultivated in the three culture mediums: the standard medium: alpha-MEM+ FGF2, the medium (with 5% HPL), the medium (with 10% HPL).

The flow cytometry revealed that all the galectins of interest (Gal-1, Gal-3, Gal-9, and Gal-13) are expressed in BM-MSCs, in above mentioned culture mediums,. It thus becomes clear that expression percents of galacetins revealed by the flow cytometry in hBM-MCSs depend on two parameters which are the composition of the medium and the type of the galectin.

Thus, we observed mainly that the best Galectins' expression was visualized in the standard medium (alpha-MEM /FGF2). Previous studies have shown that the supplementation of fibroblast growth factor 2 (FGF2) in vitro selects for the survival of a large number of of MSCs enriched in pluripotent mesenchymal precursors enhances the growth of MSCs maintains their multilineagedifferentiationpotentialduringinvitroexpansionandprolongs theirlifespan[26]. So, this data and our observation suggest that the increase of galectins in the presence of FGF2 isdue to the improvement ofthe growth theproliferation anddifferentiationpotential ofMSCs.

Then, when comparing the means of the galectins' expression by the MSCs we concluded that the best means of protein expression were those of Gal-1; this galectin had the highest means of expression in all culture mediums. Friederike Gieseke and al. confirm our result; thus, they report that Gal-1 is highly expressed in MSCs. [27]

The results of the RT-PCR are in accordance with those of the flow cytometry. Thus, the RT-PCR applied to RNA of the MSCs revealed that galectins (Gal-1, Gal-9, Gal-11, and Gal-13) are expressed by both undifferentiated and differentiated BM-MSCs. So, before the induction of the differentiation process, the MSCs cultivated in the standard medium expressed Gal-1, Gal-9, Gal-11, but not Gal-13, while the MSCs cultivated in the mediums enriched by the HPL expressed only the Gal-1. Nevertheless, after the induction of the MSCs differentiation, first we observed that Gal-1, Gal-9, Gal-11 were no more expressed in the standard culture's medium (alpha MEM+ FGF2) while we detected the expression of Gal-13 in the adipocytes. Second, when examining the expression of galectins by MSCs cultivated in mediums contain‐ ing the HPL we noticed the presence of Gal-1, Gal-9 with a low expression, Gal-11 in both adipocytes and VMLs and finally Gal-13 in adipocytes.

We noted that MSCs expressed the galectins constitutively, but this expression was modulated differently depending on the type of the galectin the culture's stage and the composition of the culture medium.

These findings are in agreement with previous reports. Thus, Friederike Gieseke and al. [27] claim that m RNAs of Gal-1, Gal-3, -8 and -9 were detected in MSCs by the RT-PCR and when performing a quantitative real time –PCR the m RNAs of Gal-3, -8 and -9 were detected to a lesser extent than Gal-1 which is similar to the results we obtained when performing the flow cytometry.

Najar M and al. [14] confirmed the constitutive expression of galectin-1 m RNA by BM-MSCs. Regarding Gal-3 Ju-Yeon Kim and al. [28] demonstrated that Gal-3 is secreted by UCB-MSC under pathological conditions whereas low level of GAL-3 was detected by Western blot analysis in conditioned medium of naïve UCB-MSC. This report is in contrast with our results since we confirmed the absence of galectins' in both membranes and RNAs of UC-MSCs.

The present study provides new insights concerning the expression of the galectins by the MSCs. According to our observations and the reports mentioned above the galectins are located both in nuclear and membrane levels. This suggests that the galectins are released by MSCs. Because of their their multilineage differentiation potential, immunomodulatory and anti-inflammatory properties, MSCs have become increasingly attractive as a therapeutic approach. Careful characterization of MSC physiology and the effects of culture mediums context on their proliferation differentiation and molecules' secretion will increase the safety and efficiency of MSCs in clinical settings.

#### **Author details**

The expansion-promoting effect of the HPL is likely to result from the high concentration of natural growth factors that HPLcontains.In factplatelet granules containmany growth factors, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor (TGFb), platelet factor 4 (PF-4), and plateletderivedepidermalgrowthfactor(PDEGF)[20,21].Thesegrowthfactors,whicharereleasedfrom

Second, we analyzed the morphology of MSC, and observed fibroblast-like cells in BM. Thus the MSCs formed a monolayer of homogenous spindle-shaped cells with a whirlpool-like array. This result is in accordance with the literature. [25]. However, two cell phenotypes were observed among fibroblast-like BM-MSC colonies: thin spindle shaped MSC and star-shaped MSC. These BM-MSC two phenotypes were described by Wolgang et Wagnera and al [25] To detect the expression of galectins Gal-1, Gal-9, Gal-11 and Gal-13 by MSCs, we used two techniques which are the flow cytometry (FC) and the RT-PCR. We searched the expression of galectins in MSCs cultivated in the three culture mediums: the standard medium: alpha-

The flow cytometry revealed that all the galectins of interest (Gal-1, Gal-3, Gal-9, and Gal-13) are expressed in BM-MSCs, in above mentioned culture mediums,. It thus becomes clear that expression percents of galacetins revealed by the flow cytometry in hBM-MCSs depend on two parameters which are the composition of the medium and the type of the galectin.

Thus, we observed mainly that the best Galectins' expression was visualized in the standard medium (alpha-MEM /FGF2). Previous studies have shown that the supplementation of fibroblast growth factor 2 (FGF2) in vitro selects for the survival of a large number of of MSCs enriched in pluripotent mesenchymal precursors enhances the growth of MSCs maintains their multilineagedifferentiationpotentialduringinvitroexpansionandprolongs theirlifespan[26]. So, this data and our observation suggest that the increase of galectins in the presence of FGF2 isdue to the improvement ofthe growth theproliferation anddifferentiationpotential ofMSCs. Then, when comparing the means of the galectins' expression by the MSCs we concluded that the best means of protein expression were those of Gal-1; this galectin had the highest means of expression in all culture mediums. Friederike Gieseke and al. confirm our result; thus, they

The results of the RT-PCR are in accordance with those of the flow cytometry. Thus, the RT-PCR applied to RNA of the MSCs revealed that galectins (Gal-1, Gal-9, Gal-11, and Gal-13) are expressed by both undifferentiated and differentiated BM-MSCs. So, before the induction of the differentiation process, the MSCs cultivated in the standard medium expressed Gal-1, Gal-9, Gal-11, but not Gal-13, while the MSCs cultivated in the mediums enriched by the HPL expressed only the Gal-1. Nevertheless, after the induction of the MSCs differentiation, first we observed that Gal-1, Gal-9, Gal-11 were no more expressed in the standard culture's medium (alpha MEM+ FGF2) while we detected the expression of Gal-13 in the adipocytes. Second, when examining the expression of galectins by MSCs cultivated in mediums contain‐ ing the HPL we noticed the presence of Gal-1, Gal-9 with a low expression, Gal-11 in both

platelet lysate, have been shown to enhance MSC expansion in vitro. [20, 21,22,23,24]

MEM+ FGF2, the medium (with 5% HPL), the medium (with 10% HPL).

report that Gal-1 is highly expressed in MSCs. [27]

46 Stem Cell Biology in Normal Life and Diseases

adipocytes and VMLs and finally Gal-13 in adipocytes.

Faouzi Jenhani1,2\*

Address all correspondence to: faouzi.jenhani@yahoo.tn, tunisiacelltherapy@ymail.com

1 Cell Immunology and Cytometry and Cell Therapy Laboratories, Blood National Center, Tunisia

2 Unit Immunology Research, Faculty of pharmacy, Monastir, Tunisia

#### **References**

[1] Tse, W. T. , Pendleton, J. D. , Beyer, W. M. , Egalka, M. C. , Guinan, E. C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation,2003, 75, 389–397.

[2] Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L, et al. Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lympho‐ cyte and natural killer function and to induce CD4 1 CD25 high FOXP31 regulatory T cells. Stem Cells 2008;26:212-222.

mal cells from small samples of bone marrow aspirates or marrow filter washouts.

Human Platelet Lysate for MSC Expression Galectins

http://dx.doi.org/10.5772/55383

49

[16] Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy X, et al. Platelet lysates pro‐ mote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-

[17] M Dominici K Le Blanc I Mueller I Slaper-Cortenbach FC Marini DJ Prockop and EM Horwitz. Minimal Criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy (2006) Vol

[18] Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytothera‐

[19] Berger,M. G. ,Veyrat-Masson,R. ,Rapatel,C. ,Descamps, S. , Chassagne,J. ,Boiret-Dupre,N. ,2006. Cellculture medium compositionandtranslationaladultbone marrow-

[21] Kilian O, Flesch I, Wenisch S, Taborski B, Jork A, Schnettler R, et al. Effects of platelet growth factors on human mesenchymal stem cells and human endothelial cells in vi‐

[22] Van den Dolder J, Mooren R, Vloon AP, Stoelinga PJ, Jansen JA. Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of

[23] Capelli C, Domenghini M, Borleri G, Bellavita P, Poma R, Carobbio A, et al. Human platelet lysate allows expansion and clinical grade production of mesenchymal stro‐ mal cells from small samples of bone marrow aspirates or marrow filter washouts.

[24] Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy X, et al. Platelet lysates pro‐ mote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-

[25] Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Wolfgang Wagnera, Frederik Weina, Anja Seckingera, Maria Frankhauserb, Ute Wirknerc, Ulf Krausea, Jonathon Blakec, Chris‐ tian Schwagerc, Volker Ecksteina, Wilhelm Ansorgec, and Anthony D. Hoa. Experi‐

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[2] Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L, et al. Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lympho‐ cyte and natural killer function and to induce CD4 1 CD25 high FOXP31 regulatory T

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**Chapter 4**

**Canonical HSC Markers and Recent Achievements**

A specific feature of hematopoietic stem cells (HSC) is the potency to supply all types of blood cells throughout life by self-renewal and differentiation. Bone marrow (BM) is actively producing differentiated blood cells with enormous cellular turnover. Under homeostatic state, primitive HSC in adult BM divide only rarely and are located in specialized regulatory environment to avoid exhaustion and DNA damages that are supposed to cumulatively de‐ velop hematopoietic disorders such as myelodysplastic syndrome or leukemia. However, those quiescent HSC can be proliferative on demand, particularly on systemic infection or myelo-suppressive treatment. Therefore, elaborate mechanisms regulating the self-renewal and differentiation of BM HSC is indispensable to maintain normal hematopoiesis through‐ out life. The fluctuating feature of HSC is thought to be associated with their regulatory en‐

Technical improvement for purifying authentic HSC from heterogeneous cellular popula‐ tions is necessary to understand the features of those extremely rare and precious cells and promote their therapeutic application. Many studies have attempted to identify their specif‐ ic markers, and now flow cytometry- based strategies have made it possible to sort HSC with high purity in mice. However, the source and the stage of HSC change along ontogeny, which consequently influence not only their functional abilities but also their surface immu‐ nophenotypes. In the light of fluctuating nature of HSC, it should be very important to un‐ derstand their phenotype specific to reconstitution activity of the immune system after

Hematopoietic cells and endothelial cells are both generated from mesodermal precursor cells in ontogeny [1]. Thereafter, HSC pool is formed in several anatomical sites such as aor‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Sudo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

Takao Sudo, Takafumi Yokota, Tomohiko Ishibashi,

Michiko Ichii, Yukiko Doi, Kenji Oritani and

Additional information is available at the end of the chapter

Yuzuru Kanakura

**1. Introduction**

http://dx.doi.org/10.5772/54474

vironment, generally called "HSC niche".

myelo-suppressive events.

[28] Ju-Yeon Kim and al: Galectin-3 secreted by human umbilical cord blood-derived mesenchymal stem cells reduces amyloid-b42 neurotoxicity in vitro Ju-Yeon Kim a,b, Dong Hyun Kim a, Dal-Soo Kim a, Ji Hyun Kim a, Sang Young Jeong a, Hong Bae Jeon a, Eun Hui Lee b, Yoon Sun Yang a, Wonil Oh a, Jong Wook Chang a,\* FEBS Letters 584 (2010) 3601–3608.

## **Canonical HSC Markers and Recent Achievements**

Takao Sudo, Takafumi Yokota, Tomohiko Ishibashi, Michiko Ichii, Yukiko Doi, Kenji Oritani and Yuzuru Kanakura

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54474

#### **1. Introduction**

[27] Friederike Gieseke and al : Friederike Gieseke, Judith Böhringer Rita Bussolari Massi‐ mo Dominci Rupert Handgretinger and Ingo Müller. Human multipotent mesenchy‐ mal stromal cells employ galectin-1 to inhibit immune effector cells. Jul 19, 2010; doi:

[28] Ju-Yeon Kim and al: Galectin-3 secreted by human umbilical cord blood-derived mesenchymal stem cells reduces amyloid-b42 neurotoxicity in vitro Ju-Yeon Kim a,b, Dong Hyun Kim a, Dal-Soo Kim a, Ji Hyun Kim a, Sang Young Jeong a, Hong Bae Jeon a, Eun Hui Lee b, Yoon Sun Yang a, Wonil Oh a, Jong Wook Chang a,\* FEBS

10. 1182/Blood-2010-02-270777.

50 Stem Cell Biology in Normal Life and Diseases

Letters 584 (2010) 3601–3608.

A specific feature of hematopoietic stem cells (HSC) is the potency to supply all types of blood cells throughout life by self-renewal and differentiation. Bone marrow (BM) is actively producing differentiated blood cells with enormous cellular turnover. Under homeostatic state, primitive HSC in adult BM divide only rarely and are located in specialized regulatory environment to avoid exhaustion and DNA damages that are supposed to cumulatively de‐ velop hematopoietic disorders such as myelodysplastic syndrome or leukemia. However, those quiescent HSC can be proliferative on demand, particularly on systemic infection or myelo-suppressive treatment. Therefore, elaborate mechanisms regulating the self-renewal and differentiation of BM HSC is indispensable to maintain normal hematopoiesis through‐ out life. The fluctuating feature of HSC is thought to be associated with their regulatory en‐ vironment, generally called "HSC niche".

Technical improvement for purifying authentic HSC from heterogeneous cellular popula‐ tions is necessary to understand the features of those extremely rare and precious cells and promote their therapeutic application. Many studies have attempted to identify their specif‐ ic markers, and now flow cytometry- based strategies have made it possible to sort HSC with high purity in mice. However, the source and the stage of HSC change along ontogeny, which consequently influence not only their functional abilities but also their surface immu‐ nophenotypes. In the light of fluctuating nature of HSC, it should be very important to un‐ derstand their phenotype specific to reconstitution activity of the immune system after myelo-suppressive events.

Hematopoietic cells and endothelial cells are both generated from mesodermal precursor cells in ontogeny [1]. Thereafter, HSC pool is formed in several anatomical sites such as aor‐

© 2013 Sudo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ta-gonad-mesonephros (AGM) region, placenta, fetal liver, and BM. At the early stage of on‐ togeny, HSC frequently undergo symmetrical and/or asymmetrical division to form entire hematopoietic system compared to adult HSC. Those early HSC and endothelial cells bear some common surface antigens, of which expression levels on HSC decline along aging. In‐ terestingly, some of the endothelial-related surface molecules revive on HSC after BM in‐ jury, when the cells actively divide to regenerate BM cells.

CD34-

(21%) of CD150+ CD48-

of CD150+ CD48-

in Table 1.

 KSL cells, which are extremely rare, representing only 0.001-0.01% of BM mononu‐ clear cells, are almost pure primitive hematopoietic cells that have long-term multilineage repopulating potency [10]. More recently, the endothelial protein C receptor CD201 was found as a new endothelial-related HSC marker which marks approximately 70% of the SP cells. The marker seems to be useful to purify LT-HSC among the SP cells because only the

In recent years, Kiel *et al.* demonstrated that a simple combination of SLAM family markers (CD150, CD244 and CD48) could enrich primitive murine HSC. That is, one out of every 4.8

constitution [12]. Furthermore, they observed that one out of every 2.1 (47%) of CD150+ CD48- LSK cells had long-term multilineage reconstituting potential. Approximately 15~20%

the mouse life span, have more long-term repopulating potential than other cells [13,14]. We can now purify dormant LT-HSC from murine BM using the SLAM family markers in com‐ bination with LSK gating. The information regarding murine HSC markers is summarized

**Markers References**

 Thy-1Lo Sca-1+ Spangrude *et al. Science* (1988) [4] CD34-/Lo LSK Osawa *et al*. *Science* (1996) [7] Side population (SP) Goodell *et al. J Exp Med* (1996) [9]

Tip-SP LSK Matsuzaki *et al. Immunity* (2004) [10]

SP Uchida *et al. Exp Hematol* (2003) [15]

LSK Kiel *et al. Cell* (2005) [12]

CD150+ LSK Foudi *et al. Nat Biotechnol* (2009) [13]

LSK Wilson *et al. Cell* (2008) [14]

cells from young adult murine BM gave long-term multilineage re‐

LSK cells, which divide only 5-6 times during

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

53

CD201 + subpopulation exhibited repopulating ability [11].

LSK or CD34- CD150+ CD48-

Lin-

CD48-

BrdU and histone 2B-retaining CD48-

Lin, lineage; Rho, Rhodamine-123; LSK, Lin-

Histone 2B-retaining CD48-

**Table 1.** Markers for adult murine hematopoietic stem cells.

Lin- Rho-

CD150+ CD41-

**3. HSC markers during developmental stages**

CD150+ CD34-

HSC markers during developmental stages are not identical to those of adult HSC. In the embryo, functional HSC that can reconstitute hematopoiesis in adult recipients are firstly found in the aorta-gonad-mesonephros (AGM) region at approximately embryonic day 10 (E10) [16-18]. Many reports have demonstrated that those earliest authentic HSC bud from endothelial-related cells, which involve the concept of "hemangioblast" or "hemogenic en‐ dothelium" [19-23]. In fact, emerging HSC and endothelial cells share various surface mark‐

Sca-1+ c-kit+

We recently reported endothelial cell-selective adhesion molecule (ESAM) as a new marker for HSC [2]. Interestingly, ESAM levels on HSC clearly mirror the shift of HSC between qui‐ escence and activation, and the up-regulation amplitude is prominent in comparison to oth‐ er HSC-related antigens [3]. Furthermore, we found that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis [3]. In this chapter, we review a wealth of information about traditional HSC markers, and introduce our recent findings.

#### **2. Development of the strategy for purifying HSC from murine BM**

HSC are defined by their capacity for both self-renewal and differentiation into all the blood cell types. In 1988, Spangrude *et al.* reported lineage (Lin; Mac-1, Gr-1, B220, CD4, and CD8)- Thy-1Lo Sca-1+ cells in mouse BM as a multipotent HSC population. When these cells were transplanted into lethally irradiated mice, only thirty cells were sufficient to save 50% of the recipient mice and reconstitute B, T, and myeloid cells [4]. In 1991, Ogawa *et al.* reported that half of the c-kit+ BM cells do not express Lin (Mac-1, Gr-1, Ter119, and B220) markers, and ckit- population do not include hematopoietic progenitor cells [5]. From then on, Lin- Sca-1+ ckit+ (LSK ) cells has been used as the population in which HSC are highly concentrated [6,7]. HSC can be functionally classified as either long-term (LT-HSC) or short-term (ST-HSC) ac‐ cording to their capacity to give rise to life-long or transient hematopoiesis. Osawa *et al.* showed that CD34+ LSK cells are capable of only short-term multilineage differentiation. In contrast, CD34-/Lo LSK cells have long-term multilineage reconstitution capacity. They also showed that CD34-/Lo LSK cells can differentiate into CD34+ LSK cells [7]. LSK fraction also can be divided into two populations by expression level of Flk-2. While LT-HSC are en‐ riched in the Flk-2- LSK fraction, the Flk-2+ LSK cells are mainly ST-HSC [8].

While the techniques of purifying HSC by use of surface markers had been promoted, Goodell *et al.* reported the method for purifying HSC without use of surface markers. Hoechst33342 is a fluorescent dye which binds to DNA of live cells. When Hoechst fluores‐ cence on whole BM was examined simultaneously at two emission wavelength (red and blue), one population of cells with increased ability to efflux Hoechst dye was observed. Goodell *et al.* named it "side population (SP)", and showed that a majority of HSC were en‐ riched in the SP by competitive repopulating experiments [9]. Subsequently, Matsuzaki *et al.* described a method of further purifying HSC by combining staining with antibodies to sur‐ face molecules with the Hoechst dye efflux. They showed the fraction of cells with the stron‐ gest dye efflux activity (termed as "Tip"-SP) has the highest marrow-repopulating activity. While 20% of "Tip"-SP cells are primitive hematopoietic cells, more than 90% of "Tip"-SP CD34- KSL cells, which are extremely rare, representing only 0.001-0.01% of BM mononu‐ clear cells, are almost pure primitive hematopoietic cells that have long-term multilineage repopulating potency [10]. More recently, the endothelial protein C receptor CD201 was found as a new endothelial-related HSC marker which marks approximately 70% of the SP cells. The marker seems to be useful to purify LT-HSC among the SP cells because only the CD201 + subpopulation exhibited repopulating ability [11].

In recent years, Kiel *et al.* demonstrated that a simple combination of SLAM family markers (CD150, CD244 and CD48) could enrich primitive murine HSC. That is, one out of every 4.8 (21%) of CD150+ CD48 cells from young adult murine BM gave long-term multilineage re‐ constitution [12]. Furthermore, they observed that one out of every 2.1 (47%) of CD150+ CD48- LSK cells had long-term multilineage reconstituting potential. Approximately 15~20% of CD150+ CD48- LSK or CD34- CD150+ CD48- LSK cells, which divide only 5-6 times during the mouse life span, have more long-term repopulating potential than other cells [13,14]. We can now purify dormant LT-HSC from murine BM using the SLAM family markers in com‐ bination with LSK gating. The information regarding murine HSC markers is summarized in Table 1.


**Table 1.** Markers for adult murine hematopoietic stem cells.

ta-gonad-mesonephros (AGM) region, placenta, fetal liver, and BM. At the early stage of on‐ togeny, HSC frequently undergo symmetrical and/or asymmetrical division to form entire hematopoietic system compared to adult HSC. Those early HSC and endothelial cells bear some common surface antigens, of which expression levels on HSC decline along aging. In‐ terestingly, some of the endothelial-related surface molecules revive on HSC after BM in‐

We recently reported endothelial cell-selective adhesion molecule (ESAM) as a new marker for HSC [2]. Interestingly, ESAM levels on HSC clearly mirror the shift of HSC between qui‐ escence and activation, and the up-regulation amplitude is prominent in comparison to oth‐ er HSC-related antigens [3]. Furthermore, we found that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis [3]. In this chapter, we review a wealth of

information about traditional HSC markers, and introduce our recent findings.

**2. Development of the strategy for purifying HSC from murine BM**

kit- population do not include hematopoietic progenitor cells [5]. From then on, Lin-

showed that CD34-/Lo LSK cells can differentiate into CD34+

LSK fraction, the Flk-2+

HSC are defined by their capacity for both self-renewal and differentiation into all the blood cell types. In 1988, Spangrude *et al.* reported lineage (Lin; Mac-1, Gr-1, B220, CD4, and CD8)-

transplanted into lethally irradiated mice, only thirty cells were sufficient to save 50% of the recipient mice and reconstitute B, T, and myeloid cells [4]. In 1991, Ogawa *et al.* reported that half of the c-kit+ BM cells do not express Lin (Mac-1, Gr-1, Ter119, and B220) markers, and c-

kit+ (LSK ) cells has been used as the population in which HSC are highly concentrated [6,7]. HSC can be functionally classified as either long-term (LT-HSC) or short-term (ST-HSC) ac‐ cording to their capacity to give rise to life-long or transient hematopoiesis. Osawa *et al.*

contrast, CD34-/Lo LSK cells have long-term multilineage reconstitution capacity. They also

can be divided into two populations by expression level of Flk-2. While LT-HSC are en‐

While the techniques of purifying HSC by use of surface markers had been promoted, Goodell *et al.* reported the method for purifying HSC without use of surface markers. Hoechst33342 is a fluorescent dye which binds to DNA of live cells. When Hoechst fluores‐ cence on whole BM was examined simultaneously at two emission wavelength (red and blue), one population of cells with increased ability to efflux Hoechst dye was observed. Goodell *et al.* named it "side population (SP)", and showed that a majority of HSC were en‐ riched in the SP by competitive repopulating experiments [9]. Subsequently, Matsuzaki *et al.* described a method of further purifying HSC by combining staining with antibodies to sur‐ face molecules with the Hoechst dye efflux. They showed the fraction of cells with the stron‐ gest dye efflux activity (termed as "Tip"-SP) has the highest marrow-repopulating activity. While 20% of "Tip"-SP cells are primitive hematopoietic cells, more than 90% of "Tip"-SP

cells in mouse BM as a multipotent HSC population. When these cells were

LSK cells are capable of only short-term multilineage differentiation. In

LSK cells are mainly ST-HSC [8].

 Sca-1+ c-

LSK cells [7]. LSK fraction also

jury, when the cells actively divide to regenerate BM cells.

52 Stem Cell Biology in Normal Life and Diseases

Thy-1Lo Sca-1+

showed that CD34+

riched in the Flk-2-

#### **3. HSC markers during developmental stages**

HSC markers during developmental stages are not identical to those of adult HSC. In the embryo, functional HSC that can reconstitute hematopoiesis in adult recipients are firstly found in the aorta-gonad-mesonephros (AGM) region at approximately embryonic day 10 (E10) [16-18]. Many reports have demonstrated that those earliest authentic HSC bud from endothelial-related cells, which involve the concept of "hemangioblast" or "hemogenic en‐ dothelium" [19-23]. In fact, emerging HSC and endothelial cells share various surface mark‐ ers such as CD34 and VE-cadherin that do not mark adult murine LT-HSC [24-26]. On the contrary, the emerging HSC do not express either Sca-1 or CD45, a pan-hematopoietic mark‐ er [19,27]. Interestingly, those developing HSC express CD41/Integrin-αv, a marker for meg‐ akaryocytes [28].

**4. Niche signals regulating HSC pool**

controlled the number of HSC by regulating SNO cells [37].

On the other hand, Kiel *et al.* reported that many CD150+ CD48- CD41- Lin-

**5. Differences between murine and human HSC markers**

long-term HSC of human BM are enriched in CD34+ CD38-

[26,47,48].

cadherin+

CD150+

CD48-

tain HSC hibernation [46].

HSC are CD34-

CD38+

We think it seems meaningful to deal with the "HSC niche" briefly here, although another chapter in this book provides more detailed information about its function. Molecular cross‐ talk between HSC and their niches has been considered to be important to provide signals for self-renewing division that maintain HSC pool. Although precise mechanisms regulating HSC status still remain unknown, there are accumulating evidences to involve several spe‐

In 1994, human osteoblasts were shown to maintained hematopoiesis by constitutively pro‐ ducing G-CSF in vitro [41]. In the first decade of the 21st century, a notion that connects os‐ teoblasts with the HSC niche rapidly developed. Parathyroid hormone (PTH), which is a main regulator of calcium homeostasis, was reported to increase in the number of both os‐ teoblasts and HSC, suggesting osteoblasts as the candidate for HSC niche [42]. In addition, it was also reported that BrdU label retaining cells (LRC) were attached to spindle-shaped N-

contact with sinusoidal endothelial cells in spleen or BM, suggesting that endothelial cells are also essential components of the HSC niche. With regard to cytokine-chemokine sinal‐ ings, the CXC chemokine ligand 12 (CXCL12) -CXC chemokine receptor 4 (CXCR4) pathway was found to be important. In vitro, HSC expressing CXCR4 migrate in response to CXCL12 which is the ligand for CXCR4 [43]. Nagasawa's laboratory reported that a majority of

that the numbers of HSC in CAR cell-depleted mice were reduced in comparison with con‐ trol mice. These data are supportive of the idea that CXCL12-CXCR4 pathway is essential for HSC pool [39,44]. Recently, Yamazaki *et al.* reported TGF- β as a candidate niche signal in the control of HSC hibernation [45]. The same group advocated that glial cells, regulating activation of TGF-β signal, might be a component of the HSC niche in adult BM and main‐

A critical issue that has been an obstacle in applying the information of murine HSC to hu‐ man is the lack of common HSC markers between the two species. Researchers described above have made great efforts to purify authentic HSC from murine hematopoietic organs. Owing to those achievements, we can now sort LT-HSC with very high purity from the murine BM. However, human HSC cannot be purified with the same markers. Human HSC do not express Sca-1 or CD150 that are the established HSC markers in mice. In addition, the

osteoblasts (SNO) cells, and that bone morphogenetic protein (BMP) signalling

CD41- HSC were in contact with CXCL12-abundant reticular (CAR) cells, and

LT-HSC were in

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

55

population, while murine BM

cific cells, or cytokines and chemokines secreted from stromal cells in this process.

Although HSC do not emerge in the fetal liver de novo, the organ is the main site of HSC expansion before birth. Circulating HSC seed in the fetal liver, where they robustly expand and differentiate. Indeed, numbers of HSC increase ~40-fold in the fetal liver between E12 and E16 [29]. Unlike the emerging HSC in the AGM region, HSC in fetal liver express CD45 and Sca-1. Morrison *et al.* showed that HSC are highly enriched in Thy1Lo Sca-1+ Lin- Mac1+ fraction of fetal liver cells [30]. His group later demonstrated that the SLAM family markers (CD150+ CD48- ) are also useful to enrich for HSC in E14.5 fetal liver just as in adult BM by the fact that 37% of CD150+ CD48- Sca-1+ Lin- Mac1+ fetal liver cells had long-term reconsti‐ tuting capacity [31]. Although the expression levels of AA4.1 and VE-cadherin are very high at the early stage of fetal hematopoiesis, they become gradually down-regulated after E12- E13 [32,33]. Interestingly, the phenotype of HSC in fetal liver rapidly changes after E16, when their number is reaching to a plateau level [30]. Recently, our group reported ESAM as a novel HSC marker in fetal liver (see below). HSC markers during mouse ontogeny are summarized in Table 2.


**Table 2.** Markers for hematopoietic stem cells during mouse ontogeny.

Adult and fetal HSC are not the same with regard to not only surface phenotypes but also cell-cycle status. Recent studies have shown that the long-term reconstituting activity of adult BM is sustained mostly in very quiescent HSC [13,14]. However, cycling HSC from the fetal liver give rise to higher levels of reconstitution than HSC obtained from adult BM [30,36]. The microenvironments, known as "HSC niches", are believed to influence cell-cycle status of HSC, and adult HSC niches in BM seem to be different from HSC niches in the fetal liver [12,37-40]. More precise analyses of hematopoietic environment in the embryo should give us valuable information regarding what are the imperative conditions for HSC expan‐ sion and how the alteration of surface molecules on HSC is functionally involved in that process. Furthermore, such cell surface antigens that mirror the HSC state are invaluable for understanding the relationship between HSC and their niches.

### **4. Niche signals regulating HSC pool**

ers such as CD34 and VE-cadherin that do not mark adult murine LT-HSC [24-26]. On the contrary, the emerging HSC do not express either Sca-1 or CD45, a pan-hematopoietic mark‐ er [19,27]. Interestingly, those developing HSC express CD41/Integrin-αv, a marker for meg‐

Although HSC do not emerge in the fetal liver de novo, the organ is the main site of HSC expansion before birth. Circulating HSC seed in the fetal liver, where they robustly expand and differentiate. Indeed, numbers of HSC increase ~40-fold in the fetal liver between E12 and E16 [29]. Unlike the emerging HSC in the AGM region, HSC in fetal liver express CD45 and Sca-1. Morrison *et al.* showed that HSC are highly enriched in Thy1Lo Sca-1+ Lin- Mac1+ fraction of fetal liver cells [30]. His group later demonstrated that the SLAM family markers

tuting capacity [31]. Although the expression levels of AA4.1 and VE-cadherin are very high at the early stage of fetal hematopoiesis, they become gradually down-regulated after E12- E13 [32,33]. Interestingly, the phenotype of HSC in fetal liver rapidly changes after E16, when their number is reaching to a plateau level [30]. Recently, our group reported ESAM as a novel HSC marker in fetal liver (see below). HSC markers during mouse ontogeny are

VE-

c-kit+

**Fetal age Location Markers References**

cadherin+ Sca-1- AA4.1+ ESAM+

AA4.1+ VE-cadherin+/- CD150+ CD48-

Sca-1+ Mac1+ Tie-2+ Flt3-

CD34+ CD45+ CD31+

ESAM+

Adult and fetal HSC are not the same with regard to not only surface phenotypes but also cell-cycle status. Recent studies have shown that the long-term reconstituting activity of adult BM is sustained mostly in very quiescent HSC [13,14]. However, cycling HSC from the fetal liver give rise to higher levels of reconstitution than HSC obtained from adult BM [30,36]. The microenvironments, known as "HSC niches", are believed to influence cell-cycle status of HSC, and adult HSC niches in BM seem to be different from HSC niches in the fetal liver [12,37-40]. More precise analyses of hematopoietic environment in the embryo should give us valuable information regarding what are the imperative conditions for HSC expan‐ sion and how the alteration of surface molecules on HSC is functionally involved in that process. Furthermore, such cell surface antigens that mirror the HSC state are invaluable for

) are also useful to enrich for HSC in E14.5 fetal liver just as in adult BM by

fetal liver cells had long-term reconsti‐

Petrenko *et al. Immunity* (1999) [33], Hsu *et al. Blood* (2000) [34], Baumann *et al. Blood* (2004) [24], Fraser *et al. Exp Hematol* (2002) [25], Ogawa *Exp Hematol* (2002) [26], de Brujin *et al. Immunity* (2002) [19], Mikkola *et al. Blood* (2003) [28], Matsubara *et al. J Exp Med* (2005) [27], Kim *et al. Blood* (2005) [32], Kim *et al. Blood* (2006) [31], Mansson *et al. Immunity* (2007) [35], Yokota *et al.* Blood (2009) [2]

akaryocytes [28].

54 Stem Cell Biology in Normal Life and Diseases

(CD150+ CD48-

summarized in Table 2.

the fact that 37% of CD150+ CD48- Sca-1+ Lin- Mac1+

E8.5-E10.5 AGM region CD41+ CD34+ CD45-

**Table 2.** Markers for hematopoietic stem cells during mouse ontogeny.

understanding the relationship between HSC and their niches.

E11.5-E16.5 Fetal liver CD41-

We think it seems meaningful to deal with the "HSC niche" briefly here, although another chapter in this book provides more detailed information about its function. Molecular cross‐ talk between HSC and their niches has been considered to be important to provide signals for self-renewing division that maintain HSC pool. Although precise mechanisms regulating HSC status still remain unknown, there are accumulating evidences to involve several spe‐ cific cells, or cytokines and chemokines secreted from stromal cells in this process.

In 1994, human osteoblasts were shown to maintained hematopoiesis by constitutively pro‐ ducing G-CSF in vitro [41]. In the first decade of the 21st century, a notion that connects os‐ teoblasts with the HSC niche rapidly developed. Parathyroid hormone (PTH), which is a main regulator of calcium homeostasis, was reported to increase in the number of both os‐ teoblasts and HSC, suggesting osteoblasts as the candidate for HSC niche [42]. In addition, it was also reported that BrdU label retaining cells (LRC) were attached to spindle-shaped Ncadherin+ osteoblasts (SNO) cells, and that bone morphogenetic protein (BMP) signalling controlled the number of HSC by regulating SNO cells [37].

On the other hand, Kiel *et al.* reported that many CD150+ CD48- CD41- Lin- LT-HSC were in contact with sinusoidal endothelial cells in spleen or BM, suggesting that endothelial cells are also essential components of the HSC niche. With regard to cytokine-chemokine sinal‐ ings, the CXC chemokine ligand 12 (CXCL12) -CXC chemokine receptor 4 (CXCR4) pathway was found to be important. In vitro, HSC expressing CXCR4 migrate in response to CXCL12 which is the ligand for CXCR4 [43]. Nagasawa's laboratory reported that a majority of CD150+ CD48- CD41- HSC were in contact with CXCL12-abundant reticular (CAR) cells, and that the numbers of HSC in CAR cell-depleted mice were reduced in comparison with con‐ trol mice. These data are supportive of the idea that CXCL12-CXCR4 pathway is essential for HSC pool [39,44]. Recently, Yamazaki *et al.* reported TGF- β as a candidate niche signal in the control of HSC hibernation [45]. The same group advocated that glial cells, regulating activation of TGF-β signal, might be a component of the HSC niche in adult BM and main‐ tain HSC hibernation [46].

#### **5. Differences between murine and human HSC markers**

A critical issue that has been an obstacle in applying the information of murine HSC to hu‐ man is the lack of common HSC markers between the two species. Researchers described above have made great efforts to purify authentic HSC from murine hematopoietic organs. Owing to those achievements, we can now sort LT-HSC with very high purity from the murine BM. However, human HSC cannot be purified with the same markers. Human HSC do not express Sca-1 or CD150 that are the established HSC markers in mice. In addition, the long-term HSC of human BM are enriched in CD34+ CD38 population, while murine BM HSC are CD34- CD38+ [26,47,48].

Early studies in the 1980s proved by using monoclonal antibody technique that CD34+ popu‐ lation of human BM includes immature hematopoietic progenitors [49-51]. Berenson *et al.* showed that autologous CD34+ cells enriched from baboon BM were able to reconstitute nor‐ mal hematopoiesis after lethal irradiation. Animals transplanted with CD34 cells, however, did not recover sufficient hematopoiesis [52]. Afterwards, over the past two decades, CD34 positive has been used as a reliable marker for human HSC or hematopoietic progenitor cells (HPC). Indeed, transplantation of CD34+ cells obtained from donor BM, peripheral blood, or cord blood (CB) can provide long-term and multilineage hematopoietic reconstitu‐ tion in recipients.

dition. While activated HSC increase the expression level of Sca-1, CD150, Tie2, Endo‐ glin, Mac-1, and CD34, they clearly decrease that of c-kit and N-cadherin [26,56,57]. Some endothelial-related antigens, which mark actively dividing fetal HSC but do not mark quiescent adult HSC, are up-regulated again on the activated HSC after BM injury. The characteristics of those activated HSC are reminiscent of fetal HSC. Since no obvious phenotypes have been documented regarding CD34 or CD150-deficient mice, how the up-regulation of those molecules contributes to the functions and/or characteristics of ac‐ tivated HSC remains unknown [12,58]. Tie2 and Endoglin, which are the receptors for angiopoietin and TGF, respectively, might transduce important signals to regulate divid‐ ing speed of HSC. If we could accurately monitor the fluctuation of HSC status with a set of surface markers, that should yield significant insight regarding HSC biology and HSC applications for clinical purposes. As a very recent achievement, our group has

demonstrated that ESAM is a useful marker for activated HSC.

[61,62]. We found the ESAMHi population of Rag1- c-kitHi Sca-1+

cantly different between Rag1- c-kitHi Sca-1+

.

creased with age after reaching adulthood 2

pression is clearly detectable on human CB CD34+

E10.5 AGM cells, only ESAM+

transcripts were detected in Lin-

manuscript in preparation).

myeloid cells 2

that ESAM+ Sca-1+ Lin-

Mac1+

**7. An endothelial-related antigen ESAM as a new novel HSC marker**

We previously reported sorting strategy of HSC and early lymphoid progenitors (ELP) from Rag1/GFP knockin mice [59,60]. We searched for genes whose expression levels are signifi‐

micro-array data. Among the HSC related genes ESAM drew our attention because its tran‐ scripts were conspicuous in the HSC fraction whereas the expression was drastically downregulated in the ELP fraction. ESAM molecule is an immunoglobulin superfamily protein that is exposed on cell surface and originally identified as an endothelial cell-specific protein

was highly enriched for LT-HSC compared with ESAM-/Lo subset. Among Rag1/GFP- Tie2Hi

ESAM is also expressed on adult murine HSC-enriched fraction in BM. Ooi *et al.* reported

tional HSC-enriched LSK cells, and that ESAM expression on HSC was conserved among various mouse strains [63]. ESAM levels on HSC are variable according to developing stages or advancing age. Interestingly, the intensity of ESAM expression on HSC gradually in‐

The usefulness of ESAM as a HSC marker has been further enhanced by the findings that its expression in human HSC is also detected. Ooi *et al.* reported that robust levels of ESAM

transcripts in unfractionated CB cells were very low [63]. We have confirmed that ESAM ex‐

flow cytometry [64]. In addition, our group has also observed that the marker is effective as well for adult human HSC in both BM and mobilized peripheral blood. (Ishibashi *et al.*

CD38-

novel murine HSC marker throughout life including developmental stages.

CD34+

cells could effectively produce both CD19+

BM cells could more effectively enrich for LT-HSC than the conven‐

HSC and Rag1Lo c-kitHi Sca-1+ ELP by analyzing

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

57

. Based on these observations, ESAM can be a

CD90+ human HSC, while the levels of ESAM

cells by using its specific antibody and

fraction of E14.5 fetal liver

lymphoid cells and

As CD34 marks human HSC as well as more differentiated progenitor cells, researchers have sought additional markers to further enrich CD34+ population for LT-HSC. Baum *et al.* reported that CD90/Thy-1+ population in Lin- CD34+ cells contained pluripotent hemato‐ poietic progenitors [53]. Recently a series of studies of John Dick's laboratory have success‐ fully improved the techniques to more purify human HSC. His group reported that human HSC activity was restricted to CD49f+ fraction, and that single Lin- CD34+ CD38- CD45RA-Thy-1+ Rhodamin123Lo CD49f+ cells in CB cells accomplished multilineage engraftment in immune-deficient mice [54].

While LT-HSC can be enriched mainly in the CD34+ population, the possibility that CD34 cells also contain LT-HSC has been reported. Bhatia *et al.* showed human CD34- population in Lin cells of BM and CB also contained LT-HSC [55]. It should be important to compare the features of primate CD34- HSC with those of murine CD34- LSK cells. In addition, a new positive marker for human HSC could resolve the relationship between the CD34+ and CD34- HSC. Markers for human HSC are summarized in Table 3.


**Table 3.** Markers for human hematopoietic stem cells.

#### **6. Differences between quiescent and activated HSC markers**

After mice are treated with cytotoxic agents or irradiation, most of cell-cycling hemato‐ poietic cells are killed and dormant primitive HSC start to proliferate. The patterns of surface molecules expressed on activated HSC change from those under steady-state con‐ dition. While activated HSC increase the expression level of Sca-1, CD150, Tie2, Endo‐ glin, Mac-1, and CD34, they clearly decrease that of c-kit and N-cadherin [26,56,57]. Some endothelial-related antigens, which mark actively dividing fetal HSC but do not mark quiescent adult HSC, are up-regulated again on the activated HSC after BM injury. The characteristics of those activated HSC are reminiscent of fetal HSC. Since no obvious phenotypes have been documented regarding CD34 or CD150-deficient mice, how the up-regulation of those molecules contributes to the functions and/or characteristics of ac‐ tivated HSC remains unknown [12,58]. Tie2 and Endoglin, which are the receptors for angiopoietin and TGF, respectively, might transduce important signals to regulate divid‐ ing speed of HSC. If we could accurately monitor the fluctuation of HSC status with a set of surface markers, that should yield significant insight regarding HSC biology and HSC applications for clinical purposes. As a very recent achievement, our group has demonstrated that ESAM is a useful marker for activated HSC.

Early studies in the 1980s proved by using monoclonal antibody technique that CD34+

mal hematopoiesis after lethal irradiation. Animals transplanted with CD34-

showed that autologous CD34+

56 Stem Cell Biology in Normal Life and Diseases

tion in recipients.

in Lin-

CD34-

Lin-

CD34+ CD38-

reported that CD90/Thy-1+

Thy-1+ Rhodamin123Lo CD49f+

immune-deficient mice [54].

cells (HPC). Indeed, transplantation of CD34+

have sought additional markers to further enrich CD34+

While LT-HSC can be enriched mainly in the CD34+

CD34+ Lin-

Lin- CD34-

CD34+ CD38-

**Table 3.** Markers for human hematopoietic stem cells.

CD45RA-

Lin-

HSC. Markers for human HSC are summarized in Table 3.

**6. Differences between quiescent and activated HSC markers**

After mice are treated with cytotoxic agents or irradiation, most of cell-cycling hemato‐ poietic cells are killed and dormant primitive HSC start to proliferate. The patterns of surface molecules expressed on activated HSC change from those under steady-state con‐

lation of human BM includes immature hematopoietic progenitors [49-51]. Berenson *et al.*

did not recover sufficient hematopoiesis [52]. Afterwards, over the past two decades, CD34 positive has been used as a reliable marker for human HSC or hematopoietic progenitor

blood, or cord blood (CB) can provide long-term and multilineage hematopoietic reconstitu‐

As CD34 marks human HSC as well as more differentiated progenitor cells, researchers

poietic progenitors [53]. Recently a series of studies of John Dick's laboratory have success‐ fully improved the techniques to more purify human HSC. His group reported that human HSC activity was restricted to CD49f+ fraction, and that single Lin- CD34+ CD38- CD45RA-

cells also contain LT-HSC has been reported. Bhatia *et al.* showed human CD34- population

 cells of BM and CB also contained LT-HSC [55]. It should be important to compare the features of primate CD34- HSC with those of murine CD34- LSK cells. In addition, a new positive marker for human HSC could resolve the relationship between the CD34+ and

**Markers References**

CD34+ Berenson *et al. J Clin Invest* (1988) [52] CD34+ CD38- Terstappen *et al. Blood* (1991) [48]

CD38- Bhatia *et al. Nat Med* (1998) [55]

Thy-1+ RhoLo CD49f+ Notta *et al. Science* (2011) [54]

RhoLo McKenzie *et al. Blood* (2007) [47]

Thy-1+ Baum *et al. Proc Natl Acad Sci USA* (1992) [53]

population in Lin- CD34+

cells enriched from baboon BM were able to reconstitute nor‐

cells in CB cells accomplished multilineage engraftment in

cells obtained from donor BM, peripheral

population for LT-HSC. Baum *et al.*

cells contained pluripotent hemato‐

population, the possibility that CD34-

popu‐

cells, however,

### **7. An endothelial-related antigen ESAM as a new novel HSC marker**

We previously reported sorting strategy of HSC and early lymphoid progenitors (ELP) from Rag1/GFP knockin mice [59,60]. We searched for genes whose expression levels are signifi‐ cantly different between Rag1- c-kitHi Sca-1+ HSC and Rag1Lo c-kitHi Sca-1+ ELP by analyzing micro-array data. Among the HSC related genes ESAM drew our attention because its tran‐ scripts were conspicuous in the HSC fraction whereas the expression was drastically downregulated in the ELP fraction. ESAM molecule is an immunoglobulin superfamily protein that is exposed on cell surface and originally identified as an endothelial cell-specific protein [61,62]. We found the ESAMHi population of Rag1- c-kitHi Sca-1+ fraction of E14.5 fetal liver was highly enriched for LT-HSC compared with ESAM-/Lo subset. Among Rag1/GFP- Tie2Hi E10.5 AGM cells, only ESAM+ cells could effectively produce both CD19+ lymphoid cells and Mac1+ myeloid cells 2 .

ESAM is also expressed on adult murine HSC-enriched fraction in BM. Ooi *et al.* reported that ESAM+ Sca-1+ Lin- BM cells could more effectively enrich for LT-HSC than the conven‐ tional HSC-enriched LSK cells, and that ESAM expression on HSC was conserved among various mouse strains [63]. ESAM levels on HSC are variable according to developing stages or advancing age. Interestingly, the intensity of ESAM expression on HSC gradually in‐ creased with age after reaching adulthood 2 . Based on these observations, ESAM can be a novel murine HSC marker throughout life including developmental stages.

The usefulness of ESAM as a HSC marker has been further enhanced by the findings that its expression in human HSC is also detected. Ooi *et al.* reported that robust levels of ESAM transcripts were detected in Lin- CD34+ CD38- CD90+ human HSC, while the levels of ESAM transcripts in unfractionated CB cells were very low [63]. We have confirmed that ESAM ex‐ pression is clearly detectable on human CB CD34+ cells by using its specific antibody and flow cytometry [64]. In addition, our group has also observed that the marker is effective as well for adult human HSC in both BM and mobilized peripheral blood. (Ishibashi *et al.* manuscript in preparation).

#### **8. ESAM monitors HSC status between quiescence and self-renewal**

As mentioned above, the expression pattern of surface antigens on activated HSC after BM injury substantially differs from that on quiescent HSC. Administration of an anti-cancer drug 5-FU causes apoptosis of dividing hematopoietic progenitors, while the treatment re‐ tains quiescent LT-HSC and induces their proliferation afterward. We have observed that re‐ markable increase of ESAM expression levels transiently occurs on BM HSC after a 5-FU treatment. Furthermore, we have proved that the long-term hematopoietic reconstituting ac‐ tivity is almost exclusive to LSK cells bearing up-regulated ESAM expression [3].

Although expression levels of CD34, Tie2, and Endoglin on LSK show modest increases af‐ ter 5-FU injection, up-regulation of ESAM is remarkable (Figure 1). Why does ESAM need to revive so vividly on HSC after BM injury? One possible reason is that HSC might directly receive necessary signals which regulate self-renewal or differentiation via interaction with ESAM. Another possibility is that high amounts of ESAM might change the polarity or mo‐ bility of HSC, which consequently facilitate them to settle in adequate supporting niches (Figure 2). The latter assumption is likely because Wegmann and colleagues reported that ESAM deficiency causes insufficient Rho signalling in endothelial cells, which regulates the stabilization of endothelial tight junctions [65]. Rho is also expressed in hematopoietic pro‐ genitors and involved in their polarity and mobility [66]. It is noteworthy that more than 80% of ESAMHi HSC were located around perivascular areas in 5-FU-treated BM3 .

In any case, ESAM is likely to play an indispensable role during the recovery from BM in‐ jury. Because, while ESAM deficient mice do not show significant hematopoietic defects in homeostatic stage, the mice fall into severe and prolonged pancytopenia after myelo-sup‐ pressive treatment. In particular, they suffer from severe anemia and frequently die before hematopoietic recovery. Our findings indicate that ESAM not only marks activated HSC but also functionally supports their proliferation and differentiation.

**Figure 2.** Tentative models of ESAM function. (A) In this model, activated HSC directly receive necessary signals which regulate self-renewal or differentiation via interaction with ESAM. (B) In this model, HSC change their polarity or mobi‐ lity, and consequently, they can move to appropriate niches. ESAM may function as an adhesion factor between HSC

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

59

In this chapter, we summarized achievements for identification of murine and human HSC, and introduced endothelial-related antigen ESAM as a useful HSC marker. While we can now purify murine LT-HSC with high efficiency, characterization of human HSC is less well understood because of insufficient information about surface antigens. Over two decades CD34-positive has been believed to be a reliable marker for human HSC/HPC. Although there are now accumulating evidences regarding surface markers to further enrich human LT-HSC in the CD34+ fraction, more information about human HSC-related antigens should be useful to improve strategies of HSC application to the clinical medicine. Although ESAM was originally identified with an endothelial specific molecule, we have demonstrated that it is a positive marker for both murine and human HSC. Because ESAM seems to play an essential role for hematopoietic recovery after BM injury, it would be significant to elucidate downstream signals of ESAM, and the possibility of ESAM as niche components. In addi‐ tion, we now know that the up-regulation of ESAM is observed on cultured murine embry‐ onic stem (ES) cells cultured in the OP9 system which recapitulate very primitive stages of

and their niches.

**9. Concluding remarks**

**Figure 1.** Overview of cell surface expression levels on quiescent steady-state HSC and activated HSC.

**Figure 2.** Tentative models of ESAM function. (A) In this model, activated HSC directly receive necessary signals which regulate self-renewal or differentiation via interaction with ESAM. (B) In this model, HSC change their polarity or mobi‐ lity, and consequently, they can move to appropriate niches. ESAM may function as an adhesion factor between HSC and their niches.

#### **9. Concluding remarks**

**8. ESAM monitors HSC status between quiescence and self-renewal**

58 Stem Cell Biology in Normal Life and Diseases

tivity is almost exclusive to LSK cells bearing up-regulated ESAM expression [3].

80% of ESAMHi HSC were located around perivascular areas in 5-FU-treated BM3

**Figure 1.** Overview of cell surface expression levels on quiescent steady-state HSC and activated HSC.

also functionally supports their proliferation and differentiation.

In any case, ESAM is likely to play an indispensable role during the recovery from BM in‐ jury. Because, while ESAM deficient mice do not show significant hematopoietic defects in homeostatic stage, the mice fall into severe and prolonged pancytopenia after myelo-sup‐ pressive treatment. In particular, they suffer from severe anemia and frequently die before hematopoietic recovery. Our findings indicate that ESAM not only marks activated HSC but

.

As mentioned above, the expression pattern of surface antigens on activated HSC after BM injury substantially differs from that on quiescent HSC. Administration of an anti-cancer drug 5-FU causes apoptosis of dividing hematopoietic progenitors, while the treatment re‐ tains quiescent LT-HSC and induces their proliferation afterward. We have observed that re‐ markable increase of ESAM expression levels transiently occurs on BM HSC after a 5-FU treatment. Furthermore, we have proved that the long-term hematopoietic reconstituting ac‐

Although expression levels of CD34, Tie2, and Endoglin on LSK show modest increases af‐ ter 5-FU injection, up-regulation of ESAM is remarkable (Figure 1). Why does ESAM need to revive so vividly on HSC after BM injury? One possible reason is that HSC might directly receive necessary signals which regulate self-renewal or differentiation via interaction with ESAM. Another possibility is that high amounts of ESAM might change the polarity or mo‐ bility of HSC, which consequently facilitate them to settle in adequate supporting niches (Figure 2). The latter assumption is likely because Wegmann and colleagues reported that ESAM deficiency causes insufficient Rho signalling in endothelial cells, which regulates the stabilization of endothelial tight junctions [65]. Rho is also expressed in hematopoietic pro‐ genitors and involved in their polarity and mobility [66]. It is noteworthy that more than

> In this chapter, we summarized achievements for identification of murine and human HSC, and introduced endothelial-related antigen ESAM as a useful HSC marker. While we can now purify murine LT-HSC with high efficiency, characterization of human HSC is less well understood because of insufficient information about surface antigens. Over two decades CD34-positive has been believed to be a reliable marker for human HSC/HPC. Although there are now accumulating evidences regarding surface markers to further enrich human LT-HSC in the CD34+ fraction, more information about human HSC-related antigens should be useful to improve strategies of HSC application to the clinical medicine. Although ESAM was originally identified with an endothelial specific molecule, we have demonstrated that it is a positive marker for both murine and human HSC. Because ESAM seems to play an essential role for hematopoietic recovery after BM injury, it would be significant to elucidate downstream signals of ESAM, and the possibility of ESAM as niche components. In addi‐ tion, we now know that the up-regulation of ESAM is observed on cultured murine embry‐ onic stem (ES) cells cultured in the OP9 system which recapitulate very primitive stages of

hematopoietic development [67] (Doi et al. manuscript in preparation). ESAM might have some roles in embryonic hematopoiesis at very early stages. As an on-going study, we are now investigating whether ESAM can be a useful biomarker for inducing hematopoietic cells from ES or induced pluripotent stem cells.

[10] Matsuzaki Y, Kinjo K, Mulligan RC, et al. Unexpectedly efficient homing capacity of

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

61

[11] Balazs AB, Fabian AJ, Esmon CT, et al. Endothelial protein C receptor (CD201) ex‐ plicitly identifies hematopoietic stem cells in murine bone marrow. Blood.

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#### **Author details**

Takao Sudo, Takafumi Yokota\* , Tomohiko Ishibashi, Michiko Ichii, Yukiko Doi, Kenji Oritani and Yuzuru Kanakura

\*Address all correspondence to: yokotat@bldon.med.osaka-u.ac.jp

Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan

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Department of Hematology and Oncology, Osaka University Graduate School of Medicine,

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**Chapter 5**

**Hematopoietic Stem Cells and Response to Interferon**

Homeostasis in the bone marrow is dependent on the ability of hematopoietic stem cells (HSCs) to self-renew faithfully, differentiate into various lineages of the hematopoietic system, and form blood cells of several types (Figure 1) [1,2]. Under homeostatic conditions, HSCs are thought to be quiescent, and they are referred to as long-term reconstituting HSCs (LT-HSC) or dormant HSCs (dHSCs) [3,4]. Blood and immune cells are produced by the more differen‐ tiated short-term reconstituting HSCs (ST-HSCs) or multipotent progenitors (MPPs). Genetic and molecular studies of HSC self-renewal have identified candidate regulatory factors, in‐ cluding cell-intrinsic regulators, such as transcription factors and cell surface receptors, and cell-extrinsic regulators, such as the bone marrow niche and cytokines. Under certain condi‐ tions, such as inflammatory stress, HSCs differentiate into progenitor cells with less ability to self-renew, and they can be stimulated to divide and/or differentiate into all cell types in the peripheral blood [5,6]. Under inflammatory conditions, such as during bacterial infection or sepsis, an apparent expansion of lineage-negative Sca-1+c-Kit+ bone marrow cells (KSL) has been observed [7–11]. HSCs and progenitor cells are involved in the expansion of KSL; and expansion of the KSL population in the bone marrow has been associated with a loss of dor‐ mant LT-HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation within that population. The process of the transition of HSCs from dormancy to activity is

Interferon is produced by cells of the immune system in response to challenge by agents, such as viruses, bacteria, and tumor cells. Type I IFNs are induced by the genomes of many RNA viruses during viral infection, and they suppress viral replication and have immunomodula‐ tory activity. IFNs are used clinically to treat viral diseases and malignancies, such as chronic myeloid leukemia (CML)[12]. In addition, under steady state conditions in the absence of in‐ fection, a small amount of intrinsic IFN is produced constitutively [13]. Recently, Essers et al. demonstrated that chronic activation of the IFN-α pathway impairs the function of HSCs and

> © 2013 Masumi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Atsuko Masumi

**1. Introduction**

http://dx.doi.org/10.5772/54689

Additional information is available at the end of the chapter

mediated by type I interferon (IFN) and type II IFN.


## **Hematopoietic Stem Cells and Response to Interferon**

### Atsuko Masumi

[55] Bhatia M, Bonnet D, Murdoch B, et al. A newly discovered class of human hemato‐

[56] Haug JS, He XC, Grindley JC, et al. N-cadherin expression level distinguishes re‐ served versus primed states of hematopoietic stem cells. Cell Stem Cell.

[57] Randall TD, Weissman IL. Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood.

[58] Cheng J, Baumhueter S, Cacalano G, et al. Hematopoietic defects in mice lacking the

[59] Igarashi H, Gregory SC, Yokota T, et al. Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow. Immunity. 2002;17:117-130. [60] Yokota T, Kouro T, Hirose J, et al. Unique properties of fetal lymphoid progenitors identified according to RAG1 gene expression. Immunity. 2003;19:365-375.

[61] Hirata K, Ishida T, Penta K, et al. Cloning of an immunoglobulin family adhesion molecule selectively expressed by endothelial cells. J Biol Chem.

[62] Nasdala I, Wolburg-Buchholz K, Wolburg H, et al. A transmembrane tight junction protein selectively expressed on endothelial cells and platelets. J Biol Chem.

[63] Ooi AG, Karsunky H, Majeti R, et al. The adhesion molecule esam1 is a novel hema‐

[64] Yokota T, Oritani K, Butz S, et al. Markers for Hematopoietic Stem Cells: Histories and Recent Achievements. Advances in Hematopoietic Stem Cell Research: InTech;

[65] Wegmann F, Petri B, Khandoga AG, et al. ESAM supports neutrophil extravasation, activation of Rho, and VEGF-induced vascular permeability. J Exp Med.

[66] Fonseca AV, Freund D, Bornhauser M, et al. Polarization and migration of hemato‐ poietic stem and progenitor cells rely on the RhoA/ROCK I pathway and an active reorganization of the microtubule network. J Biol Chem. 2010;285:31661-31671. [67] Nakano T, Kodama H, Honjo T. Generation of lymphohematopoietic cells from em‐

topoietic stem cell marker. Stem Cells. 2009;27:653-661.

bryonic stem cells in culture. Science. 1994;265:1098-1101.

poietic cells with SCID-repopulating activity. Nat Med. 1998;4:1038-1045.

2008;2:367-379.

64 Stem Cell Biology in Normal Life and Diseases

1997;89:3596-3606.

2001;276:16223-16231.

2002;277:16294-16303.

2012:77-88.

2006;203:1671-1677.

sialomucin CD34. Blood. 1996;87:479-490.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54689

#### **1. Introduction**

Homeostasis in the bone marrow is dependent on the ability of hematopoietic stem cells (HSCs) to self-renew faithfully, differentiate into various lineages of the hematopoietic system, and form blood cells of several types (Figure 1) [1,2]. Under homeostatic conditions, HSCs are thought to be quiescent, and they are referred to as long-term reconstituting HSCs (LT-HSC) or dormant HSCs (dHSCs) [3,4]. Blood and immune cells are produced by the more differen‐ tiated short-term reconstituting HSCs (ST-HSCs) or multipotent progenitors (MPPs). Genetic and molecular studies of HSC self-renewal have identified candidate regulatory factors, in‐ cluding cell-intrinsic regulators, such as transcription factors and cell surface receptors, and cell-extrinsic regulators, such as the bone marrow niche and cytokines. Under certain condi‐ tions, such as inflammatory stress, HSCs differentiate into progenitor cells with less ability to self-renew, and they can be stimulated to divide and/or differentiate into all cell types in the peripheral blood [5,6]. Under inflammatory conditions, such as during bacterial infection or sepsis, an apparent expansion of lineage-negative Sca-1+c-Kit+ bone marrow cells (KSL) has been observed [7–11]. HSCs and progenitor cells are involved in the expansion of KSL; and expansion of the KSL population in the bone marrow has been associated with a loss of dor‐ mant LT-HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation within that population. The process of the transition of HSCs from dormancy to activity is mediated by type I interferon (IFN) and type II IFN.

Interferon is produced by cells of the immune system in response to challenge by agents, such as viruses, bacteria, and tumor cells. Type I IFNs are induced by the genomes of many RNA viruses during viral infection, and they suppress viral replication and have immunomodula‐ tory activity. IFNs are used clinically to treat viral diseases and malignancies, such as chronic myeloid leukemia (CML)[12]. In addition, under steady state conditions in the absence of in‐ fection, a small amount of intrinsic IFN is produced constitutively [13]. Recently, Essers et al. demonstrated that chronic activation of the IFN-α pathway impairs the function of HSCs and

© 2013 Masumi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



**Chapter 6**

**Stem Cell Predictive Hemotoxicology**

Stem cells, those elusive entities that have the capacity for producing, maintaining and reconstituting the integrity of a biological system, also demonstrate the potential to predict partial or life-threatening damage in response to drugs, environmental compounds and other agents. It is ironic however, that in the animal or human, prior to the manifestation of such potential biological damage most, if not all of the stem cells might have been eradicated. To predict possible damage, surrogate *in vitro* stem cell assays have been developed that utilize specific properties and characteristics that divulge, through a measured response, how the

*In vitro* assays that detect toxicity to stem cells of a biological system allow potentially life-threatening damage to be predicted prior to human clinical trials taking place and environmental agents from causing harm. Discussions about stem cells usually focuses not on primary cells, but rather on embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, their ability to produce virtually any type of functional cell and their use in cellular therapy and regenerative medicine. The ES and iPS types of stem cells are, in fact, the least understood of all stem cell types. For many companies investigating or considering using these stem cells routinely as surrogate *in vitro* models for toxicity test‐ ing many questions remain, including (1) what is the relevance of these cells, (2) how do they compare with primary stem cell populations, and (3) can they be validated? In many cases, it is not the stem cells themselves, but rather the cells derived from ES and iPS cells that are of interest. Several companies already produce ES- or iPS-derived cardi‐ omyocytes, hepatocytes, neural cells and many other cell types not only for toxicity test‐

ing, but also for basic research, cellular therapy and regenerative medicine.

How can stem cells be used to predict toxicity? The answer to this question lies in the characteristics and properties of stem cells and how they respond to different situations.

and reproduction in any medium, provided the original work is properly cited.

© 2013 Harper and Rich; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Additional information is available at the end of the chapter

Holli Harper and Ivan N. Rich

system will react to different agents.

http://dx.doi.org/10.5772/54430

**1. Introduction**

## **Stem Cell Predictive Hemotoxicology**

Holli Harper and Ivan N. Rich

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54430

### **1. Introduction**

Stem cells, those elusive entities that have the capacity for producing, maintaining and reconstituting the integrity of a biological system, also demonstrate the potential to predict partial or life-threatening damage in response to drugs, environmental compounds and other agents. It is ironic however, that in the animal or human, prior to the manifestation of such potential biological damage most, if not all of the stem cells might have been eradicated. To predict possible damage, surrogate *in vitro* stem cell assays have been developed that utilize specific properties and characteristics that divulge, through a measured response, how the system will react to different agents.

*In vitro* assays that detect toxicity to stem cells of a biological system allow potentially life-threatening damage to be predicted prior to human clinical trials taking place and environmental agents from causing harm. Discussions about stem cells usually focuses not on primary cells, but rather on embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, their ability to produce virtually any type of functional cell and their use in cellular therapy and regenerative medicine. The ES and iPS types of stem cells are, in fact, the least understood of all stem cell types. For many companies investigating or considering using these stem cells routinely as surrogate *in vitro* models for toxicity test‐ ing many questions remain, including (1) what is the relevance of these cells, (2) how do they compare with primary stem cell populations, and (3) can they be validated? In many cases, it is not the stem cells themselves, but rather the cells derived from ES and iPS cells that are of interest. Several companies already produce ES- or iPS-derived cardi‐ omyocytes, hepatocytes, neural cells and many other cell types not only for toxicity test‐ ing, but also for basic research, cellular therapy and regenerative medicine.

How can stem cells be used to predict toxicity? The answer to this question lies in the characteristics and properties of stem cells and how they respond to different situations.

© 2013 Harper and Rich; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

To understand this better, stem cell systems can be divided into "definitive" and "nondefinitive" systems as illustrated in Fig. 1. Definitive stem cell systems are responsible for maintaining a specific biological system. They can be divided into continuously pro‐ liferating systems such as the blood-forming or lympho-hematopoietic system, the gastrointestinal system, hair and skin, reproductive organs and cells of the eye cornea. Although not necessarily a continuously proliferating system, the mesenchymal stem cell (MSC), also called the multipotent mesenchymal stromal cell [1] system can been includ‐ ed, because in culture, the MSCs proliferate and can be passaged over a long period of time. Definitive stem cells systems can also demonstrate partial proliferation. These in‐ clude, but are not limited to, the liver, lung, kidney, heart, pancreas, and the neural/ neuronal system. From a toxicological viewpoint, these are not usually considered stem cell systems. Yet, the different types of lineage cells present in these organs and the abili‐ ty to maintain a specific cell mass has all the intricacies of a stem cell system, especially during development, even though the cell turnover in the adult may be very low. Nondefinitive stem cells systems are represented by the ES and iPS cell systems, which can, theoretically, give rise to any of the definitive stem cell systems. Indeed, it is a prerequi‐ site that the production of functionally, mature cells from ES or iPS cells first pass through a definitive stem cell compartment.

ever happens at the stem cell level will ultimately affect all downstream events. These char‐

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 81

**Figure 1. Definitive and Non-Definitive Stem Cell Systems.** Definitive stem cell systems can be further divided into continuously and partially proliferating systems. Non-definitive stem cell systems such as embryonic stem cells and in‐ duced pluripotent stem cells can produce definitive stem cell systems, which in turn, give rise to mature functional

All definitive stem cell systems have a common organization shown in Fig. 2. There is a continuum of stem cells within the stem cell compartment that exhibit different degrees of primitiveness or "stemness", which in turn, implies changing proliferating potential or potency as a stem cell moves through the compartment to the point of determination. These characteristic properties actually provide the information that allows stem cells to be predictors of potential toxicity. Once a stem cell becomes a progenitor cell, prolifera‐ tion continues and actually increases for a certain time so that the compartment can be amplified, until it ceases completely and the differentiation and maturation processes takes over. These changes have important implications for the types of assays that can

From Fig. 2, it is clear that proliferation occurs prior to differentiation. Although there is considerable overlap between proliferation and differentiation, they are two separate proc‐ esses that cannot be measured using the same assay readout. Since stem cells only proliferate, it follows that a proliferation assay is required to detect the presence and response of stem cells to a compound or agent. Using a differentiation assay to detect the effect of a compound or agent that targets one or more steps in the proliferation process can influence the interpretation

cells.

be used *in vitro* to detect potential toxicity.

acteristics enable stem cells to be the most important predictors of potential toxicity.

#### **2. Stem cell characteristics and properties used for toxicity testing**

Stem cells of primary, definitive systems always represent a very small proportion of the tissue or organ cellularity. This proportion is between 0.1 and 0.01% or less. The basic definition of a stem cell is that it possesses the capacity for self-renewal. In fact, stem cell systems are usually termed self-renewal cell systems, meaning that one stem cell can produce two daughter cells that are exact replicas of the parent. However, self-renewal is a difficult property to measure. The capacity for either serial *in vivo* repopulation or *in vitro* serial re-plating is considered a property of stem cells that implicates not only the presence of stem cells, but also their selfrenewal capability. The fact that serial *in vitro* re-plating or *in vivo* repopulation cannot be performed ad infinitum is not only an indication for a stem cell hierarchy [2-7], but also for an alternative hypothesis to stem cell self-renewal. This hypothesis states that tissues and organs are endowed with a specific number of stem cells. Once used up, the system ceases to function [8]. Regardless of the hypothesis, this important property can be utilized in a toxicological setting by employing secondary re-plating technology. This allows not only the presence of residual stem cells to be detected that have not been affected by a compound, but any change in sensitivity to a compound that might be important during repeated dose administration.

Stem cells have two other important properties that can be applied to toxicity testing. The first is that they are undifferentiated. The second is that stem cells proliferate. Stem cells can be "determined" into one or more lineages of mature functional cells. When a stem cell be‐ comes determined, it ceases to be a stem cell and becomes a progenitor cell that proliferates and differentiates. The fact that stem cells can be induced to differentiate means that what‐ ever happens at the stem cell level will ultimately affect all downstream events. These char‐ acteristics enable stem cells to be the most important predictors of potential toxicity.

To understand this better, stem cell systems can be divided into "definitive" and "nondefinitive" systems as illustrated in Fig. 1. Definitive stem cell systems are responsible for maintaining a specific biological system. They can be divided into continuously pro‐ liferating systems such as the blood-forming or lympho-hematopoietic system, the gastrointestinal system, hair and skin, reproductive organs and cells of the eye cornea. Although not necessarily a continuously proliferating system, the mesenchymal stem cell (MSC), also called the multipotent mesenchymal stromal cell [1] system can been includ‐ ed, because in culture, the MSCs proliferate and can be passaged over a long period of time. Definitive stem cells systems can also demonstrate partial proliferation. These in‐ clude, but are not limited to, the liver, lung, kidney, heart, pancreas, and the neural/ neuronal system. From a toxicological viewpoint, these are not usually considered stem cell systems. Yet, the different types of lineage cells present in these organs and the abili‐ ty to maintain a specific cell mass has all the intricacies of a stem cell system, especially during development, even though the cell turnover in the adult may be very low. Nondefinitive stem cells systems are represented by the ES and iPS cell systems, which can, theoretically, give rise to any of the definitive stem cell systems. Indeed, it is a prerequi‐ site that the production of functionally, mature cells from ES or iPS cells first pass

**2. Stem cell characteristics and properties used for toxicity testing**

Stem cells of primary, definitive systems always represent a very small proportion of the tissue or organ cellularity. This proportion is between 0.1 and 0.01% or less. The basic definition of a stem cell is that it possesses the capacity for self-renewal. In fact, stem cell systems are usually termed self-renewal cell systems, meaning that one stem cell can produce two daughter cells that are exact replicas of the parent. However, self-renewal is a difficult property to measure. The capacity for either serial *in vivo* repopulation or *in vitro* serial re-plating is considered a property of stem cells that implicates not only the presence of stem cells, but also their selfrenewal capability. The fact that serial *in vitro* re-plating or *in vivo* repopulation cannot be performed ad infinitum is not only an indication for a stem cell hierarchy [2-7], but also for an alternative hypothesis to stem cell self-renewal. This hypothesis states that tissues and organs are endowed with a specific number of stem cells. Once used up, the system ceases to function [8]. Regardless of the hypothesis, this important property can be utilized in a toxicological setting by employing secondary re-plating technology. This allows not only the presence of residual stem cells to be detected that have not been affected by a compound, but any change in sensitivity to a compound that might be important during repeated dose administration.

Stem cells have two other important properties that can be applied to toxicity testing. The first is that they are undifferentiated. The second is that stem cells proliferate. Stem cells can be "determined" into one or more lineages of mature functional cells. When a stem cell be‐ comes determined, it ceases to be a stem cell and becomes a progenitor cell that proliferates and differentiates. The fact that stem cells can be induced to differentiate means that what‐

through a definitive stem cell compartment.

80 Stem Cell Biology in Normal Life and Diseases

**Figure 1. Definitive and Non-Definitive Stem Cell Systems.** Definitive stem cell systems can be further divided into continuously and partially proliferating systems. Non-definitive stem cell systems such as embryonic stem cells and in‐ duced pluripotent stem cells can produce definitive stem cell systems, which in turn, give rise to mature functional cells.

All definitive stem cell systems have a common organization shown in Fig. 2. There is a continuum of stem cells within the stem cell compartment that exhibit different degrees of primitiveness or "stemness", which in turn, implies changing proliferating potential or potency as a stem cell moves through the compartment to the point of determination. These characteristic properties actually provide the information that allows stem cells to be predictors of potential toxicity. Once a stem cell becomes a progenitor cell, prolifera‐ tion continues and actually increases for a certain time so that the compartment can be amplified, until it ceases completely and the differentiation and maturation processes takes over. These changes have important implications for the types of assays that can be used *in vitro* to detect potential toxicity.

From Fig. 2, it is clear that proliferation occurs prior to differentiation. Although there is considerable overlap between proliferation and differentiation, they are two separate proc‐ esses that cannot be measured using the same assay readout. Since stem cells only proliferate, it follows that a proliferation assay is required to detect the presence and response of stem cells to a compound or agent. Using a differentiation assay to detect the effect of a compound or agent that targets one or more steps in the proliferation process can influence the interpretation and conclusion of the results. This can have far-reaching consequences on the decision to move forward with the development of a new drug candidate.

of human origin) nor animal testing provide good extrapolation to the human situation. It is not uncommon for unexpected results or toxicity to rear its head during animal studies be‐ cause of the lack of predictive information obtained during previous screening and testing [13]. Many published articles have dealt with this problem, one of the most notable being the monograph on Toxicity Testing in the 21st Century [14]. Despite the goals of the drug development pipeline and the considerable effort being undertaken by regulatory agencies [15-20] to determine the effect of environmental agents on human cells, interpretation and conclusions often fall short due to lack of understanding of the mechanism of action of the molecule, incorrect assay readout and/or incorrect target cell, to name but a few reasons. If the goal is to determine the effect on human cells, then mouse, rat, dog or even non-human primate cells will not provide the required information; human cells must be used. It goes without saying that drug development or testing xenobiotic agents cannot be performed on human subjects. It is for this reason why surrogate *in vitro* assays using primary human cells obtained from donors under the auspices of regulatory controlled internal review boards (IRBs) provide the best alternative. However, even under these circumstances, detailed knowledge of the biological system under study is necessary in order to interpret and make

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 83

Of all the biological systems of the body, the one most studied is also one of the systems that is given the least priority with respect to toxicity. The blood-forming or hematopoietic system and the gastrointestinal system are two continuously proliferating systems that are expected to be dramatically affected by anti-proliferating agents such as anti-cancer drugs. As a result, the only relevant questions are (a) how severe would toxicity be, and (b) would use of the drug

conclusions in the most objective manner.

provide a favorable therapeutic index?

**Figure 3.** The Major Stages of the Drug Development Pipeline

**Figure 2.** The Common Organization of Definitive Stem Cell Systems

Toxicity represents between 30-40% of the drug attrition rate [9,10]. It is therefore not sur‐ prising that biopharmaceutical companies are eager to employ assays that allow early pre‐ diction of toxicity prior to starting human clinical trials. Once the drug discovery phase has been concluded, the drug development phase begins (Fig. 3) by screening thousands of com‐ pounds in a battery of tests to determine absorption, distribution, metabolism and excretion (ADME) as well as preliminary toxicity (ADME/Tox). Many of the ADME/Tox assays as well as those in the lead optimization phase use transformed cell lines as cell targets, such as the NCI60 cell line panel [11,12]. Once these tests have whittled down the number of possi‐ ble drug candidates, pre-clinical animal models are used. Neither cell lines (even if they are of human origin) nor animal testing provide good extrapolation to the human situation. It is not uncommon for unexpected results or toxicity to rear its head during animal studies be‐ cause of the lack of predictive information obtained during previous screening and testing [13]. Many published articles have dealt with this problem, one of the most notable being the monograph on Toxicity Testing in the 21st Century [14]. Despite the goals of the drug development pipeline and the considerable effort being undertaken by regulatory agencies [15-20] to determine the effect of environmental agents on human cells, interpretation and conclusions often fall short due to lack of understanding of the mechanism of action of the molecule, incorrect assay readout and/or incorrect target cell, to name but a few reasons. If the goal is to determine the effect on human cells, then mouse, rat, dog or even non-human primate cells will not provide the required information; human cells must be used. It goes without saying that drug development or testing xenobiotic agents cannot be performed on human subjects. It is for this reason why surrogate *in vitro* assays using primary human cells obtained from donors under the auspices of regulatory controlled internal review boards (IRBs) provide the best alternative. However, even under these circumstances, detailed knowledge of the biological system under study is necessary in order to interpret and make conclusions in the most objective manner.

Of all the biological systems of the body, the one most studied is also one of the systems that is given the least priority with respect to toxicity. The blood-forming or hematopoietic system and the gastrointestinal system are two continuously proliferating systems that are expected to be dramatically affected by anti-proliferating agents such as anti-cancer drugs. As a result, the only relevant questions are (a) how severe would toxicity be, and (b) would use of the drug provide a favorable therapeutic index?

**Figure 3.** The Major Stages of the Drug Development Pipeline

and conclusion of the results. This can have far-reaching consequences on the decision to move

forward with the development of a new drug candidate.

82 Stem Cell Biology in Normal Life and Diseases

**Figure 2.** The Common Organization of Definitive Stem Cell Systems

Toxicity represents between 30-40% of the drug attrition rate [9,10]. It is therefore not sur‐ prising that biopharmaceutical companies are eager to employ assays that allow early pre‐ diction of toxicity prior to starting human clinical trials. Once the drug discovery phase has been concluded, the drug development phase begins (Fig. 3) by screening thousands of com‐ pounds in a battery of tests to determine absorption, distribution, metabolism and excretion (ADME) as well as preliminary toxicity (ADME/Tox). Many of the ADME/Tox assays as well as those in the lead optimization phase use transformed cell lines as cell targets, such as the NCI60 cell line panel [11,12]. Once these tests have whittled down the number of possi‐ ble drug candidates, pre-clinical animal models are used. Neither cell lines (even if they are

Hemotoxicity testing is traditionally performed during the last stage of drug development, namely pre-clinical animal testing. Circulating blood parameters are measured and at necropsy, bone marrow, spleen and even liver hematopathology are performed. Primary stem and progenitor cells cannot be morphologically identified. Morphological identification of cells is only possible once the cells start to differentiate and mature. Consequently, traditional hemotoxicity testing provides little, if any, predictive value since most of the toxic effects have occurred on more primitive cells.

As mentioned above, although proliferation is required to produce colonies, the number of cells produced as a quantitative measure of proliferation cannot be ascertained. Although proliferation is assumed, the CFC assay actually detects differentiation ability or potential.

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 85

Over several years, the European Center for the Validation of Alternative Methods (ECVAM) undertook a series of studies in which a number of drugs and chemicals were tested using the CFC assay. These studies are noteworthy because they represented the first attempt to validate a prediction model for assessing the maximum tolerated dose (MTD, equivalent to the IC90 value) for drugs that induce neutropenia [26,27] using the CFC assay. The studies were performed in different laboratories and were later extended to compounds that caused thrombocytopenia [28]. Potential neutropenia was detected by the effect on the granulocytemacrophage colony-forming cell or GM-CFC (also called CFC or CFC-GM), while thrombo‐ cytopenia was detected by the effect on the megakaryocyte colony-forming cell or Mk-CFC (also called CFC-Mk). A decrease or inhibition in the number of colonies counted derived from GM-CFC or Mk-CFC predicted a reduction in neutrophils or platelets in the circulation. The authors demonstrated that the model could correctly predict the MTD of 20 out of 23 drugs

There are two points worth emphasizing. First, not all compounds will produce an estimated IC90 value and may not even produce an IC50 value, when tested using the CFC assay. Does that mean that these compounds will not produce neutropenia or thrombocytopenia? It is interesting to note that the same CFC assay that is used to predict toxicity causing neutropenia or thrombocytopenia, is also used in an opposite manner to predict time to neutrophil or platelet engraftment after bone marrow, mobilized peripheral blood or umbilical cord blood stem cell transplantation for cellular therapy [39-31]. In either case, the GM-CFC or MK-CFC populations provide no information on the response of the more sensitive and more important stem cells. After all, it is the hematopoietic stem cells that give rise to both of these populations. This leads to the second point, namely that many compounds target one or more steps in the proliferation process, either at a molecular and/or cellular level. Although both GM-CFC and Mk-CFC populations are proliferating progenitor cell populations, they are not always the primary targets. When a compound affects more than one lineage, the primary effect is not on those lineages individually, but on the common cell that gives rise to those lineages, namely the stem cells [32]. From a practical viewpoint, however, the CFC assay posses daunting problems. The ECVAM studies summarized previously were exceptional in that the authors took the trouble to try and verify and standardize the readout of the assay that is inherently subjective and lacks the necessary external standards and controls by which the assay could be properly validated. In studies performed by the National Marrow Donor Program (NMDP), the results showed very high variability in CFU colony counting for cord blood [33]. This high variability, primarily due to the inaccuracy of dispensing methylcellulose and colony counting, together with the lack of high throughput capability does not provide the biopharmaceutical industry, environmental agencies or other areas of toxicology, risk or efficacy assessment with a routine and trustworthy assay platform. To negate all of these problems, the HALO Predic‐

This has important consequences for toxicity testing.

tested (87% predictive rate).

tive Hemotoxicity Platform was developed.

Much of our knowledge about the characterization, properties and responses of hematopoietic stem cells and the system as a whole has been provided through the use of drugs and other agents (e.g. radiation) using both *in vivo* and later, *in vitro* assays. The information obtained has allowed the organization and hierarchy within the different compartments of the hema‐ topoietic and lymphopoietic systems to be elucidated. By utilizing the knowledge that has accrued over more than six decades, analysis of the lympho-hematopoietic stem and progen‐ itor cells provide the highest degree of predictive toxicity of any biological system.

#### **3. The Colony-Forming Cell (CFC) assay and ECVAM studies**

In 1966, Bradley and Metcalf in Melbourne, Australia [21] and Pluznik and Sachs in Rehovot, Israel, [22] independently published what is now known as the colony-forming unit (CFU) or colony-forming cell (CFC) assay. In its original form, mouse bone marrow target cells were suspended in agar containing a conditioned medium that we now know contained granulo‐ cyte-macrophage colony stimulating factor or GM-CSF as well as other soluble factors. In the semi-solid medium, the cells underwent proliferation and later differentiation to produce colonies of cells that were identified either as neutrophils, macrophages or a combination of the two cell types. The number of colonies counted under an inverted microscope was proportional to both the number of cells plated and the dose of the conditioned medium added. In the same year, Cole and Paul [23] in Glasgow, Scotland reported the first *in vitro* suspension culture of murine erythropoietic cells from the yolk sac and fetal liver. Culture of erythropoietic cells under clonal conditions did not occur until 1971, when the Axelrad group [24] in Toronto, Canada, demonstrated that erythroid colonies could be produced using a plasma clot techni‐ que. In 1974, Iscove and colleagues [25] in Basel, Switzerland introduced the methylcellulose CFC assay that is still used today. Since that time, colony assays have been developed to detect multiple cell populations of every blood cell lineage, including several different stem cell populations. In addition, conditioned media has been replaced with recombinant growth factors and cytokines.

In Section 2, emphasis was placed on the importance between proliferation and differentiation. The cell populations detected using the CFC assay must all be proliferating populations, otherwise the production of colonies would not occur. However, to identify the type of colony, the *in vitro* culture must be allowed to proceed long enough so that the cells produced can themselves be identified as being derived from a morphologically unidentifiable stem, progenitor or precursor cell, all of which are capable of proliferation, but to different extents. As mentioned above, although proliferation is required to produce colonies, the number of cells produced as a quantitative measure of proliferation cannot be ascertained. Although proliferation is assumed, the CFC assay actually detects differentiation ability or potential. This has important consequences for toxicity testing.

Hemotoxicity testing is traditionally performed during the last stage of drug development, namely pre-clinical animal testing. Circulating blood parameters are measured and at necropsy, bone marrow, spleen and even liver hematopathology are performed. Primary stem and progenitor cells cannot be morphologically identified. Morphological identification of cells is only possible once the cells start to differentiate and mature. Consequently, traditional hemotoxicity testing provides little, if any, predictive value since most of the toxic effects have

Much of our knowledge about the characterization, properties and responses of hematopoietic stem cells and the system as a whole has been provided through the use of drugs and other agents (e.g. radiation) using both *in vivo* and later, *in vitro* assays. The information obtained has allowed the organization and hierarchy within the different compartments of the hema‐ topoietic and lymphopoietic systems to be elucidated. By utilizing the knowledge that has accrued over more than six decades, analysis of the lympho-hematopoietic stem and progen‐

In 1966, Bradley and Metcalf in Melbourne, Australia [21] and Pluznik and Sachs in Rehovot, Israel, [22] independently published what is now known as the colony-forming unit (CFU) or colony-forming cell (CFC) assay. In its original form, mouse bone marrow target cells were suspended in agar containing a conditioned medium that we now know contained granulo‐ cyte-macrophage colony stimulating factor or GM-CSF as well as other soluble factors. In the semi-solid medium, the cells underwent proliferation and later differentiation to produce colonies of cells that were identified either as neutrophils, macrophages or a combination of the two cell types. The number of colonies counted under an inverted microscope was proportional to both the number of cells plated and the dose of the conditioned medium added. In the same year, Cole and Paul [23] in Glasgow, Scotland reported the first *in vitro* suspension culture of murine erythropoietic cells from the yolk sac and fetal liver. Culture of erythropoietic cells under clonal conditions did not occur until 1971, when the Axelrad group [24] in Toronto, Canada, demonstrated that erythroid colonies could be produced using a plasma clot techni‐ que. In 1974, Iscove and colleagues [25] in Basel, Switzerland introduced the methylcellulose CFC assay that is still used today. Since that time, colony assays have been developed to detect multiple cell populations of every blood cell lineage, including several different stem cell populations. In addition, conditioned media has been replaced with recombinant growth

In Section 2, emphasis was placed on the importance between proliferation and differentiation. The cell populations detected using the CFC assay must all be proliferating populations, otherwise the production of colonies would not occur. However, to identify the type of colony, the *in vitro* culture must be allowed to proceed long enough so that the cells produced can themselves be identified as being derived from a morphologically unidentifiable stem, progenitor or precursor cell, all of which are capable of proliferation, but to different extents.

itor cells provide the highest degree of predictive toxicity of any biological system.

**3. The Colony-Forming Cell (CFC) assay and ECVAM studies**

occurred on more primitive cells.

84 Stem Cell Biology in Normal Life and Diseases

factors and cytokines.

Over several years, the European Center for the Validation of Alternative Methods (ECVAM) undertook a series of studies in which a number of drugs and chemicals were tested using the CFC assay. These studies are noteworthy because they represented the first attempt to validate a prediction model for assessing the maximum tolerated dose (MTD, equivalent to the IC90 value) for drugs that induce neutropenia [26,27] using the CFC assay. The studies were performed in different laboratories and were later extended to compounds that caused thrombocytopenia [28]. Potential neutropenia was detected by the effect on the granulocytemacrophage colony-forming cell or GM-CFC (also called CFC or CFC-GM), while thrombo‐ cytopenia was detected by the effect on the megakaryocyte colony-forming cell or Mk-CFC (also called CFC-Mk). A decrease or inhibition in the number of colonies counted derived from GM-CFC or Mk-CFC predicted a reduction in neutrophils or platelets in the circulation. The authors demonstrated that the model could correctly predict the MTD of 20 out of 23 drugs tested (87% predictive rate).

There are two points worth emphasizing. First, not all compounds will produce an estimated IC90 value and may not even produce an IC50 value, when tested using the CFC assay. Does that mean that these compounds will not produce neutropenia or thrombocytopenia? It is interesting to note that the same CFC assay that is used to predict toxicity causing neutropenia or thrombocytopenia, is also used in an opposite manner to predict time to neutrophil or platelet engraftment after bone marrow, mobilized peripheral blood or umbilical cord blood stem cell transplantation for cellular therapy [39-31]. In either case, the GM-CFC or MK-CFC populations provide no information on the response of the more sensitive and more important stem cells. After all, it is the hematopoietic stem cells that give rise to both of these populations. This leads to the second point, namely that many compounds target one or more steps in the proliferation process, either at a molecular and/or cellular level. Although both GM-CFC and Mk-CFC populations are proliferating progenitor cell populations, they are not always the primary targets. When a compound affects more than one lineage, the primary effect is not on those lineages individually, but on the common cell that gives rise to those lineages, namely the stem cells [32]. From a practical viewpoint, however, the CFC assay posses daunting problems. The ECVAM studies summarized previously were exceptional in that the authors took the trouble to try and verify and standardize the readout of the assay that is inherently subjective and lacks the necessary external standards and controls by which the assay could be properly validated. In studies performed by the National Marrow Donor Program (NMDP), the results showed very high variability in CFU colony counting for cord blood [33]. This high variability, primarily due to the inaccuracy of dispensing methylcellulose and colony counting, together with the lack of high throughput capability does not provide the biopharmaceutical industry, environmental agencies or other areas of toxicology, risk or efficacy assessment with a routine and trustworthy assay platform. To negate all of these problems, the HALO Predic‐ tive Hemotoxicity Platform was developed.

#### **4. Predictive stem cell hemotoxicity testing**

Whereas the CFU assay may be used to predict neutropenia, thrombocytopenia, anemia and the MTD indicated by the IC90 values [27-29], stem cells assays allow potential hemotoxicity to be taken to a different system-wide "global" level. The reason is provided in Fig. 2 and in more detail in Fig. 4, which shows the different lympho-hematopoietic cell populations that can be detected using a hemotoxicity screening and testing platform specifically developed for this purpose. This platform, called HALO, will be described in more detail in the next section. Figures 2 and 4 demonstrate that functionally mature cells from definitive continu‐ ously proliferating and partially proliferating cell systems, are derived from stem cells. As such, any perturbation or damage to the stem cell compartment will ultimately affect all downstream cell populations. In other words, examining the effect on stem cells allows the "global" effect on the system to be predicted. Since more is known about the organization, hierarchy and regulation of the lympho-hematopoietic system than probably any other biological system in the body, this knowledge can be used to predict and explain potentially deleterious effects to the system. Changes in the response to hematopoietic stem cells will affect all three primary hematopoietic lineages, namely the erythropoietic, myelomonocytic and magakaryopoietic lineages. Changes in the response to lympho-hematopoietic stem cells, i.e. those stem cells that can give rise to both the lymphopoietic and hematopoietic cells, will be expected to affect most, if not all cell lineages, including the T- and B-cell lineages and therefore the immune system as a whole.

Predictive stem cell hemotoxicity testing is not simply the estimation of IC values so that compounds can be ranked in order of toxicity to different cell populations or species. There are several other important applications in which stem cell hemotoxicity, and indeed stem cell toxicity in general, can be used. Examples of these applications will be discussed later in this chapter. First, however, it is necessary to describe the principles, characteristics and properties of the assay that make this possible.

**Figure 4.** The Organization and Hierarchy of the Lympho-Hematopoietic System as a Model for a Definitive Continu‐

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 87

When cells proliferate or are inhibited from proliferation by drugs or other agents, the concentration of intracellular adenosine triphosphate (iATP) changes proportionately. This biochemical marker is an indicator of cellular and mitochondrial integrity and there‐ fore viability of the cells. Indeed, iATP is used as a metabolic viability assay (as opposed to a dye exclusion viability assay). Under normal conditions, stimulation of cell prolifera‐ tion requires specific growth factors and/or cytokines either alone or in combination (cocktails). For continuously proliferating systems, growth factors or cytokines need to be present continuously, albeit, in very small concentrations, in order to maintain cell sur‐ vival and production. Thus, to detect the effect of any agent on hematopoietic cells *in vi‐ tro*, the target cell population must be stimulated in order to detect changes in the cell

ously Proliferating Stem Cell System. The properties of stem cells play an integral part in predicting toxicity.

**5.1. Concepts and principles of the HALO platform**

#### **5. Materials and methods**

HALO is the acronym for Hematopoietic/Hemotoxicity Assays via Luminescence Output. This platform was originally designed and developed to provide the biopharmaceutical in‐ dustry with a high throughput, validated assay to examine the effects of virtually any com‐ pound on different cell populations of the lympho-hematopoietic system from multiple species. Initially, the assay platform was developed for fresh, primary human cells, as a sur‐ rogate assay that could be used at virtually at stage in the drug development pipeline (Fig. 3) to extrapolate to the human situation, and as an alternative to pre-clinical animal studies. The platform has since been further developed to include non-human primate, horse, pig, sheep, dog, rat and mouse, not only for toxicity studies, but also for basic research and vet‐ erinary applications.

**Figure 4.** The Organization and Hierarchy of the Lympho-Hematopoietic System as a Model for a Definitive Continu‐ ously Proliferating Stem Cell System. The properties of stem cells play an integral part in predicting toxicity.

#### **5.1. Concepts and principles of the HALO platform**

**4. Predictive stem cell hemotoxicity testing**

86 Stem Cell Biology in Normal Life and Diseases

the immune system as a whole.

of the assay that make this possible.

**5. Materials and methods**

erinary applications.

Whereas the CFU assay may be used to predict neutropenia, thrombocytopenia, anemia and the MTD indicated by the IC90 values [27-29], stem cells assays allow potential hemotoxicity to be taken to a different system-wide "global" level. The reason is provided in Fig. 2 and in more detail in Fig. 4, which shows the different lympho-hematopoietic cell populations that can be detected using a hemotoxicity screening and testing platform specifically developed for this purpose. This platform, called HALO, will be described in more detail in the next section. Figures 2 and 4 demonstrate that functionally mature cells from definitive continu‐ ously proliferating and partially proliferating cell systems, are derived from stem cells. As such, any perturbation or damage to the stem cell compartment will ultimately affect all downstream cell populations. In other words, examining the effect on stem cells allows the "global" effect on the system to be predicted. Since more is known about the organization, hierarchy and regulation of the lympho-hematopoietic system than probably any other biological system in the body, this knowledge can be used to predict and explain potentially deleterious effects to the system. Changes in the response to hematopoietic stem cells will affect all three primary hematopoietic lineages, namely the erythropoietic, myelomonocytic and magakaryopoietic lineages. Changes in the response to lympho-hematopoietic stem cells, i.e. those stem cells that can give rise to both the lymphopoietic and hematopoietic cells, will be expected to affect most, if not all cell lineages, including the T- and B-cell lineages and therefore

Predictive stem cell hemotoxicity testing is not simply the estimation of IC values so that compounds can be ranked in order of toxicity to different cell populations or species. There are several other important applications in which stem cell hemotoxicity, and indeed stem cell toxicity in general, can be used. Examples of these applications will be discussed later in this chapter. First, however, it is necessary to describe the principles, characteristics and properties

HALO is the acronym for Hematopoietic/Hemotoxicity Assays via Luminescence Output. This platform was originally designed and developed to provide the biopharmaceutical in‐ dustry with a high throughput, validated assay to examine the effects of virtually any com‐ pound on different cell populations of the lympho-hematopoietic system from multiple species. Initially, the assay platform was developed for fresh, primary human cells, as a sur‐ rogate assay that could be used at virtually at stage in the drug development pipeline (Fig. 3) to extrapolate to the human situation, and as an alternative to pre-clinical animal studies. The platform has since been further developed to include non-human primate, horse, pig, sheep, dog, rat and mouse, not only for toxicity studies, but also for basic research and vet‐ When cells proliferate or are inhibited from proliferation by drugs or other agents, the concentration of intracellular adenosine triphosphate (iATP) changes proportionately. This biochemical marker is an indicator of cellular and mitochondrial integrity and there‐ fore viability of the cells. Indeed, iATP is used as a metabolic viability assay (as opposed to a dye exclusion viability assay). Under normal conditions, stimulation of cell prolifera‐ tion requires specific growth factors and/or cytokines either alone or in combination (cocktails). For continuously proliferating systems, growth factors or cytokines need to be present continuously, albeit, in very small concentrations, in order to maintain cell sur‐ vival and production. Thus, to detect the effect of any agent on hematopoietic cells *in vi‐ tro*, the target cell population must be stimulated in order to detect changes in the cell population response to the agent. The agent is usually added in a dose-dependent man‐ ner to the target cells, which are then incubated for a specific period of time. Thereafter, the cultures are removed from the incubator and the cells lysed to release the iATP. The latter then becomes a limiting substrate for a luciferin-luciferase reaction to produce bio‐ luminescence in the form of light as shown in the equation below.

important consequences. Cell interaction reduces the time for the onset of cell prolifera‐ tion by approximately 24 hours. This means that measurement of cell proliferation can be measured within 5 to 7 days. Indeed, for all of the studies described here, human cells were incubated for 5 days. Non-human primate cells are usually incubated for the same time, but all other animal cells only require 4 days of incubation. The second con‐ sequence of allowing cell interaction to occur is the two-fold increase in assay sensitivity. As with most cell cultures, cells are incubated at 37ºC in a fully humidified atmosphere containing CO2. Incubating cells under low oxygen tension of 5% O2, which is approx. equivalent to the venous oxygen tension, reduces oxygen toxicity due to free radical pro‐ duction and improves plating efficiency [34,35] for all lympho-hematopoietic cell popula‐

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 89

Four basic controls were always included for toxicity studies. A background control included cells, but no growth factors. A vehicle control was similar to the background control, but included the vehicle used to dissolve the compound. Growth of the target cell population without any compound or vehicle constituted the growth factor control. A similar control that included the vehicle was designated the growth factor + vehicle control. Drugs and other

Prior to measuring any sample, the instrument was calibrated and the assay standardized using an external ATP standard and controls. The procedures have been described previously [32] and detailed procedures can also be obtained [36,37]. Calibration and standardization

There are other advantages for calibrating and standardizing the assay. First results can be compared over time. Second, the output of a plate luminometer is in Relative Lumi‐ nescence Units or RLU. The results are relative because different instruments demon‐ strate different ranges of RLU. These ranges may vary from 0 to 100 for one manufacturer or 0 to several million for another. This means that it would be very diffi‐ cult to directly compare results within and between laboratories using RLU values. Per‐ forming an ATP standard curve allows all the results to be interpolated from RLU

HALO was originally developed from the "classic" CFC assay because the latter was the only cell-based assay that could detect primitive hematopoietic cell populations. Since HALO is a proliferation assay, while the CFC detects differentiation of the same cells, and because proliferation occurs prior to differentiation, it follows that one assay can verify the other. Indeed, several publications have shown a direct correlation between

**5.5. Instrument calibration, assay standardization and sample processing**

were also part of the assay validation process (see Section 5.6).

values into standardized ATP concentrations (µM).

**5.6. Assay verification and validation**

the two assays [32,38,39].

tions as well as other cell types.

agents were investigated over 6 – 9 doses.

**5.4. Controls and dosing**

$$\text{LiATP} + \text{Luiciferin} + \text{O}\_2 \xrightarrow[\text{Mg}^{2+}]{\text{Lucíferase}} \text{Oxylucíferin} + \text{AMP} + \text{PP}\_i + \text{CO}\_2 + \text{LIGHT}\_i$$

The light is measured in a plate luminometer. The amount of light produced correlates directly with any change in the iATP concentration and therefore with the state of proliferation or inhibition of the cells.

#### **5.2. Cell sources**

Cells from any hematopoietic or lymphopoietic organ can be used. For most of the studies described here, fresh or cryopreserved human bone marrow or peripheral blood cells were collected with prior authorization by an Internal Review Board (IRB). Human cells were obtained from Lonza (Walkerville, MD) or Allcells (Berkely, CA). A mononuclear cell (MNC) fraction was prepared using density gradient centrifugation. A nucleated cell count was performed using a Z2 particle counter (Beckman Coulter), while dye exclusion viability was performed using 7-aminoactinoycin D (7-AAD) and flow cytometry. Metabolic viability was performed using LIVEGlo (HemoGenix, Colorado Springs, CO).

#### **5.3. Cell culture**

For all toxicity studies, MNC were diluted so that the final cell concentration was either 7,500 or 10,000 cells/well. Either 96-well or 384-well, solid white-wall plates were used and all dispensing was performed using a liquid handler (Beckman Coulter, EPICS XL-MCL). After the cell suspension was prepared, it was added to a Master Mix containing reagents including growth factors and/or cytokines to stimulate the target cell population being studied. Five different hematopoietic stem cell populations have so far been devel‐ oped for this assay, the most important being the Colony-Forming Cell – Granulocyte, Erythroid, Macrophage, Megakaryocyte or CFC-GEMM (referred to in Fig. 4 as CFC-GEMM 1). This particular stem cell population is stimulated with erythropoietin (EPO), granulocyte-macrophage and granulocyte colony stimulating factors (GM-CSF, G-CSF), Interleukins 3 and 6 (IL-3, IL-6), stem cell factor (SCF), Flt3-Ligand (Flt3-L) and thrombo‐ poietin (TPO). Compared to a "classic" CFC assay, HALO does not incorporate methyl‐ cellulose and is therefore not a clonal assay. Instead HALO uses Suspension Expansion Culture (SEC) Technology, which has several advantages over methylcellulose assays. First, SEC assays allow more accurate dispensing using liquid handlers. This is in con‐ trast to inaccurately dispensing methylcellulose with syringes and needles. Second, the use of liquid handlers allows for true high throughput capability with accurate dispens‐ ing even in 384-well plates. Third, as opposed to methylcellulose, where little or no cell interaction occurs, SEC technology allows cells to interact with each other. This has two important consequences. Cell interaction reduces the time for the onset of cell prolifera‐ tion by approximately 24 hours. This means that measurement of cell proliferation can be measured within 5 to 7 days. Indeed, for all of the studies described here, human cells were incubated for 5 days. Non-human primate cells are usually incubated for the same time, but all other animal cells only require 4 days of incubation. The second con‐ sequence of allowing cell interaction to occur is the two-fold increase in assay sensitivity. As with most cell cultures, cells are incubated at 37ºC in a fully humidified atmosphere containing CO2. Incubating cells under low oxygen tension of 5% O2, which is approx. equivalent to the venous oxygen tension, reduces oxygen toxicity due to free radical pro‐ duction and improves plating efficiency [34,35] for all lympho-hematopoietic cell popula‐ tions as well as other cell types.

#### **5.4. Controls and dosing**

population response to the agent. The agent is usually added in a dose-dependent man‐ ner to the target cells, which are then incubated for a specific period of time. Thereafter, the cultures are removed from the incubator and the cells lysed to release the iATP. The latter then becomes a limiting substrate for a luciferin-luciferase reaction to produce bio‐

The light is measured in a plate luminometer. The amount of light produced correlates directly with any change in the iATP concentration and therefore with the state of proliferation or

Cells from any hematopoietic or lymphopoietic organ can be used. For most of the studies described here, fresh or cryopreserved human bone marrow or peripheral blood cells were collected with prior authorization by an Internal Review Board (IRB). Human cells were obtained from Lonza (Walkerville, MD) or Allcells (Berkely, CA). A mononuclear cell (MNC) fraction was prepared using density gradient centrifugation. A nucleated cell count was performed using a Z2 particle counter (Beckman Coulter), while dye exclusion viability was performed using 7-aminoactinoycin D (7-AAD) and flow cytometry. Metabolic viability was

For all toxicity studies, MNC were diluted so that the final cell concentration was either 7,500 or 10,000 cells/well. Either 96-well or 384-well, solid white-wall plates were used and all dispensing was performed using a liquid handler (Beckman Coulter, EPICS XL-MCL). After the cell suspension was prepared, it was added to a Master Mix containing reagents including growth factors and/or cytokines to stimulate the target cell population being studied. Five different hematopoietic stem cell populations have so far been devel‐ oped for this assay, the most important being the Colony-Forming Cell – Granulocyte, Erythroid, Macrophage, Megakaryocyte or CFC-GEMM (referred to in Fig. 4 as CFC-GEMM 1). This particular stem cell population is stimulated with erythropoietin (EPO), granulocyte-macrophage and granulocyte colony stimulating factors (GM-CSF, G-CSF), Interleukins 3 and 6 (IL-3, IL-6), stem cell factor (SCF), Flt3-Ligand (Flt3-L) and thrombo‐ poietin (TPO). Compared to a "classic" CFC assay, HALO does not incorporate methyl‐ cellulose and is therefore not a clonal assay. Instead HALO uses Suspension Expansion Culture (SEC) Technology, which has several advantages over methylcellulose assays. First, SEC assays allow more accurate dispensing using liquid handlers. This is in con‐ trast to inaccurately dispensing methylcellulose with syringes and needles. Second, the use of liquid handlers allows for true high throughput capability with accurate dispens‐ ing even in 384-well plates. Third, as opposed to methylcellulose, where little or no cell interaction occurs, SEC technology allows cells to interact with each other. This has two

+ CO2+*LIGHT*

luminescence in the form of light as shown in the equation below.

performed using LIVEGlo (HemoGenix, Colorado Springs, CO).

Oxyluciferin + AMP + PPi

Mg2+ Luciferase

*i*ATP + Luciferin + O2 →

88 Stem Cell Biology in Normal Life and Diseases

inhibition of the cells.

**5.2. Cell sources**

**5.3. Cell culture**

Four basic controls were always included for toxicity studies. A background control included cells, but no growth factors. A vehicle control was similar to the background control, but included the vehicle used to dissolve the compound. Growth of the target cell population without any compound or vehicle constituted the growth factor control. A similar control that included the vehicle was designated the growth factor + vehicle control. Drugs and other agents were investigated over 6 – 9 doses.

#### **5.5. Instrument calibration, assay standardization and sample processing**

Prior to measuring any sample, the instrument was calibrated and the assay standardized using an external ATP standard and controls. The procedures have been described previously [32] and detailed procedures can also be obtained [36,37]. Calibration and standardization were also part of the assay validation process (see Section 5.6).

There are other advantages for calibrating and standardizing the assay. First results can be compared over time. Second, the output of a plate luminometer is in Relative Lumi‐ nescence Units or RLU. The results are relative because different instruments demon‐ strate different ranges of RLU. These ranges may vary from 0 to 100 for one manufacturer or 0 to several million for another. This means that it would be very diffi‐ cult to directly compare results within and between laboratories using RLU values. Per‐ forming an ATP standard curve allows all the results to be interpolated from RLU values into standardized ATP concentrations (µM).

#### **5.6. Assay verification and validation**

HALO was originally developed from the "classic" CFC assay because the latter was the only cell-based assay that could detect primitive hematopoietic cell populations. Since HALO is a proliferation assay, while the CFC detects differentiation of the same cells, and because proliferation occurs prior to differentiation, it follows that one assay can verify the other. Indeed, several publications have shown a direct correlation between the two assays [32,38,39].

Validation, on the other hand, is quite a different matter. Assay validation is defined as "establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes" [40]. When an assay is properly validated the accuracy (proportion of correct outcomes), sensitivity (proportion of correctly identified positive samples), selectivity (pro‐ portion of correctly identified negative samples), precision (intra and inter-laboratory varia‐ bility) and robustness (the ability of the assay to withstand changes and transferability) all combine to give the user the assurance that the results obtained are correct. The ECVAM studies described in Section 2 above were, and still are, the closest the CFC assay has come to being validated. There have been many attempts to validate the CFC assay, but all have failed. Certainly the assay has shown, from a subjective viewpoint, some of the attributes. However, since there are no standards and controls by which the CFC assay can provide documented and quantitative evidence for each of the required parameters, the assay has never been properly validated. Like many assays that have been used for decades, the CFC assay has been "grandfathered" in and used despite the problematic trustworthiness and meaning of the results obtained [33,37].

**5.7. Statistics**

All of the results provided were produced using 8 replicate wells/point. Compound dose response curves were fitted to a 4- or 5-parameter logistic curve fit using SoftMax Pro software (Molecular Devices) from results exported directed from the plate luminometer and calculated automatically. To estimate IC values, raw data were converted to a percentage of the growth factor + vehicle control. Additional statistics, curve fitting or graphing was performed using

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 91

From a practical viewpoint, stem and progenitor cells are distinguished by at least two different characteristics. First, stem and progenitor cell populations are stimulated using different cocktails of growth factors and cytokines. In this way, specific cell populations can be targeted and studied, even though the cell suspension may contain other cell types. Combined with the culture conditions, this allows detection and measurement of specific cell populations. The other distinguishing characteristic is the difference in proliferation ability and potential between stem and progenitor cells. Even within the stem cell compartment, differences in proliferation potential will indicate the primitiveness or "stemness" of populations. This characteristic is shown in Fig. 5 for normal bone marrow cells. Since the stem cells are more primitive than the progenitor cells, it would be expected that their proliferation potential would be greater. Figure 5 shows that the two stem cell populations exhibit, not only greater ATP concentration values, but also greater linear regression cell dose response slopes than the hematopoietic or lymphopoietic progenitor cells. It is the slope of the cell dose response that measures proliferation potential. The greater the slope, the higher the proliferation potential, and the more primitive the cell population. Indeed, this is the basic principle for measuring potency of hematopoietic stem cell therapeutic products for transplantation [37]. In this way, it is possible to distinguish different stem cell populations, in this case the hematopoietic stem cell, CFC-GEMM 1, from the more primitive lympho-hematopoietic stem cell, HPP-SP (high proliferative potential – stem and progenitor cell). The HPP-SP stem cell will be discussed in more detail in Section 6.4. The three cell dose response clusters showing the differences in proliferation potential in Fig. 5 for stem cells, hematopoietic progenitor cells and lympho‐ poietic progenitor cells would be expected based on the organization of the blood-forming system shown in Fig. 4. Figure 6 demonstrates the expected proliferation ability of the seven different cell populations in response to mitomycin-C, with the stem cells showing the greatest ability to proliferate followed by the three hematopoietic lineages and lymphopoietic lineages. The steepness of the linear regression slope of the cell dose response for a cell population provides a measure of the proliferation potential. Stem cells exhibit the greatest proliferation potential of all cells. Within the stem cell compartment, stem cells with different potentials for proliferation also indicate their primitiveness. Proliferation ability is measured at a single

Prism software (GraphPad) or OriginPro (OriginLab).

**6.1. Distinguishing the response of stem cells from progenitor cells**

**6. Results and discussion**

cell dose (see Fig. 6).

HALO, from the outset, was designed to be validated. The assay was developed to incorporate the range values specified in the FDA Guidance on Bioanalytical Method Validation [40]. In summary, these values are as follows:


In addition, the assay has also been validated against the Registry of Cytotoxicity Prediction Model, which will be discussed in more detail in Section 5B.

#### **5.7. Statistics**

Validation, on the other hand, is quite a different matter. Assay validation is defined as "establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes" [40]. When an assay is properly validated the accuracy (proportion of correct outcomes), sensitivity (proportion of correctly identified positive samples), selectivity (pro‐ portion of correctly identified negative samples), precision (intra and inter-laboratory varia‐ bility) and robustness (the ability of the assay to withstand changes and transferability) all combine to give the user the assurance that the results obtained are correct. The ECVAM studies described in Section 2 above were, and still are, the closest the CFC assay has come to being validated. There have been many attempts to validate the CFC assay, but all have failed. Certainly the assay has shown, from a subjective viewpoint, some of the attributes. However, since there are no standards and controls by which the CFC assay can provide documented and quantitative evidence for each of the required parameters, the assay has never been properly validated. Like many assays that have been used for decades, the CFC assay has been "grandfathered" in and used despite the problematic trustworthiness and meaning of the

HALO, from the outset, was designed to be validated. The assay was developed to incorporate the range values specified in the FDA Guidance on Bioanalytical Method Validation [40]. In

**•** Sensitivity & Selectivity by Receiver Operator Characteristics (ROC): Area Under Curve

**•** Log-log linear regression slope for ATP standard curve: 0.937 ± 15% (slope range: 0.796 –

In addition, the assay has also been validated against the Registry of Cytotoxicity Prediction

results obtained [33,37].

90 Stem Cell Biology in Normal Life and Diseases

**•** Assay linearity: => 5 logs.

**•** Accuracy: ~95%.

**•** Robustness: ~95%.

1.07)

summary, these values are as follows:

**•** Assay ATP sensitivity: ~ 0.001µM.

**•** Assay cell linearity: 1,000 - > 25,000 cells/well.

**•** Assay cell sensitivity: 20-25 cells/well, depending on cell purity).

**•** Precision: = < 15%. Lower limit of quantification (LLOQ): 20%.

**•** High throughput capability (Z-factor [57]): > 0.76.

(AUC) 0.73 – 0.752 (lowest possible value: 0.5; highest possible value, 1).

**•** Lowest ATP value indicating unsustainable cell proliferation: ~ 0.04µM.

**•** ATP value below which cells are not metabolically viable: ~0.01µM.

Model, which will be discussed in more detail in Section 5B.

All of the results provided were produced using 8 replicate wells/point. Compound dose response curves were fitted to a 4- or 5-parameter logistic curve fit using SoftMax Pro software (Molecular Devices) from results exported directed from the plate luminometer and calculated automatically. To estimate IC values, raw data were converted to a percentage of the growth factor + vehicle control. Additional statistics, curve fitting or graphing was performed using Prism software (GraphPad) or OriginPro (OriginLab).

#### **6. Results and discussion**

#### **6.1. Distinguishing the response of stem cells from progenitor cells**

From a practical viewpoint, stem and progenitor cells are distinguished by at least two different characteristics. First, stem and progenitor cell populations are stimulated using different cocktails of growth factors and cytokines. In this way, specific cell populations can be targeted and studied, even though the cell suspension may contain other cell types. Combined with the culture conditions, this allows detection and measurement of specific cell populations. The other distinguishing characteristic is the difference in proliferation ability and potential between stem and progenitor cells. Even within the stem cell compartment, differences in proliferation potential will indicate the primitiveness or "stemness" of populations. This characteristic is shown in Fig. 5 for normal bone marrow cells. Since the stem cells are more primitive than the progenitor cells, it would be expected that their proliferation potential would be greater. Figure 5 shows that the two stem cell populations exhibit, not only greater ATP concentration values, but also greater linear regression cell dose response slopes than the hematopoietic or lymphopoietic progenitor cells. It is the slope of the cell dose response that measures proliferation potential. The greater the slope, the higher the proliferation potential, and the more primitive the cell population. Indeed, this is the basic principle for measuring potency of hematopoietic stem cell therapeutic products for transplantation [37]. In this way, it is possible to distinguish different stem cell populations, in this case the hematopoietic stem cell, CFC-GEMM 1, from the more primitive lympho-hematopoietic stem cell, HPP-SP (high proliferative potential – stem and progenitor cell). The HPP-SP stem cell will be discussed in more detail in Section 6.4. The three cell dose response clusters showing the differences in proliferation potential in Fig. 5 for stem cells, hematopoietic progenitor cells and lympho‐ poietic progenitor cells would be expected based on the organization of the blood-forming system shown in Fig. 4. Figure 6 demonstrates the expected proliferation ability of the seven different cell populations in response to mitomycin-C, with the stem cells showing the greatest ability to proliferate followed by the three hematopoietic lineages and lymphopoietic lineages.

The steepness of the linear regression slope of the cell dose response for a cell population provides a measure of the proliferation potential. Stem cells exhibit the greatest proliferation potential of all cells. Within the stem cell compartment, stem cells with different potentials for proliferation also indicate their primitiveness. Proliferation ability is measured at a single cell dose (see Fig. 6).

lymphopoietic lineages.

From a practical viewpoint, stem and progenitor cells are distinguished by at least two different characteristics. First, stem and progenitor cell populations are stimulated using different cocktails of growth factors and cytokines. In this way, specific cell populations can be targeted and studied, even though the cell suspension may contain other cell types. Combined with the culture conditions, this allows detection and measurement of specific cell populations. The other distinguishing characteristic is the difference in proliferation ability and potential between stem and progenitor cells. Even within the stem cell compartment, differences in proliferation potential will indicate the primitiveness or "stemness" of populations. This characteristic is shown in Fig. 5 for normal bone marrow cells. Since the stem cells are more primitive than the progenitor cells, it would be expected that their proliferation potential would be greater. Figure 5 shows that the two stem cell populations exhibit, not only greater ATP concentration values, but also greater linear regression cell dose response slopes than the hematopoietic or lymphopoietic progenitor cells. It is the slope of the cell dose response that measures proliferation potential. The greater the slope, the higher the proliferation potential, and the more primitive the cell population. Indeed, this is the basic principle for measuring potency of hematopoietic stem cell therapeutic products for transplantation [37]. In this way, it is possible to distinguish different stem cell populations, in this case the hematopoietic stem cell, CFC-GEMM 1, from the more primitive lympho-hematopoietic stem cell, HPP-SP (high proliferative potential – stem and progenitor cell). The HPP-SP stem cell will be discussed in more detail in Section 5C. The three cell dose response clusters showing the differences in proliferation potential in Fig. 5 for stem cells, hematopoietic progenitor cells and lymphopoietic progenitor cells would be expected based on the organization of the blood-forming system

**6.2. Drug and compound screening for stem cell toxicity**

tested were also used in the ECVAM studies [26,27].

ranked, a different and more plausible picture is obtained (Fig. 8).

that could possibly result in a false interpretation and conclusion.

In its most basic form, a single drug or compound is tested in a dose dependent manner on a target cell population. If the agent is cytotoxic to the cells, a negative sigmoidal dose re‐ sponse (Fig. 6) will result from which the estimated percent inhibitory concentrations (IC) can be calculated. Figure 7 shows the dose response curves from 13 drugs and compounds tested on hematopoietic stem cells (CFC-GEMM 1) derived from fresh, human bone marrow using the MNC fraction. Although different cell types are included in this fraction, stimula‐ tion of this particular stem cell population using a specific growth factor cocktail provides the relevant information. For each of the compounds tested a 4-parameter logistic curve fit was plotted from which the IC values could be calculated. Table 1 shows all of the com‐ pounds ranked in order of IC50 (µM) value from the most to the least toxic. The IC90 value (equivalent to the maximum tolerated dose, MTD) is also provided. Many of the compounds

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 93

Table 1 shows some compounds designated as NV or NE. The term NV indicates that an IC20 values was obtained, but no IC50 or IC90 value. The term NE means "no effect" in that no IC values could be estimated. As a result, methotrexate, which is an anti-cancer agent and expected to produce a more dramatic effect on stem cells, is actually ranked near the end of the list. Furthermore, compounds that do not allow an IC value to be calculated might actually produce some effect. The problem with ranking compounds based on their IC values is that it does not take into account the "form" of the dose response curve, which can actually provide more information than the IC value alone. Figure 7 shows a large num‐ ber of different dose response curves. One of the most important parameters provided by the 4-parameter logistic curve fit is coefficient or parameter B, which describes the transition of the curve to the midpoint of the dose response. This is a measure of steepness or slope. In some cases the slope is shallow, while in other cases it is almost vertical. How can this and other parameters of the dose response curve be taken into account so that they are inde‐ pendent of the IC value? The answer lies in calculating the area under the curve (AUC) for the range of doses used. When the AUC is performed and plotted so that the compounds are

In this case, the AUC values for both stem cells (CFC-GEMM 1) and granulocyte-macro‐ phage colony-forming cells (GM-CFC) are shown. When the results for CFC-GEMM 1 are compared with those in Table 1, the results generally follow the IC50 values. Howev‐ er, the toxicity of methotrexate is significantly increased and cycloheximide is more toxic than paclitaxel. The results for the GM progenitor cells have been included to demon‐ strate that progenitor cells exhibit lower toxicities than stem cells. Unless there is evi‐ dence to demonstrate that a compound acts on a specific hematopoietic lineage, it is more prudent to analyze potential toxicity to the stem cell compartment first, rather than focusing on a particular lineage, since the latter will only provide limited information

Figure 5. Measuring Proliferation Potential of Cell Populations **Figure 5.** Measuring Proliferation Potential of Cell Populations

6).

**6.2. Drug and compound screening for stem cell toxicity** 

information that could possible result in a false interpretation and conclusion.

In its most basic form, a single drug or compound is tested in a dose dependent manner on a target cell population. If the agent is cytotoxic to the cells, a negative sigmoidal dose response (Fig. 6) will result from which the estimated percent inhibitory concentrations (IC) can be calculated. Figure 7 shows the dose response curves from 13 drugs and compounds tested on hematopoietic stem cells (CFC-GEMM 1) derived from fresh, human bone marrow using the MNC fraction. Although different cell types are included in this fraction, stimulation of this particular stem cell population using a specific growth factor cocktail provides the relevant information. For each of the compounds tested a 4-parameter logistic curve fit was plotted from which the IC values could be calculated. Table 1 shows all of the compounds ranked in order of IC50 (M) value from the most to the least toxic. The IC90 value (equivalent to the maximum tolerated dose, MTD) is also provided. Many of the compounds tested were also used

Table 1 shows some compounds designated as NV or NE. The term NV indicates that an IC20 values was obtained, but no IC50 or IC90 value. The term NE means "no effect" in that no IC values could be estimated. As a result, methotrexate, which is an anticancer agent and expected to produce a more dramatic effect on stem cells, is actually ranked near the end of the list. Furthermore, compounds that do not allow an IC value to be calculated might actually produce some effect. The problem with ranking compounds based on their IC values is that it does not take into account the "form" of the dose response curve, which can actually provide more information than the IC value alone. Figure 13 shows a large number of different dose response curves. One of the most important parameters provided by the 4-parameter logistic curve fit is coefficient or parameter B, which describes the transition of the curve to the midpoint of the dose response. This is a measure of steepness or slope. In some cases the slope is shallow, while in other cases it is almost vertical. How can this and other parameters of the dose response curve be taken into account so that they are independent of the IC value? The answer lies in calculating the area under the curve (AUC) for the range of doses used. When the AUC is performed and plotted so that the compounds are ranked, a different and more plausible picture is

In this case, the AUC values for both stem cells (CFC-GEMM 1) and granulocyte-macrophage colony-forming cells (GM-CFC) are shown. When the results for CFC-GEMM 1 are compared with those in Table 1, the results generally follow the IC50 values. However, the toxicity of methotrexate is significantly increased and cycloheximide is more toxic than paclitaxel. The results for the GM progenitor cells have been included to demonstrate that progenitor cells exhibit lower toxicities than stem cells. Unless there is evidence to demonstrate that a compound acts on a specific hematopoietic lineage, it is more prudent to analyze potential toxicity to the stem cell compartment first, rather than focusing on a particular lineage, since the latter will only provide limited

Figure 6. Demonstration of Proliferation Ability between Cell Populations **Figure 6.** Demonstration of Proliferation Ability between Cell Populations

in the ECVAM studies [26,27].

obtained (Fig. 8).

#### **6.2. Drug and compound screening for stem cell toxicity**

From a practical viewpoint, stem and progenitor cells are distinguished by at least two different characteristics. First, stem and progenitor cell populations are stimulated using different cocktails of growth factors and cytokines. In this way, specific cell populations can be targeted and studied, even though the cell suspension may contain other cell types. Combined with the culture conditions, this allows detection and measurement of specific cell populations. The other distinguishing characteristic is the difference in proliferation ability and potential between stem and progenitor cells. Even within the stem cell compartment, differences in proliferation potential will indicate the primitiveness or "stemness" of populations. This characteristic is shown in Fig. 5 for normal bone marrow cells. Since the stem cells are more primitive than the progenitor cells, it would be expected that their proliferation potential would be greater. Figure 5 shows that the two stem cell populations exhibit, not only greater ATP concentration values, but also greater linear regression cell dose response slopes than the hematopoietic or lymphopoietic progenitor cells. It is the slope of the cell dose response that measures proliferation potential. The greater the slope, the higher the proliferation potential, and the more primitive the cell population. Indeed, this is the basic principle for measuring potency of hematopoietic stem cell therapeutic products for transplantation [37]. In this way, it is possible to distinguish different stem cell populations, in this case the hematopoietic stem cell, CFC-GEMM 1, from the more primitive lympho-hematopoietic stem cell, HPP-SP (high proliferative potential – stem and progenitor cell). The HPP-SP stem cell will be discussed in more detail in Section 5C. The three cell dose response clusters showing the differences in proliferation potential in Fig. 5 for stem cells, hematopoietic progenitor cells and lymphopoietic progenitor cells would be expected based on the organization of the blood-forming system shown in Fig. 4. Figure 6 demonstrates the expected proliferation ability of the seven different cell populations in response to mitomycin-C, with the stem cells showing the greatest ability to proliferate followed by the three hematopoietic lineages and

lymphopoietic lineages.

92 Stem Cell Biology in Normal Life and Diseases

6).

Figure 5. Measuring Proliferation Potential of Cell Populations

Figure 6. Demonstration of Proliferation Ability between Cell Populations

**6.2. Drug and compound screening for stem cell toxicity** 

information that could possible result in a false interpretation and conclusion.

In its most basic form, a single drug or compound is tested in a dose dependent manner on a target cell population. If the agent is cytotoxic to the cells, a negative sigmoidal dose response (Fig. 6) will result from which the estimated percent inhibitory concentrations (IC) can be calculated. Figure 7 shows the dose response curves from 13 drugs and compounds tested on hematopoietic stem cells (CFC-GEMM 1) derived from fresh, human bone marrow using the MNC fraction. Although different cell types are included in this fraction, stimulation of this particular stem cell population using a specific growth factor cocktail provides the relevant information. For each of the compounds tested a 4-parameter logistic curve fit was plotted from which the IC values could be calculated. Table 1 shows all of the compounds ranked in order of IC50 (M) value from the most to the least toxic. The IC90 value (equivalent to the maximum tolerated dose, MTD) is also provided. Many of the compounds tested were also used

Table 1 shows some compounds designated as NV or NE. The term NV indicates that an IC20 values was obtained, but no IC50 or IC90 value. The term NE means "no effect" in that no IC values could be estimated. As a result, methotrexate, which is an anticancer agent and expected to produce a more dramatic effect on stem cells, is actually ranked near the end of the list. Furthermore, compounds that do not allow an IC value to be calculated might actually produce some effect. The problem with ranking compounds based on their IC values is that it does not take into account the "form" of the dose response curve, which can actually provide more information than the IC value alone. Figure 13 shows a large number of different dose response curves. One of the most important parameters provided by the 4-parameter logistic curve fit is coefficient or parameter B, which describes the transition of the curve to the midpoint of the dose response. This is a measure of steepness or slope. In some cases the slope is shallow, while in other cases it is almost vertical. How can this and other parameters of the dose response curve be taken into account so that they are independent of the IC value? The answer lies in calculating the area under the curve (AUC) for the range of doses used. When the AUC is performed and plotted so that the compounds are ranked, a different and more plausible picture is

In this case, the AUC values for both stem cells (CFC-GEMM 1) and granulocyte-macrophage colony-forming cells (GM-CFC) are shown. When the results for CFC-GEMM 1 are compared with those in Table 1, the results generally follow the IC50 values. However, the toxicity of methotrexate is significantly increased and cycloheximide is more toxic than paclitaxel. The results for the GM progenitor cells have been included to demonstrate that progenitor cells exhibit lower toxicities than stem cells. Unless there is evidence to demonstrate that a compound acts on a specific hematopoietic lineage, it is more prudent to analyze potential toxicity to the stem cell compartment first, rather than focusing on a particular lineage, since the latter will only provide limited

**Figure 5.** Measuring Proliferation Potential of Cell Populations

in the ECVAM studies [26,27].

**Figure 6.** Demonstration of Proliferation Ability between Cell Populations

obtained (Fig. 8).

In its most basic form, a single drug or compound is tested in a dose dependent manner on a target cell population. If the agent is cytotoxic to the cells, a negative sigmoidal dose re‐ sponse (Fig. 6) will result from which the estimated percent inhibitory concentrations (IC) can be calculated. Figure 7 shows the dose response curves from 13 drugs and compounds tested on hematopoietic stem cells (CFC-GEMM 1) derived from fresh, human bone marrow using the MNC fraction. Although different cell types are included in this fraction, stimula‐ tion of this particular stem cell population using a specific growth factor cocktail provides the relevant information. For each of the compounds tested a 4-parameter logistic curve fit was plotted from which the IC values could be calculated. Table 1 shows all of the com‐ pounds ranked in order of IC50 (µM) value from the most to the least toxic. The IC90 value (equivalent to the maximum tolerated dose, MTD) is also provided. Many of the compounds tested were also used in the ECVAM studies [26,27].

The steepness of the linear regression slope of the cell dose response for a cell population provides a measure of the proliferation potential. Stem cells exhibit the greatest proliferation potential of all cells. Within the stem cell compartment, stem cells with different potentials for proliferation also indicate their primitiveness. Proliferation ability is measure at a single cell dose (see Fig. Table 1 shows some compounds designated as NV or NE. The term NV indicates that an IC20 values was obtained, but no IC50 or IC90 value. The term NE means "no effect" in that no IC values could be estimated. As a result, methotrexate, which is an anti-cancer agent and expected to produce a more dramatic effect on stem cells, is actually ranked near the end of the list. Furthermore, compounds that do not allow an IC value to be calculated might actually produce some effect. The problem with ranking compounds based on their IC values is that it does not take into account the "form" of the dose response curve, which can actually provide more information than the IC value alone. Figure 7 shows a large num‐ ber of different dose response curves. One of the most important parameters provided by the 4-parameter logistic curve fit is coefficient or parameter B, which describes the transition of the curve to the midpoint of the dose response. This is a measure of steepness or slope. In some cases the slope is shallow, while in other cases it is almost vertical. How can this and other parameters of the dose response curve be taken into account so that they are inde‐ pendent of the IC value? The answer lies in calculating the area under the curve (AUC) for the range of doses used. When the AUC is performed and plotted so that the compounds are ranked, a different and more plausible picture is obtained (Fig. 8).

> In this case, the AUC values for both stem cells (CFC-GEMM 1) and granulocyte-macro‐ phage colony-forming cells (GM-CFC) are shown. When the results for CFC-GEMM 1 are compared with those in Table 1, the results generally follow the IC50 values. Howev‐ er, the toxicity of methotrexate is significantly increased and cycloheximide is more toxic than paclitaxel. The results for the GM progenitor cells have been included to demon‐ strate that progenitor cells exhibit lower toxicities than stem cells. Unless there is evi‐ dence to demonstrate that a compound acts on a specific hematopoietic lineage, it is more prudent to analyze potential toxicity to the stem cell compartment first, rather than focusing on a particular lineage, since the latter will only provide limited information that could possibly result in a false interpretation and conclusion.

**Compound Effect Rank IC50 (μM) IC90 (μM)** Camptothecin Anti-cancer 1 0.02 0.14 Paclitaxol Anti-cancer 2 0.18 4.81 Cycloheximide Pesticide 3 0.23 0.86 Cyclosporin A Immunosuppressant 4 2.57 8.2 5-Fluorouracil Anti-cancer 5 5.79 29.7

(Thorazine) Anti-psychotic <sup>6</sup> 5.88 7.03 Rifampicin Anti-bacterial 7 12.8 NV Zedovuidine(AZT) Anti-viral 8 30.4 NV Choramphenicol Anti-bacterial 9 94.7 NV Indomethacin Anti-inflammatory 10 394.2 947.5 Methotrexate Anti-cancer 11 NV NV Acyclovir Anti-viral 12 NE NE Warfarin Anti-coagulant 13 NE NE NV indicates No Value for these IC values. An IC20 value would have been estimated by the software program.

NE indicate No Effect. In this case, the dose response for the compound did not produce an IC values.

**Ranking of Stem and Progenitor Cell Toxicity**

Stem Cells GM Progenitor Cells

Figure 8. Ranking of Stem Cell and Granulocyte-Macrophage Progenitor (GM-CFC) Toxicity According to the Calculated Area Under the Curve

**Acyclovir**

**AZT**

**Indomethacin**

**Chloramphenicol**

Least Toxic

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 95

**Warfarin**

The Registry of Cytotoxicity (RC) is a list of 347 compounds for which the IC50 values using a neutral red uptake assay for human keratinocytes and mouse 3T3 cells and the oral LD50 values for rat or mouse are known. When validating an *in vitro* assay against the RC, a sample of reference compounds is tested. The resulting IC50 values from the *in vitro* assay are then plotted against the LD50 values for the same compounds. A linear regression should be obtained exhibiting equation constants within a specific range. If this occurs, the *in vitro* assay is considered a validated cytotoxic test. The results validating HALO against the RC Prediction Model were first reported in 2005 [33]. One of the most interesting aspects of this prediction model is that once an assay has been validated, it can be used to convert *in vitro* IC values into clinically relevant doses that can be used as starting doses for pre-clinical animal models or human clinical trials. An example of this is shown in Table 2 where the results of converting IC20, IC50 and IC90 range values derived from the effects of 18 compounds on CFC-GEMM 1 bone marrow cells is shown. The predicted doses derived from the IC values are given in both milligrams/kilogram (mg/kg) and milligrams/meter2 (mg/m2). Doses used in the clinic to treat patients are also shown in mg/kg or mg/m2 where available. With the exception of two drugs, namely acyclovir and warfarin, nearly all of the doses predicted by the *in vitro* CFC-GEMM 1 assay using ATP bioluminescence are in the same order of magnitude or very close to the doses used to treat patients. In some cases lower doses were predicted (e.g. 5-fluorouracil), while in other cases slightly higher doses were predicted (e.g. cyclosporine A, indomethacin, cisplatin and mitomycin-C). Thus these predicted starting

Figure 5 shows that the response of stem cells to toxic agents can vary dramatically. In some cases, agents cause complete eradication of all stem and progenitor cells at high doses. In other cases, there is partial cytotoxicity at which, even at high doses, stem and progenitor cells are not eradicated. This is an indication that some stem cells survive or are possibly resistant to the drug or compound. If stem cells are not noticeably affected at high doses, there is a good chance that when the drug or compound is removed, the system will reconstitute itself. If no stem cells are available, this will not occur. However, there are other aspects to

values may be used in early toxicity and efficacy studies to "bracket" the lower and higher dose ranges.

**Table 1.** Ranking of Stem Cell Toxicity According to IC50 Values

Most Toxic

(AUC) for the Dose Responses shown in Figure 7.

**Paclitaxol**

ed Area Under the Curve (AUC) for the Dose Responses shown in Figure 7.

**Cyclosporin A**

**Chlorpromazine**

**5-FU**

**Rifampicine**

**Figure 8.** Ranking of Stem Cell and Granulocyte-Macrophage Progenitor (GM-CFC) Toxicity According to the Calculat‐

**Methotrexate**

**Camptothecin**

**0**

**10,000**

**20,000**

**30,000**

**Area Under the Dose Response Curve**

**40,000**

**50,000**

**Cycloheximide**

**6.4. Residual stem cells after toxicity** 

this phenomenon that are important.

**6.3. The registry of cytotoxicity prediction model [41]** 

Chlorpromazine

**Figure 7.** The Effect of 13 Compounds on Hematopoietic CFC-GEMM Stem Cells. Diagram showing the dose response plots produced automatically by SoftMax Pro software after the data was collected by the SpectraMax L plate lumin‐ ometer. The parameters that define the 4-parameter logistic curve to which the dose responses of the compounds are fitted are as follows: Parameter A, asymptote (flat part of the curve) at low Y-values; Parameter D, asymptote at the highest Y-values; Parameter or coefficient B, the transition from the asymptotes to the center of the curve; Parameter or coefficient C, is the midpoint between parameters A and D, also called the IC50 or EC50. Data that cannot be prop‐ erly fitted will result in ambiguous results.


NV indicates No Value for these IC values. An IC20 value would have been estimated by the software program.

NE indicate No Effect. In this case, the dose response for the compound did not produce an IC values.

**Figure 7.** The Effect of 13 Compounds on Hematopoietic CFC-GEMM Stem Cells. Diagram showing the dose response plots produced automatically by SoftMax Pro software after the data was collected by the SpectraMax L plate lumin‐ ometer. The parameters that define the 4-parameter logistic curve to which the dose responses of the compounds are fitted are as follows: Parameter A, asymptote (flat part of the curve) at low Y-values; Parameter D, asymptote at the highest Y-values; Parameter or coefficient B, the transition from the asymptotes to the center of the curve; Parameter or coefficient C, is the midpoint between parameters A and D, also called the IC50 or EC50. Data that cannot be prop‐

erly fitted will result in ambiguous results.

94 Stem Cell Biology in Normal Life and Diseases

#### **Ranking of Stem and Progenitor Cell Toxicity**

Figure 8. Ranking of Stem Cell and Granulocyte-Macrophage Progenitor (GM-CFC) Toxicity According to the Calculated Area Under the Curve (AUC) for the Dose Responses shown in Figure 7. **Figure 8.** Ranking of Stem Cell and Granulocyte-Macrophage Progenitor (GM-CFC) Toxicity According to the Calculat‐ ed Area Under the Curve (AUC) for the Dose Responses shown in Figure 7.

values may be used in early toxicity and efficacy studies to "bracket" the lower and higher dose ranges.

The Registry of Cytotoxicity (RC) is a list of 347 compounds for which the IC50 values using a neutral red uptake assay for human keratinocytes and mouse 3T3 cells and the oral LD50 values for rat or mouse are known. When validating an *in vitro* assay against the RC, a sample of reference compounds is tested. The resulting IC50 values from the *in vitro* assay are then plotted against the LD50 values for the same compounds. A linear regression should be obtained exhibiting equation constants within a specific range. If this occurs, the *in vitro* assay is considered a validated cytotoxic test. The results validating HALO against the RC Prediction Model were first reported in 2005 [33]. One of the most interesting aspects of this prediction model is that once an assay has been validated, it can be used to convert *in vitro* IC values into clinically relevant doses that can be used as starting doses for pre-clinical animal models or human clinical trials. An example of this is shown in Table 2 where the results of converting IC20, IC50 and IC90 range values derived from the effects of 18 compounds on CFC-GEMM 1 bone marrow cells is shown. The predicted doses derived from the IC values are given in both milligrams/kilogram (mg/kg) and milligrams/meter2 (mg/m2). Doses used in the clinic to treat patients are also shown in mg/kg or mg/m2 where available. With the exception of two drugs, namely acyclovir and warfarin, nearly all of the doses predicted by the *in vitro* CFC-GEMM 1 assay using ATP bioluminescence are in the same order of magnitude or very close to the doses used to treat patients. In some cases lower doses were predicted (e.g. 5-fluorouracil), while in other cases slightly higher doses were predicted (e.g. cyclosporine A, indomethacin, cisplatin and mitomycin-C). Thus these predicted starting

Figure 5 shows that the response of stem cells to toxic agents can vary dramatically. In some cases, agents cause complete eradication of all stem and progenitor cells at high doses. In other cases, there is partial cytotoxicity at which, even at high doses, stem and progenitor cells are not eradicated. This is an indication that some stem cells survive or are possibly resistant to the drug or compound. If stem cells are not noticeably affected at high doses, there is a good chance that when the drug or compound is removed, the system will reconstitute itself. If no stem cells are available, this will not occur. However, there are other aspects to

**6.3. The registry of cytotoxicity prediction model [41]** 

**6.4. Residual stem cells after toxicity** 

this phenomenon that are important.

#### **6.3. The registry of cytotoxicity prediction model [41]**

The Registry of Cytotoxicity (RC) is a list of 347 compounds, for which the IC50 values using a neutral red uptake assay for human keratinocytes and mouse 3T3 cells and the oral LD50 values for rat or mouse, are known. When validating an *in vitro* assay against the RC, a sample of reference compounds is tested. The resulting IC50 values from the *in vitro* assay are then plotted against the LD50 values for the same compounds. A linear regression should be obtained exhibiting equation constants within a specific range. If this occurs, the *in vitro* assay is considered a validated cytotoxic test. The results validat‐ ing HALO against the RC Prediction Model were first reported in 2005 [33]. One of the most interesting aspects of this prediction model is that once an assay has been validat‐ ed, it can be used to convert *in vitro* IC values into clinically relevant doses that can be used as starting doses for pre-clinical animal models or human clinical trials. An exam‐ ple of this is shown in Table 2 where the results of converting IC20, IC50 and IC90 range values derived from the effects of 18 compounds on CFC-GEMM 1 bone marrow cells is shown. The predicted doses derived from the IC values are given in both milli‐ grams/kilogram (mg/kg) and milligrams/meter2 (mg/m2 ). Doses used in the clinic to treat patients are also shown in mg/kg or mg/m2 where available. With the exception of two drugs, namely acyclovir and warfarin, nearly all of the doses predicted by the *in vitro* CFC-GEMM 1 assay using ATP bioluminescence are in the same order of magnitude or very close to the doses used to treat patients. In some cases lower doses were predicted (e.g. 5-fluorouracil), while in other cases slightly higher doses were predicted (e.g. cyclo‐ sporine A, indomethacin, cisplatin and mitomycin-C). Thus these predicted starting val‐ ues may be used in early toxicity and efficacy studies to "bracket" the lower and higher dose ranges.

**Predicted Dosing Range from In Vitro Stem Cell Assay**

Drug/Compound Dose in mg/kg Dose in mg/m2 Doses or Dose Range

Cyclosporin A 14.2 – 31.2 524 – 1,155 5 – 10 Indomethacin 32 – 73 1,190 – 2,700 0.2 – 2 Zedovudine (AZT) 4.3 – 12.2 161 – 452 1 – 7.4

Acyclovir NV NV 5 – 500

Choramphenicol 16 – 24 594 – 896 12.5/30 – 50 Rifampicin 24 – 26 894 – 955 10 Warfarin NV NV 0.1 – 5

dose in mg/m2 is obtained by multiplying the dose in mg/kg by a specific factor described in [42].

Chlorpromozine (Thorazine)

Starting Doses

Doxorubicin 2.6 – 6.9 97 – 255 25/50/60/75 Daunorubicin 0.5 – 2.6 19.6 – 97 30/45/60 5-Fluorouracil 2.0 – 7.0 79 – 259 400 – 2,600 Paclitaxel 2.0 – 17.5 72 – 647 75 – 250 Imatinib (Gleevec) 3.6 – 30.5 132 – 1,125 400/600 Methotrexate 5.8 215 10 – 8,000

6.8 – 7.7 253 – 285 1 – 4.5

Camptothecin 0.36 – 1.52 13.3 – 56 25/320/470

SJG-136 0.1 – 0.3 4 – 10 6 – 40 Cisplatin 6.3 – 9.8 233 – 363 30 – 100 Mitomycin-C 1.2 – 6.0 47 – 220 6/10 – 20 The IC values obtained from the validated *in vitro* assay are entered into the equation: Y = 0.435 \* Log (IC value) + 0.625 [41]. The dose in mg/kg is then obtained by multiplying the value for Y with the molecular weight of the compound. The

**Table 2.** Using the Registry of Cytotoxicity Prediction Model to Convert *In Vitro* IC Values into Clinically Relevant

To demonstrate this, we developed an *in vitro* secondary re-plating assay for primitive stem cells called high proliferation potential – stem and progenitor cells (HPP-SP). This stem cell population, within the stem cell compartment (Fig. 4), is approximately at the divergence of the lymphopoietic and hematopoietic systems. The majority of HPP-SP stem cells are quies‐ cent. They can be induced or "primed" into proliferation with IL-3, IL-6, SCF and Fl3-L. This stem cell population is designated HPP-SP 1. Once the HPP-SP 1 cells begin proliferation, they can be expanded with a similar cocktail of growth factors and cytokines to that for CFC-GEMM 1, but with the addition of interleukins 2 and 7 (IL-2, IL-7). This fully stimulated primitive stem cell population is designated HPP-SP 2. In this two-stage assay, the HPP-SP 1, present in the MNC fraction of bone marrow are cultured in the presence of the drug or compound in a dosedependent manner. Thereafter, the cells are removed from culture, washed and re-plated in a secondary culture system in which the HPP-SP 2 population is measured. By performing a secondary re-plating step, the assay is substantiating the presence of primitive stem cells

**Published Drug Doses Used to Treat Patients**

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430

> Doses or Dose Range in mg/m2

97

in mg/kg

#### **6.4. Residual stem cells after toxicity**

Figure 7 shows that the response of stem cells to toxic agents can vary dramatically. In some cases, agents cause complete eradication of all stem and progenitor cells at high doses. In other cases, there is partial cytotoxicity at which, even at high doses, stem and progenitor cells are not eradicated. This is an indication that some stem cells survive or are possibly resistant to the drug or compound. If stem cells are not noticeably affected at high doses, there is a good chance that when the drug or compound is removed, the system will reconstitute itself. If no stem cells are available, this will not occur. However, there are other aspects to this phenom‐ enon that are important.

Primitive stem cells are usually in a quiescent state; they are not proliferating and therefore not in cell cycle. This does not mean that they cannot be affected by an agent. Small molecules can enter a cell even if it is quiescent. When required to initiate the proliferation process and begin cell division, the process may be aborted because the agent inhibits the process. This is a potential dangerous situation for two reasons. First, the "backup plan" for reconstituting the system may not function. Second, if cells do begin to proliferate and divide, they may be more sensitive to the agent. The consequence of this is that repeated administration of the drug or compound will continually reduce the proportion of residual stem cells present.


**6.3. The registry of cytotoxicity prediction model [41]**

96 Stem Cell Biology in Normal Life and Diseases

grams/kilogram (mg/kg) and milligrams/meter2 (mg/m2

dose ranges.

**6.4. Residual stem cells after toxicity**

enon that are important.

The Registry of Cytotoxicity (RC) is a list of 347 compounds, for which the IC50 values using a neutral red uptake assay for human keratinocytes and mouse 3T3 cells and the oral LD50 values for rat or mouse, are known. When validating an *in vitro* assay against the RC, a sample of reference compounds is tested. The resulting IC50 values from the *in vitro* assay are then plotted against the LD50 values for the same compounds. A linear regression should be obtained exhibiting equation constants within a specific range. If this occurs, the *in vitro* assay is considered a validated cytotoxic test. The results validat‐ ing HALO against the RC Prediction Model were first reported in 2005 [33]. One of the most interesting aspects of this prediction model is that once an assay has been validat‐ ed, it can be used to convert *in vitro* IC values into clinically relevant doses that can be used as starting doses for pre-clinical animal models or human clinical trials. An exam‐ ple of this is shown in Table 2 where the results of converting IC20, IC50 and IC90 range values derived from the effects of 18 compounds on CFC-GEMM 1 bone marrow cells is shown. The predicted doses derived from the IC values are given in both milli‐

patients are also shown in mg/kg or mg/m2 where available. With the exception of two drugs, namely acyclovir and warfarin, nearly all of the doses predicted by the *in vitro* CFC-GEMM 1 assay using ATP bioluminescence are in the same order of magnitude or very close to the doses used to treat patients. In some cases lower doses were predicted (e.g. 5-fluorouracil), while in other cases slightly higher doses were predicted (e.g. cyclo‐ sporine A, indomethacin, cisplatin and mitomycin-C). Thus these predicted starting val‐ ues may be used in early toxicity and efficacy studies to "bracket" the lower and higher

Figure 7 shows that the response of stem cells to toxic agents can vary dramatically. In some cases, agents cause complete eradication of all stem and progenitor cells at high doses. In other cases, there is partial cytotoxicity at which, even at high doses, stem and progenitor cells are not eradicated. This is an indication that some stem cells survive or are possibly resistant to the drug or compound. If stem cells are not noticeably affected at high doses, there is a good chance that when the drug or compound is removed, the system will reconstitute itself. If no stem cells are available, this will not occur. However, there are other aspects to this phenom‐

Primitive stem cells are usually in a quiescent state; they are not proliferating and therefore not in cell cycle. This does not mean that they cannot be affected by an agent. Small molecules can enter a cell even if it is quiescent. When required to initiate the proliferation process and begin cell division, the process may be aborted because the agent inhibits the process. This is a potential dangerous situation for two reasons. First, the "backup plan" for reconstituting the system may not function. Second, if cells do begin to proliferate and divide, they may be more sensitive to the agent. The consequence of this is that repeated administration of the drug or

compound will continually reduce the proportion of residual stem cells present.

). Doses used in the clinic to treat

The IC values obtained from the validated *in vitro* assay are entered into the equation: Y = 0.435 \* Log (IC value) + 0.625 [41]. The dose in mg/kg is then obtained by multiplying the value for Y with the molecular weight of the compound. The dose in mg/m2 is obtained by multiplying the dose in mg/kg by a specific factor described in [42].

**Table 2.** Using the Registry of Cytotoxicity Prediction Model to Convert *In Vitro* IC Values into Clinically Relevant Starting Doses

To demonstrate this, we developed an *in vitro* secondary re-plating assay for primitive stem cells called high proliferation potential – stem and progenitor cells (HPP-SP). This stem cell population, within the stem cell compartment (Fig. 4), is approximately at the divergence of the lymphopoietic and hematopoietic systems. The majority of HPP-SP stem cells are quies‐ cent. They can be induced or "primed" into proliferation with IL-3, IL-6, SCF and Fl3-L. This stem cell population is designated HPP-SP 1. Once the HPP-SP 1 cells begin proliferation, they can be expanded with a similar cocktail of growth factors and cytokines to that for CFC-GEMM 1, but with the addition of interleukins 2 and 7 (IL-2, IL-7). This fully stimulated primitive stem cell population is designated HPP-SP 2. In this two-stage assay, the HPP-SP 1, present in the MNC fraction of bone marrow are cultured in the presence of the drug or compound in a dosedependent manner. Thereafter, the cells are removed from culture, washed and re-plated in a secondary culture system in which the HPP-SP 2 population is measured. By performing a secondary re-plating step, the assay is substantiating the presence of primitive stem cells present in the first "priming" step of culture. The proliferation at both stages is determined using ATP bioluminescence technology. The results using busulphan and daunorubicin are shown in Figs. 9A and 9B, respectively. The effect of busulphan (Fig. 9A) on HPP-SP 1 demonstrates partial cytotoxicity to the stem cells and the presence of residual stem cells. However, when the treated cells are removed from primary culture and placed into secondary cultured to reveal their expansion potential, there are few residual cells that are available for expansion and the high doses used in the primary culture eradicated any remaining cells. There was also little change in the IC50 values. This indicates that busulphan continued to act on primitive stem cells leaving no residual stem cells (secondary culture results minus primary culture results) for possible repopulation. Daunorubicin (Fig. 9B) is highly toxic to stem cells with an IC50 value in the nanomolar range compared to the micromolar range for busulphan. At low doses of daunorubicin, residual stem cells would be available, but secondary culture demonstrates that both these and the residual cells have increased their sensitivity by approx. 3 fold, indicating that repeated drug administration would incur increased sensitivity of the stem cells to the drug.

response at the cellular level. To investigate this, we developed an assay in which drugs could

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 99

Figure 10A shows the response when verapamil is titrated against cyclosporin A, while Fig. 10B shows the effect when cyclosporin A is titrated against verapamil. Both drugs inhibit 3A4 CYP450 enzyme. Individually, both drugs are cytotoxic to CFC-GEMM 1 stem cells. However, when titrated against each other, cytotoxicity may be observed initially, but may be followed by an opposite effect at higher doses. The cells appear to overcome the inhibitory effects. In terms of DDI, this would indicate that one or other drug is present at concentrations that could cause serious harm to the patient. This unusual dose response behavior produces a U-shaped or inverted dose response curve that has been observed for many compounds, including dopamine [45] and endostatins [46]. Although often attributed to solubility, these effects appear to be pharmacologically and physiologically important, but in most cases, the mecha‐ nism is not understood. This is the first indication that DDI can occur at the stem cell level. Considering the importance of assessing toxicity to stem cells and the predictive value afforded by these cells, it is obvious that more has to be learnt before the consequences of these reactions

One of the most interesting aspects of drug treatment is the field of chronotherapy; the administration of drugs in accordance with circadian rhythms. Although studied for decades, the role of circadian rhythms to reduce toxicity and improve drug efficacy has been largely ignored by the biopharmaceutical industry. The primary reason for this is because chrono‐ therapeutic studies are difficult, time-consuming and expensive to perform. Nevertheless, many areas of chronotherapy, especially using anti-cancer drugs. have proved to be successful [47-49]. Many cellular functions are dependent upon circadian rhythms. It is not the purpose

be titrated against each other to determine potential DDI on stem cells.

**Figure 10.** Examples of Drug-Drug Interactions at the Stem Cell Level

on a stem cell system can be understood.

**6.6. Circadian rhythm and stem cells**

**Figure 9.** Assessing Residual Stem Cell Activity and Change in Stem Cell Sensitivity to Agents by Measuring the Re‐ sponse of Primitive Stem Cells in a Two-Step Secondary Re-Plating In Vitro Assay.

#### **6.5. Stem cells and drug-drug interactions**

Drug-drug interaction (DDI) can lead to dangerous consequences if not investigated properly. Traditionally, DDI are investigated using cultured hepatocytes since the liver is the organ primarily responsible for detoxification. The main enzymes investigated during DDI studies are those of the cytochrome P450 (CYP450) system present in the endoplasmic reticulum of the cells. CYP450 enzymes are present not only in hepatocytes, but in virtually all cells. There are a large number of CYP450 enzymes and assays are available for many of these. Depending on the drug or compound, one or more CYP450 enzymes can be induced or inhibited [43,44]. The response by different enzymes provides an indication as to whether an interaction between different drugs will occur. However, measurement of CYP450 activities does not indicate a

response at the cellular level. To investigate this, we developed an assay in which drugs could be titrated against each other to determine potential DDI on stem cells.

**Figure 10.** Examples of Drug-Drug Interactions at the Stem Cell Level

present in the first "priming" step of culture. The proliferation at both stages is determined using ATP bioluminescence technology. The results using busulphan and daunorubicin are shown in Figs. 9A and 9B, respectively. The effect of busulphan (Fig. 9A) on HPP-SP 1 demonstrates partial cytotoxicity to the stem cells and the presence of residual stem cells. However, when the treated cells are removed from primary culture and placed into secondary cultured to reveal their expansion potential, there are few residual cells that are available for expansion and the high doses used in the primary culture eradicated any remaining cells. There was also little change in the IC50 values. This indicates that busulphan continued to act on primitive stem cells leaving no residual stem cells (secondary culture results minus primary culture results) for possible repopulation. Daunorubicin (Fig. 9B) is highly toxic to stem cells with an IC50 value in the nanomolar range compared to the micromolar range for busulphan. At low doses of daunorubicin, residual stem cells would be available, but secondary culture demonstrates that both these and the residual cells have increased their sensitivity by approx. 3 fold, indicating that repeated drug administration would incur increased sensitivity of the

**Figure 9.** Assessing Residual Stem Cell Activity and Change in Stem Cell Sensitivity to Agents by Measuring the Re‐

Drug-drug interaction (DDI) can lead to dangerous consequences if not investigated properly. Traditionally, DDI are investigated using cultured hepatocytes since the liver is the organ primarily responsible for detoxification. The main enzymes investigated during DDI studies are those of the cytochrome P450 (CYP450) system present in the endoplasmic reticulum of the cells. CYP450 enzymes are present not only in hepatocytes, but in virtually all cells. There are a large number of CYP450 enzymes and assays are available for many of these. Depending on the drug or compound, one or more CYP450 enzymes can be induced or inhibited [43,44]. The response by different enzymes provides an indication as to whether an interaction between different drugs will occur. However, measurement of CYP450 activities does not indicate a

sponse of Primitive Stem Cells in a Two-Step Secondary Re-Plating In Vitro Assay.

**6.5. Stem cells and drug-drug interactions**

stem cells to the drug.

98 Stem Cell Biology in Normal Life and Diseases

Figure 10A shows the response when verapamil is titrated against cyclosporin A, while Fig. 10B shows the effect when cyclosporin A is titrated against verapamil. Both drugs inhibit 3A4 CYP450 enzyme. Individually, both drugs are cytotoxic to CFC-GEMM 1 stem cells. However, when titrated against each other, cytotoxicity may be observed initially, but may be followed by an opposite effect at higher doses. The cells appear to overcome the inhibitory effects. In terms of DDI, this would indicate that one or other drug is present at concentrations that could cause serious harm to the patient. This unusual dose response behavior produces a U-shaped or inverted dose response curve that has been observed for many compounds, including dopamine [45] and endostatins [46]. Although often attributed to solubility, these effects appear to be pharmacologically and physiologically important, but in most cases, the mecha‐ nism is not understood. This is the first indication that DDI can occur at the stem cell level. Considering the importance of assessing toxicity to stem cells and the predictive value afforded by these cells, it is obvious that more has to be learnt before the consequences of these reactions on a stem cell system can be understood.

#### **6.6. Circadian rhythm and stem cells**

One of the most interesting aspects of drug treatment is the field of chronotherapy; the administration of drugs in accordance with circadian rhythms. Although studied for decades, the role of circadian rhythms to reduce toxicity and improve drug efficacy has been largely ignored by the biopharmaceutical industry. The primary reason for this is because chrono‐ therapeutic studies are difficult, time-consuming and expensive to perform. Nevertheless, many areas of chronotherapy, especially using anti-cancer drugs. have proved to be successful [47-49]. Many cellular functions are dependent upon circadian rhythms. It is not the purpose of this section to describe or even summarize this field. The intention is to instead provide an example in which the circadian rhythm of cells, especially hematopoietic stem cells [50-52], can be used to predict the best time of day to administer an anti-cancer drug, which in this case, is 5-fluorouracil (5-FU) [38].

For each time point, cells were thawed and treated with 5-FU at six doses to measure the response of CFC-GEMM 1, BFU-E, GM-CFC and Mk-CFC. The slope of each negative sigmoi‐ dal dose response curve was then calculated from the 4-parameter logistic curve fit. The dose response slope values were then analyzed by cosinor analysis for each time point and for each cell population to obtain the circadian rhythms as a function of 5-FU treatment. The results are shown in Fig. 10B. Each of the hematopoietic cell populations exhibited its own circadian rhythm in response to 5-FU. When these circadian rhythms were correlated with either the continuous infusion of 5-FU that is normally used to treat patients and that of chronomodu‐ lated infusion of 5-FU as reported by Dogliotti and colleagues in 1998 [54], the results shown in Fig. 10C were obtained. For each of the administration types, the percent overall patient response rate, toxicity and tumor response are shown. These were overlaid onto the circadian rhythm for the CFC-GEMM 1 stem cell response to 5-FU and demonstrated that the lowest toxicity and highest overall and tumor response occurred when 5-FU was administered in a chronomodulated manner in the early morning hours rather than at any other time of the day. The nadir of the CFC-GEMM 1 circadian rhythm to 5-FU occurred at 14:00 hours in the afternoon. This was approximately the same time at which the highest toxicity to 5-FU was found. As expected, these results did not correlate nearly as well for the hematopoietic progenitor cells. In addition, the results clearly demonstrate that the potential for toxicity can be dramatically reduced if the circadian rhythm of the target cells is taken into account. From the brief description here, it follows that to ascertain the best time of day to administer a drug a considerable amount of work must be undertaken. The question is whether the patient response and well-being outweigh the time and cost to perform these types of studies.

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 101

To use *in vitro* stem cell assays to predict potential toxicity to the hematopoietic system, and any stem cell system for that matter, knowledge of the biology, physiology, regulation and response is required for an *in vitro* to *in vivo* concordance to be justified. This concordance plays an integral role in predicting toxicity since it allows for *in vitro* surrogate assays to be used in place of animals and therefore comply with the principle of the 3Rs (replacement, reduction and refinement) [55]. More importantly, to allows extrapolation to the human situation. Previous literature on stem cell and hematopoietic research demonstrates that *in vitro* assays show a high concordance with *in vivo* data. Using the HALO platform, Olaharski et al. demonstrated an *in vitro* to *in vivo* concordance of greater than 80% [56]. This high degree of concordance provides the basis to predict the response of the lympho-hematopoietic and other stem cell systems to potential toxic insults. This has been described previously [32], but it is worth reiterating some of these paradigms. First, virtually any compound can be toxic to stem cells. Second, toxicity to the most primitive, definitive stem cells will affect all cells of the system. Third, since stem cells only proliferate and proliferation occurs prior to differentiation, stem cell cytotoxicity will affect all downstream cell types. Fourth, if more than one cell lineage is affected by toxicity, the target is not the cells that constitute the lineages, but the stem cells producing the lineages. Finally, stem cells are more sensitive to toxicity than the progenitor

**7. Conclusions and future trends**

These studies were performed using normal peripheral blood mononuclear cells. Blood was obtained from the same donor every 4 hours over a 24 hours period. The MNCs were fractio‐ nated at each time point and cryopreserved into aliquots. Prior to cryopreservation, an aliquot of fresh cells was used to measure the proliferation ability of hematopoietic stem cells (CFC-GEMM 1), erythropoietic progenitor cells (burst-forming units – erythroid, BFU-E), GM-CFC and megakaryopoietic progenitor cells (megakaryopoietic colony-forming cells, Mk-CFC) at each time point using HALO. After collection of the cells, an aliquot from each time point was thawed and the circadian rhythms compared to fresh cells. A cosinor curve fitting analysis was performed to produce all the circadian rhythms shown in Fig. 11 [53]. The results for hematopoietic stem cells (Fig. 11A) and all progenitor cells (not shown) demonstrate that even after cryopreservation, the cell populations maintain their circadian rhythm. This was a prerequisite to use cryopreserved cells for the remainder of the study.

**Figure 11.** Using the Circadian Rhythm of Hematopoietic Stem Cells to Predict the Best Time of Day to Administer 5- Fluorouracil to Reduce Toxicity and Improve Efficacy of the Drug.

For each time point, cells were thawed and treated with 5-FU at six doses to measure the response of CFC-GEMM 1, BFU-E, GM-CFC and Mk-CFC. The slope of each negative sigmoi‐ dal dose response curve was then calculated from the 4-parameter logistic curve fit. The dose response slope values were then analyzed by cosinor analysis for each time point and for each cell population to obtain the circadian rhythms as a function of 5-FU treatment. The results are shown in Fig. 10B. Each of the hematopoietic cell populations exhibited its own circadian rhythm in response to 5-FU. When these circadian rhythms were correlated with either the continuous infusion of 5-FU that is normally used to treat patients and that of chronomodu‐ lated infusion of 5-FU as reported by Dogliotti and colleagues in 1998 [54], the results shown in Fig. 10C were obtained. For each of the administration types, the percent overall patient response rate, toxicity and tumor response are shown. These were overlaid onto the circadian rhythm for the CFC-GEMM 1 stem cell response to 5-FU and demonstrated that the lowest toxicity and highest overall and tumor response occurred when 5-FU was administered in a chronomodulated manner in the early morning hours rather than at any other time of the day. The nadir of the CFC-GEMM 1 circadian rhythm to 5-FU occurred at 14:00 hours in the afternoon. This was approximately the same time at which the highest toxicity to 5-FU was found. As expected, these results did not correlate nearly as well for the hematopoietic progenitor cells. In addition, the results clearly demonstrate that the potential for toxicity can be dramatically reduced if the circadian rhythm of the target cells is taken into account. From the brief description here, it follows that to ascertain the best time of day to administer a drug a considerable amount of work must be undertaken. The question is whether the patient response and well-being outweigh the time and cost to perform these types of studies.

#### **7. Conclusions and future trends**

of this section to describe or even summarize this field. The intention is to instead provide an example in which the circadian rhythm of cells, especially hematopoietic stem cells [50-52], can be used to predict the best time of day to administer an anti-cancer drug, which in this

These studies were performed using normal peripheral blood mononuclear cells. Blood was obtained from the same donor every 4 hours over a 24 hours period. The MNCs were fractio‐ nated at each time point and cryopreserved into aliquots. Prior to cryopreservation, an aliquot of fresh cells was used to measure the proliferation ability of hematopoietic stem cells (CFC-GEMM 1), erythropoietic progenitor cells (burst-forming units – erythroid, BFU-E), GM-CFC and megakaryopoietic progenitor cells (megakaryopoietic colony-forming cells, Mk-CFC) at each time point using HALO. After collection of the cells, an aliquot from each time point was thawed and the circadian rhythms compared to fresh cells. A cosinor curve fitting analysis was performed to produce all the circadian rhythms shown in Fig. 11 [53]. The results for hematopoietic stem cells (Fig. 11A) and all progenitor cells (not shown) demonstrate that even after cryopreservation, the cell populations maintain their circadian rhythm. This was a

**Figure 11.** Using the Circadian Rhythm of Hematopoietic Stem Cells to Predict the Best Time of Day to Administer 5-

B.

C.

prerequisite to use cryopreserved cells for the remainder of the study.

A.

Fluorouracil to Reduce Toxicity and Improve Efficacy of the Drug.

case, is 5-fluorouracil (5-FU) [38].

100 Stem Cell Biology in Normal Life and Diseases

To use *in vitro* stem cell assays to predict potential toxicity to the hematopoietic system, and any stem cell system for that matter, knowledge of the biology, physiology, regulation and response is required for an *in vitro* to *in vivo* concordance to be justified. This concordance plays an integral role in predicting toxicity since it allows for *in vitro* surrogate assays to be used in place of animals and therefore comply with the principle of the 3Rs (replacement, reduction and refinement) [55]. More importantly, to allows extrapolation to the human situation. Previous literature on stem cell and hematopoietic research demonstrates that *in vitro* assays show a high concordance with *in vivo* data. Using the HALO platform, Olaharski et al. demonstrated an *in vitro* to *in vivo* concordance of greater than 80% [56]. This high degree of concordance provides the basis to predict the response of the lympho-hematopoietic and other stem cell systems to potential toxic insults. This has been described previously [32], but it is worth reiterating some of these paradigms. First, virtually any compound can be toxic to stem cells. Second, toxicity to the most primitive, definitive stem cells will affect all cells of the system. Third, since stem cells only proliferate and proliferation occurs prior to differentiation, stem cell cytotoxicity will affect all downstream cell types. Fourth, if more than one cell lineage is affected by toxicity, the target is not the cells that constitute the lineages, but the stem cells producing the lineages. Finally, stem cells are more sensitive to toxicity than the progenitor cells. When considering using stem cells to predict potential toxicity, at least two considera‐ tions need to be taken into account. The first is the primitiveness of the stem cell population being measured, while the second is variation between human donors. The former will depend, among other things, upon the ability and sensitivity of the assay to detect specific stem cell populations and the latter will be dependent upon the state and demographics of the donors that can, in turn, affect the stem cells. Both are difficult to control, but can provide a more realistic view.

Clarification of the Nomenclature for MSC: the International Society for Cellular

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 103

[2] Potten, CS. Cell cycles in cell hierarchies. Int J Radiat Biol Relat Stud Phys Chem

[3] Lemischka IR. The haematopoietic stem cell and its clonal progeny: mechanism regu‐ lating the hierarchy of primitive haematopoietic cells. Cancer Surv. 1992; 15:3-18.

[4] Yahata, T, Muguruma, Y, Yumino, S, Sheng, Y, Uno, T, Matsuzawa, H, Ito, M, Kato, S, Hotta, T, Ando, K. Quiescent human hematopoietic stem cells in the bone marrow niches organize the hierarchical structure of hematopoiesis. Stem Cells. 2008; 26:

[5] Campbell, CJ, Lee, JB, Levadoux-Martin, M, Wynder, T, Xenocostas, A, Leber, B, Bha‐ tia, M. The human stem cell hierarchy is defined by a functional dependence on

[6] Levesque, JP, Winkler, IG. Hierarchy of immature hematopoietic cells related to

[7] Staal FJ, Baum C, Cowan C, Dzierzak E, Hacein-Bey-Abina S, Karlsson S, Lapidot T, Lemischka I, Mendez-Ferrer S, Mikkers H, Moore K, Moreno E, Mummery CL, Robin C, Suda T, Van Pel M, Vanden Brink G, Zwaginga JJ, Fibbe WE. Stem cell self-renew‐ al; lessons from bone marrow, gut and iPS towards clinical applications. Leukemia.

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Based on these paradigms, it is worth briefly considering how the non-definitive stem cells systems (Fig. 1), ES and iPS cells, fit into predictive stem cell toxicity testing. At the present time, these cells are used to produce functionally, mature lineage-specific cells such as hepatocytes, cardiomyocytes and neurons. These and other cell types can be produced in larger numbers and presumably at a lower cost than their primary counterparts. Embryonic stem cells are used as an *in vitro* developmental toxicity model to predict teratogenicity. The use of ES and/or iPS cells for definitive stem cell system toxicity testing is certainly on the horizon. It should be remembered however, that even to produce functionally, mature hepatocytes, cardiomycytes and other cells, the ES and iPS cells must pass through the definitive stem cell compartment specific for the cells being produced. In other words, the ES and iPS cells should produce an organization analogous to that shown in Fig. 2. If this transpires, then the face of toxicity testing, and stem cell toxicity testing in particular, as well as many other applications, could significantly change the face of biological and toxicological research in the future.

#### **Acknowledgements**

The authors would like to thank Drs. Patricia Wood and William Hrushesky at the Dorn Research Institute of the William Jennings Bryan Dorn Veterans Affairs Medical Center in Columbia, South Carolina for sharing their insights and knowledge of circadian rhythms that led to the results shown in Section 5F.

#### **Author details**

Holli Harper and Ivan N. Rich

HemoGenix, Inc, U.S.A.

#### **References**

[1] Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A; The International Society for Cellular Therapy: Clarification of the Nomenclature for MSC: the International Society for Cellular Therapy Position Statement. Cytotherapy 2005; 7:393-395.

[2] Potten, CS. Cell cycles in cell hierarchies. Int J Radiat Biol Relat Stud Phys Chem Med. 1986; 49:257-278.

cells. When considering using stem cells to predict potential toxicity, at least two considera‐ tions need to be taken into account. The first is the primitiveness of the stem cell population being measured, while the second is variation between human donors. The former will depend, among other things, upon the ability and sensitivity of the assay to detect specific stem cell populations and the latter will be dependent upon the state and demographics of the donors that can, in turn, affect the stem cells. Both are difficult to control, but can provide a

Based on these paradigms, it is worth briefly considering how the non-definitive stem cells systems (Fig. 1), ES and iPS cells, fit into predictive stem cell toxicity testing. At the present time, these cells are used to produce functionally, mature lineage-specific cells such as hepatocytes, cardiomyocytes and neurons. These and other cell types can be produced in larger numbers and presumably at a lower cost than their primary counterparts. Embryonic stem cells are used as an *in vitro* developmental toxicity model to predict teratogenicity. The use of ES and/or iPS cells for definitive stem cell system toxicity testing is certainly on the horizon. It should be remembered however, that even to produce functionally, mature hepatocytes, cardiomycytes and other cells, the ES and iPS cells must pass through the definitive stem cell compartment specific for the cells being produced. In other words, the ES and iPS cells should produce an organization analogous to that shown in Fig. 2. If this transpires, then the face of toxicity testing, and stem cell toxicity testing in particular, as well as many other applications, could significantly change the face of biological and toxicological research in the future.

The authors would like to thank Drs. Patricia Wood and William Hrushesky at the Dorn Research Institute of the William Jennings Bryan Dorn Veterans Affairs Medical Center in Columbia, South Carolina for sharing their insights and knowledge of circadian rhythms that

[1] Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A; The International Society for Cellular Therapy:

more realistic view.

102 Stem Cell Biology in Normal Life and Diseases

**Acknowledgements**

**Author details**

**References**

led to the results shown in Section 5F.

Holli Harper and Ivan N. Rich

HemoGenix, Inc, U.S.A.


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**Section 2**

**Stem Cells in Disease**
