**3. Results**

#### **3.1. Selection of coating materials for feeder-free generating iPS cells**

Using gene chip approach, we investigated the levels of adhesion molecule expression on (i) CD34+ CBCs, (ii) the resulting iPSC cells and (iii) naïve iPSC on SNL (SNL76/7, ECACC) cultured in naïve cell medium. We identified several molecules that were expressed by CD34+ CBCs and by the resulting primed and naïve iPSCs cultured on feeder cell SNL (Table 1). These data prompted us to use their ligands to anchor CBCs to dishes for reprogramming in a feeder-free system. In this context, fibronectin or a relevant material, which has an-Arg-Gly-Asp-(RGD) motif that can bind to the integrin α5/β1 dimmer expressed on CBCs, was selected as a candidate for a coating material for the generation of iPSCs.


Mean and standard deviation of signal values for the expression of indicated genes from three independent experiments.

**Table 1.** Gene chip analysis of adhesion molecules on CD34+cells, and primed and naive iPSCs cultured on SNL.

From the point of view of quality control and reagent tracking, synthetic peptides expressing the RGD motif are preferable to natural ligands. Thus, Pronectin F which mimics the peptide structure of fibronectin, was chosen and tested for reprogramming CBCs. Pronectin F was synthesized by fusing two amino acid motifs, RGD and (GAGAGS)9 in tandem to produce a-RGD-(GAGAGS)9 -RGD-(GAGAGS)9 -RGD-(GAGAGS)9 -RGD-polypeptide. This polypeptide has thirteen RGD motifs and is folded at the RGD sequence. Thus, the RGD motif is effectively exposed at the limbs of the peptide bundle, facilitating its potent binding affinity to the integrin α5/β1 dimer.

### **3.2. Generation of iPS cells on synthetic peptide (Pronectin F®)**

**2.6. Karyotype analysis**

somes.

CD34+

CD34+

**3. Results**

stored in cell pellets, under fixative conditions and 20o

120 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

**3.1. Selection of coating materials for feeder-free generating iPS cells**

selected as a candidate for a coating material for the generation of iPSCs.

After the iPS cells have reached the 80% of confluence, it must be harvested and fixed to make a cytogenetic suspension. iPS cells are growth arrested and accumulated in metaphase or prometaphase by inhibiting tubulin polymerization and thus preventing the formation of the mitotic spindle using colcemid (Sigma, #D7385). Following exposure to colcemid, iPS cells are treated with a hypotonic solution to enhance the dispersion of chromosomes and fixed with carnoy fixative (Methanol: Acetic Acid=3:1). Once fixed, the cytogenetic preparation can be

spread on slides and air-dried, to be finally banded for the correct identification of chromo‐

Using gene chip approach, we investigated the levels of adhesion molecule expression on (i)

Mean and standard deviation of signal values for the expression of indicated genes from three independent experiments.

From the point of view of quality control and reagent tracking, synthetic peptides expressing the RGD motif are preferable to natural ligands. Thus, Pronectin F which mimics the peptide

**Table 1.** Gene chip analysis of adhesion molecules on CD34+cells, and primed and naive iPSCs cultured on SNL.

CBCs, (ii) the resulting iPSC cells and (iii) naïve iPSC on SNL (SNL76/7, ECACC) cultured in naïve cell medium. We identified several molecules that were expressed by

CBCs and by the resulting primed and naïve iPSCs cultured on feeder cell SNL (Table 1). These data prompted us to use their ligands to anchor CBCs to dishes for reprogramming in a feeder-free system. In this context, fibronectin or a relevant material, which has an-Arg-Gly-Asp-(RGD) motif that can bind to the integrin α5/β1 dimmer expressed on CBCs, was

C for several months. Fixed cells are

Protocol for generating iPSC on feeder less condition is shown in Figure 1.

**Figure 1.** Protocol for generation of iPSCs from CD34+CBCs on Pronectin F-coated dishes with temperature sensitive SeV vectors. P: passage.

Human ES cell-like colonies (first prime state) were picked up at day 24 and cultured on Pronectin F-coated dishes. The colonies were subjected to heat treatment (38o C, three days) at passage three (P3). Colonies emerged from single cells in Pronectin F-coated 96-well plates under naïve conditions at P4, dome-shaped colonies at P5 under naïve conditions, ES cell-like colonies (second primed) cultured under primed culture conditions at P6,P7.

The medium was changed every other day for transformed adherent cell stage (day 1-12). However, during day 13-17, primate ES medium was changed every day. The reprogramming process was monitored by checking the morphology of the transfected cells. CD34+cells infected with SeV constructs were cultured in serum-free hematopoietic cell culture, as shown in Figure 2 (day1). Some cells attached to Pronectin F-coated dishes by day four in Figure 2 (day 4). Cobble stone-like cell colonies emerged at day nine and cell clumps with round and small cells emerged inside the colonies at day 13 on Pronectin F-coated dishes (Figure 2, day 9, day 13). Cell clumps within cobble stone-like colonies grew (Figure 2, day 17) and finally human ES cell-like colonies emerged (Figure 2, day 24) on Pronectin F-coated dishes which were then picked up for serial passage. Fifteen to twenty-two dish-shape human ES cell-like colonies were picked out of 10,000 CD34+CBCs seeded on Pronectin F-coated dish in primate ES medium. Colonies were picked approximately three weeks after viral infection. Cells from individual colonies were transferred to a Pronectin F-coated 48-well plate to select passageable ES cell-like colonies capable of passage.

Reprogrammed cell clone before single cell cloning in the naïve state was named **PF** (**P**ronectin F –coated **F**eeder-less clones). The level of SeV protein expression was determined by immu‐ nostaining with SeV HN antibody (polyclonal-rabbit, gift to DNAVEC Corp., Ibaraki).

Generation and Maintenance of iPSCs From CD34+Cord Blood Cells on Artificial Cell Attachment Substrate

Then, single cells from dish-shaped (first primed) primate ES cell-like colonies at passage three were seeded on a Pronectin F-coated 96 well plate at approximately one cell per three wells

or six days, dome-shaped mouse ES cell like-colonies were collected and expanded on Pronectin F-coated dishes. Next, cell clumps were transferred to primate ES medium under

C). After five

123

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and cultured in naïve medium under hypoxic conditions (5% O2, 5% CO2 at 37o

20% O2 again to culture them in the primed state in Figure 4.

**Figure 4.** Generation of reprogrammed cell clone from a single cell via the naïve state.

Light microscopic image and ALP staining at P3 are shown in upper and lower panels, respectively. Colonies emerged from single cells in Pronectin F-coated 96-well plates under naïve condition at P4, dome-shaped colonies at P5 under naïve condition, ES cell-like colonies (second primed) cultured under primed culture condition at P6 or long-term passaged clone at P45 are shown. And, the colonies were subjected to heat treatment (38°C, three days). Colonies emerged from single cells in Pronectin F-coated 96-well plates under naïve conditions

**Figure 2.** Phase contrast light microscopic observation of cells during reprogramming. Images captured on a Pronec‐ tin F-coated dish on days 1, 4, 9, 13, 17 and 24. day 1; Infected CD34+CBCs with Sendai virus seed on Pronectin F® coated dish. day 4; Infected CD34+CBCs were attachment and little spread on Pronectin F-coated dish. day 9: Infected CD34+CBCs expansion on Pronectin F-coated dish. day 13; Infected CD34+CBCs expansion with colony-like state. day 17; generation of small ES-like colony around spreading apart of infected CD34+CBCs. day 24; Human ES cell-like colo‐ nies emerged on Pronectin F-coated dishes.

Human ES cell-like colonies (first primed state) were picked up at day 24 and cultured on Pronectin F-coated dishes. The colonies were subjected to heat treatment (38 o C, three days) at passage three (P3) to reduce the SeV constructs (Figure 3).

**Figure 3.** Expression of SeV in reprogrammed cell clone before and after heat treatment.

Expression of SeV in ES cell-like colonies before heat treatment at passage three (SeV at P3) and after heat treatment and single cell cloning at passage.

Reprogrammed cell clone before single cell cloning in the naïve state was named **PF** (**P**ronectin F –coated **F**eeder-less clones). The level of SeV protein expression was determined by immu‐ nostaining with SeV HN antibody (polyclonal-rabbit, gift to DNAVEC Corp., Ibaraki).

Then, single cells from dish-shaped (first primed) primate ES cell-like colonies at passage three were seeded on a Pronectin F-coated 96 well plate at approximately one cell per three wells and cultured in naïve medium under hypoxic conditions (5% O2, 5% CO2 at 37o C). After five or six days, dome-shaped mouse ES cell like-colonies were collected and expanded on Pronectin F-coated dishes. Next, cell clumps were transferred to primate ES medium under 20% O2 again to culture them in the primed state in Figure 4.

**Figure 4.** Generation of reprogrammed cell clone from a single cell via the naïve state.

**Figure 2.** Phase contrast light microscopic observation of cells during reprogramming. Images captured on a Pronec‐ tin F-coated dish on days 1, 4, 9, 13, 17 and 24. day 1; Infected CD34+CBCs with Sendai virus seed on Pronectin F® coated dish. day 4; Infected CD34+CBCs were attachment and little spread on Pronectin F-coated dish. day 9: Infected CD34+CBCs expansion on Pronectin F-coated dish. day 13; Infected CD34+CBCs expansion with colony-like state. day 17; generation of small ES-like colony around spreading apart of infected CD34+CBCs. day 24; Human ES cell-like colo‐

Human ES cell-like colonies (first primed state) were picked up at day 24 and cultured on

Expression of SeV in ES cell-like colonies before heat treatment at passage three (SeV at P3)

C, three days) at

Pronectin F-coated dishes. The colonies were subjected to heat treatment (38 o

**Figure 3.** Expression of SeV in reprogrammed cell clone before and after heat treatment.

and after heat treatment and single cell cloning at passage.

passage three (P3) to reduce the SeV constructs (Figure 3).

122 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

nies emerged on Pronectin F-coated dishes.

Light microscopic image and ALP staining at P3 are shown in upper and lower panels, respectively. Colonies emerged from single cells in Pronectin F-coated 96-well plates under naïve condition at P4, dome-shaped colonies at P5 under naïve condition, ES cell-like colonies (second primed) cultured under primed culture condition at P6 or long-term passaged clone at P45 are shown. And, the colonies were subjected to heat treatment (38°C, three days). Colonies emerged from single cells in Pronectin F-coated 96-well plates under naïve conditions at P4, dome-shaped colonies at P5 under naïve conditions, ES cell-like colonies (second primed).

RT-PCR determination of naïve state in iPSC colony, second primed colonies 1; PFX#2 or 3; PFX#9, naïve state colonies before each second prime state colonies (2; PFX#2 naïve, 4; PFX#9

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125

Once established on a Pronectin-coated dish, reprogrammed cell colonies can be maintained either in a Pronectin F-, Laminin-or Matrigels-coated dish for serial passage. 100-200 cell clumps (50-100 µm diameters) were seeded on 100mm dish or in six wells of a 6-well plate and cultured until colonies reach 70-80% confluence. The split ratio was routinely 1:3. This is a

It is possible to conduct cell passaging via single cell suspension in serum-free media (mTeSR1, TeSR2 and ReproFF) in the primed condition with the use of Rock inhibitor (Y-27632, Stemgent,

As shown in Figure 6, it is notable that single cells migrate towards one another three to thirtysix hrs after passage to form cell clumps. This is a single cell passage, not a single cell cloning process. We failed to generate colonies from single cell in the primed state. That is a rationale for using naïve culture for single cell subcloning purpose. It is convenient to use singe cell suspension for passage. However, morphology of cell colony via single cell passage in longer period (P20 or over) is not uniform and is no longer round. We have not accumulated enough data how relevant this even is, but from a daily practical point of view, we perform cell

*3.3.2. Characterization of reprogrammed cells by Reverse transcriptase polymerase chain reaction (RT-*

The expression of pluripotecy related genes were determined by RT-PCR. Total RNA was purified with an RNeasy Plus Micro kit (QIAGEN 74034), according to the manufacturer's instructions, and One µg of total RNA was used for reverse transcription reactions with PrimeScript RT reagent kit (TAKARA, Japan). Result is shown in Figure 7. Primer sequences

naïve). Values were normalized using the housekeeping gene *GAPDH*.

*3.3.1. Maintenance of reprogrammed cells established in feeder free condition*

protocol for passage via cell clump, not via single cell suspension.

**Figure 6.** Photograph of forming cell colony from single iPS cell suspension on Matrigel.

#2514).

passaging via cell clumps.

used for PCR are shown in Table 3.

*PCR)*

**3.3. Maintenance and characterization of reprogrammed cells**

Long-term passaged clone (PFX#9) at P45 is shown. After single cell cloning in the naïve state, picked up cell clones were named as **PFX** (**P**ronection F-coated **F**eeder-free iPSC derived from female (**X**X) cord blood cell. We used female cord blood cells (XX) to check the status of being in the naïve stage manifested by reactivation of X-chomosome inhibition. Culturing cells in the naïve state was useful for a single cell cloning in limited dilution, but we fail to support cell culture in the naïve stage robustly for more than five passages. Therefore cells were kept culturing in the primed condition (20% O2, the ES cell medium containing bFGF) after single cell cloning in the naïve state for further appraisal and passages.

Whether dome–shape cells cultured in the naïve condition (Figure 4, P5) was indeed in the naïve state or not a reactivation of X-chromosome inhibition was determined by gene chip analysis (Table 2.) and RT-PCR (Figure 5) with each states (prime [1st, 2nd] and naïve).


**Table 2.** *Xist* gene expression analysis by gene chip for X-chromosome activite / inactivite states using four different probes.

Naïve PFXs were cultured in the naïve state and 2nd primed PFs were cultured in the naïve state. PF #13 1st prime and khES-1 1st primed were cultured in the primed state (without being in the naïve state). PF #13 and PFXs are female (XX) in origin, while human ES cell line khES01 is male in (XY) origin.

**Figure 5.** Expression of *Xist* genes in naïve and prime state iPS cell determined by RT-PCR.

RT-PCR determination of naïve state in iPSC colony, second primed colonies 1; PFX#2 or 3; PFX#9, naïve state colonies before each second prime state colonies (2; PFX#2 naïve, 4; PFX#9 naïve). Values were normalized using the housekeeping gene *GAPDH*.

#### **3.3. Maintenance and characterization of reprogrammed cells**

at P4, dome-shaped colonies at P5 under naïve conditions, ES cell-like colonies (second

Long-term passaged clone (PFX#9) at P45 is shown. After single cell cloning in the naïve state, picked up cell clones were named as **PFX** (**P**ronection F-coated **F**eeder-free iPSC derived from female (**X**X) cord blood cell. We used female cord blood cells (XX) to check the status of being in the naïve stage manifested by reactivation of X-chomosome inhibition. Culturing cells in the naïve state was useful for a single cell cloning in limited dilution, but we fail to support cell culture in the naïve stage robustly for more than five passages. Therefore cells were kept culturing in the primed condition (20% O2, the ES cell medium containing bFGF) after single

Whether dome–shape cells cultured in the naïve condition (Figure 4, P5) was indeed in the naïve state or not a reactivation of X-chromosome inhibition was determined by gene chip

**Table 2.** *Xist* gene expression analysis by gene chip for X-chromosome activite / inactivite states using four different

Naïve PFXs were cultured in the naïve state and 2nd primed PFs were cultured in the naïve state. PF #13 1st prime and khES-1 1st primed were cultured in the primed state (without being in the naïve state). PF #13 and PFXs are female (XX) in origin, while human ES cell line khES01

**Figure 5.** Expression of *Xist* genes in naïve and prime state iPS cell determined by RT-PCR.

analysis (Table 2.) and RT-PCR (Figure 5) with each states (prime [1st, 2nd] and naïve).

cell cloning in the naïve state for further appraisal and passages.

124 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

primed).

probes.

is male in (XY) origin.

### *3.3.1. Maintenance of reprogrammed cells established in feeder free condition*

Once established on a Pronectin-coated dish, reprogrammed cell colonies can be maintained either in a Pronectin F-, Laminin-or Matrigels-coated dish for serial passage. 100-200 cell clumps (50-100 µm diameters) were seeded on 100mm dish or in six wells of a 6-well plate and cultured until colonies reach 70-80% confluence. The split ratio was routinely 1:3. This is a protocol for passage via cell clump, not via single cell suspension.

It is possible to conduct cell passaging via single cell suspension in serum-free media (mTeSR1, TeSR2 and ReproFF) in the primed condition with the use of Rock inhibitor (Y-27632, Stemgent, #2514).

**Figure 6.** Photograph of forming cell colony from single iPS cell suspension on Matrigel.

As shown in Figure 6, it is notable that single cells migrate towards one another three to thirtysix hrs after passage to form cell clumps. This is a single cell passage, not a single cell cloning process. We failed to generate colonies from single cell in the primed state. That is a rationale for using naïve culture for single cell subcloning purpose. It is convenient to use singe cell suspension for passage. However, morphology of cell colony via single cell passage in longer period (P20 or over) is not uniform and is no longer round. We have not accumulated enough data how relevant this even is, but from a daily practical point of view, we perform cell passaging via cell clumps.

#### *3.3.2. Characterization of reprogrammed cells by Reverse transcriptase polymerase chain reaction (RT-PCR)*

The expression of pluripotecy related genes were determined by RT-PCR. Total RNA was purified with an RNeasy Plus Micro kit (QIAGEN 74034), according to the manufacturer's instructions, and One µg of total RNA was used for reverse transcription reactions with PrimeScript RT reagent kit (TAKARA, Japan). Result is shown in Figure 7. Primer sequences used for PCR are shown in Table 3.

*3.3.3. Evaluation for remaining SeV construct*

**Table 4.** Quantitative RT-PCR determination of residual SeV viral genomes.

normalized using the housekeeping gene *GAPDH*.

integration and non-virus of established iPSC lines.

at P2 (PF#7 before HT, PF#9 before HT), non-infected CD34+

*3.3.4. Characterization of reprogrammed cells by Immunohistological staining*

BX51, IX71, Tokyo) and a light microscope (Olympus CKX31).

green, respectively) were used to visualize the staining.

Quantitative RT-PCR determination of residual SeV viral genomes in CD34+

qRT-PCR and shown in Table 4.

**CD34+ infected**

**Cell**

The remaining SeV construct after heat treatment and single cell cloning was determined by

Generation and Maintenance of iPSCs From CD34+Cord Blood Cells on Artificial Cell Attachment Substrate

SeV 193736 19719 26850 6511 3997 5414 1135

after SeV infection (CD34 infected), first primed colony iPS#7 or iPS#9 before heat treatment

generated by retrovirus (201B7), established clones at P9 (PFX#7) or (PFX#9). Values were

The residual SeV viral genome was determined by qRT-PCR analysis for selection of non-

ES cell like-colonies were stained with the Leukocyte Alkaline Phosphatase kit (VECTOR, Burlingame, CA, SK-5300) in accordance with the manufacturer's instructions. For immuno‐ chemical staining, these cells were fixed with 4% paraformaldehyde followed by staining with antibodies against Oct3/4 (1:100 sc-5279; Santa Cruz, Biotechnology USA), Nanog (1:500, RCAB0003P; Reprocell, Tokyo, Japan), SSEA-3 (1:200 MAB4303; Millipore), SSEA-4 (1:200, MAB4304, Millipore). Photomicrographs were taken with a fluorescent microscope (Olympus

ES cell-like clone PFX#9 at P8 was stained with antibodies against Nanog, Oct3/4, SSEA-3, or SSEA-4 as indicated. Alexa 594-and Alexa 488-conjugated secondary antibodies (red and

Total RNAs from several established iPSCs lines, ESCs lines (Riken BRC) and CD34+CBCs (Riken BRC) were purified with an RNeasy Plus Mini kit (QIAGEN 74136), amplified Ovation Pico WTA System (Takara cat#3300-12), labeled with an Encore Biotin Module (Takara catalog number 4200-12) and then hybridized with a human Gene Chip (U133 plus 2.0 Array Affy‐ metrix) according to the manufacturer's instructions (Figure 9). Karyotyping G-band method of iPSCs is shown in Figure 10. The amount of metaphases obtained is sometimes inadequate for chromosome analysis, thus it is always necessary to keep growing the PFX#9 iPS cells. As

*3.3.5. Characterization of reprogrammed cells by gene chip analysis and karyotyping*

shown in Figure 10, PFX#9 iPS cell on VTN-N was normal karyotypic cell.

**PF#7 before HT PF#9 before HT CD34+ 201B7 PFX#7 (P8) PFX#9 (P8)**

CBCs three days

CBCs (CD34) or iPSC clone

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127

**Figure 7.** Expression of endogenous pluripotency related genes in reprogrammed cell determined by RT-PCR.


**Table 3.** List of genes and the primers used for RT-PCR.

### *3.3.3. Evaluation for remaining SeV construct*

**Figure 7.** Expression of endogenous pluripotency related genes in reprogrammed cell determined by RT-PCR.

(endo)

(endo)

(endo)

(endo)

undifferentiated ES cell 408

undifferentiated ES cell 445

undifferentiated ES cell 306

undifferentiated ES cell 391

SeV vectors 193

control 337

144

151

328

397

hOCT3/4-F1165 GAC AGG GGG AGG GGA GGA GCT AGG Undifferentiated ES cell

hSOX2-F1430 GGG AAA TGG GAG GGG TGC AAA AGA GG undifferentiated ES cell

hMYC-F253 GCG TCC TGG GAA GGG AGA TCC GGA GC undifferentiated ES cell

hKLF4-F1128 ACG ATC GTG GCC CCG GAA AAG GAC C undifferentiated ES cell

**Primers Size(bp)**

hOCT3/4-R1283 CTT CCC TCC AAC CAG TTG CCC CAA AC

126 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

hSOX2-R1555 TTG CGT GAG TGT GGA TGG GAT TGG TG

hMYC-R555 TTG AGG GGC ATC GTC GCG GGA GGC TG

hKLF4-R1826 TGA TTG TAG TGC TTT CTG GCT GGG CTC C DPPA4-F GGAGCCGCCTGCCCTGGAAAATTC

hTERT-R3737 GGA AAA GCT GGC CCT GGG GTG GAG C REX1-F CAG ATC CTA AAC AGC TCG CAG AAT

REX1-R GCG TAC GCA AAT TAA AGT CCA GA NANOG-F CAG CCC CGA TTC TTC CAC CAG TCC C

NANOG-R CGG AAG ATT CCC AGT CGG GTT CAC C SeV vector-F GGATCACTAGGTGATATCGAGC

SeV vector-R CATATGGACAAGTCCAAGACTTC hGAPDH F AAC AGC CTC AAG ATC ATC AGC

hGAPDH R TTG GCA GGT TTT TCT AGA CGG

**Table 3.** List of genes and the primers used for RT-PCR.

DPPA4-R TTT TTC CTG ATA TTC TAT TCC CAT hTERT-F3292 CCT GCT CAA GCT GAC TCG ACA CCG TG The remaining SeV construct after heat treatment and single cell cloning was determined by qRT-PCR and shown in Table 4.


**Table 4.** Quantitative RT-PCR determination of residual SeV viral genomes.

Quantitative RT-PCR determination of residual SeV viral genomes in CD34+ CBCs three days after SeV infection (CD34 infected), first primed colony iPS#7 or iPS#9 before heat treatment at P2 (PF#7 before HT, PF#9 before HT), non-infected CD34+ CBCs (CD34) or iPSC clone generated by retrovirus (201B7), established clones at P9 (PFX#7) or (PFX#9). Values were normalized using the housekeeping gene *GAPDH*.

The residual SeV viral genome was determined by qRT-PCR analysis for selection of nonintegration and non-virus of established iPSC lines.

#### *3.3.4. Characterization of reprogrammed cells by Immunohistological staining*

ES cell like-colonies were stained with the Leukocyte Alkaline Phosphatase kit (VECTOR, Burlingame, CA, SK-5300) in accordance with the manufacturer's instructions. For immuno‐ chemical staining, these cells were fixed with 4% paraformaldehyde followed by staining with antibodies against Oct3/4 (1:100 sc-5279; Santa Cruz, Biotechnology USA), Nanog (1:500, RCAB0003P; Reprocell, Tokyo, Japan), SSEA-3 (1:200 MAB4303; Millipore), SSEA-4 (1:200, MAB4304, Millipore). Photomicrographs were taken with a fluorescent microscope (Olympus BX51, IX71, Tokyo) and a light microscope (Olympus CKX31).

ES cell-like clone PFX#9 at P8 was stained with antibodies against Nanog, Oct3/4, SSEA-3, or SSEA-4 as indicated. Alexa 594-and Alexa 488-conjugated secondary antibodies (red and green, respectively) were used to visualize the staining.

#### *3.3.5. Characterization of reprogrammed cells by gene chip analysis and karyotyping*

Total RNAs from several established iPSCs lines, ESCs lines (Riken BRC) and CD34+CBCs (Riken BRC) were purified with an RNeasy Plus Mini kit (QIAGEN 74136), amplified Ovation Pico WTA System (Takara cat#3300-12), labeled with an Encore Biotin Module (Takara catalog number 4200-12) and then hybridized with a human Gene Chip (U133 plus 2.0 Array Affy‐ metrix) according to the manufacturer's instructions (Figure 9). Karyotyping G-band method of iPSCs is shown in Figure 10. The amount of metaphases obtained is sometimes inadequate for chromosome analysis, thus it is always necessary to keep growing the PFX#9 iPS cells. As shown in Figure 10, PFX#9 iPS cell on VTN-N was normal karyotypic cell.

**Figure 8.** Characterization of established iPSC PFX#9 clones. Expression of pluripotency-related molecules in reprog‐ rammed cell clones.

**Figure 10.** G-band karyotype analyses. PFX#9 (P45)

as shown in Figure 11.

*3.3.6. In vitro differentiation potentials of reprogrammed cells*

The three germ layers differentiation potential of reprogrammed cells was tested via embryo body (EB) formation. Established ES cell-like clones were transferred to six-well, ultralow attachment plates (Corning) and cultured in DMEM/F12 containing 20% knockout serum replacement (KSR, Invitrogen) 2 mM L-glutamine, 1% NEAA, 0.1 mM 2-ME and 0.5% penicillin and streptomycin or ReproFF medium without bFGF to form EB. The medium was changed every other day. The resulting EBs were transferred to gelatin-coated plates for 16 days. Differentiation to ectodermal, mesodermal, or endodermal tissue was confirmed by detection of molecules related to three germ layers lineage differentiation such as α-feto-protein (endoderm), βIII-tubulin (ectoderm), GFAP (ectoderm), or Vimentin (mesoderm) with antibody against α-feto-protein (1:100 dilution MAB1368; R&D Systems), βIII-tublin (1:200 T4026; Sigma), GFAP(1:50 sc-6170 santa cruz biotechnology) or Vimentin (1:100 sc-5565; Santa Cruz Biotechnology) respectively. Antibodies were visualized with Alexa Fluor 488 goat antimouse (1:1,000; Invitrogen), Alexa Fluor 594 rabbit anti-mouse (1:1,000; Invitrogen), and Alexa Fluor 594 goat anti-rabbit (1:1,000; Invitrogen). Nuclei were stained with DAPI (1:1,000; Sigma)

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129

**Figure 9.** Gene expression comparison between the mean (mean) expression of clustered pluripotent stem cell. [PFX#9(iPSC from CBC with Yamanaka 4factors-heat treat Sendai virus without feeder) and HSC of Cord Blood (CD34+CBC)] and gene expression of PFX#9, or that of khES-1, (iPSC from CBC with Yamanaka 4 factors-Sendai Virus on feeder) ((left panel)]. R2: dicision coefficient

**Figure 10.** G-band karyotype analyses. PFX#9 (P45)

**Figure 9.** Gene expression comparison between the mean (mean) expression of clustered pluripotent stem cell. [PFX#9(iPSC from CBC with Yamanaka 4factors-heat treat Sendai virus without feeder) and HSC of Cord Blood (CD34+CBC)] and gene expression of PFX#9, or that of khES-1, (iPSC from CBC with Yamanaka 4 factors-Sendai Virus

**Figure 8.** Characterization of established iPSC PFX#9 clones. Expression of pluripotency-related molecules in reprog‐

128 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

on feeder) ((left panel)]. R2: dicision coefficient

rammed cell clones.

#### *3.3.6. In vitro differentiation potentials of reprogrammed cells*

The three germ layers differentiation potential of reprogrammed cells was tested via embryo body (EB) formation. Established ES cell-like clones were transferred to six-well, ultralow attachment plates (Corning) and cultured in DMEM/F12 containing 20% knockout serum replacement (KSR, Invitrogen) 2 mM L-glutamine, 1% NEAA, 0.1 mM 2-ME and 0.5% penicillin and streptomycin or ReproFF medium without bFGF to form EB. The medium was changed every other day. The resulting EBs were transferred to gelatin-coated plates for 16 days. Differentiation to ectodermal, mesodermal, or endodermal tissue was confirmed by detection of molecules related to three germ layers lineage differentiation such as α-feto-protein (endoderm), βIII-tubulin (ectoderm), GFAP (ectoderm), or Vimentin (mesoderm) with antibody against α-feto-protein (1:100 dilution MAB1368; R&D Systems), βIII-tublin (1:200 T4026; Sigma), GFAP(1:50 sc-6170 santa cruz biotechnology) or Vimentin (1:100 sc-5565; Santa Cruz Biotechnology) respectively. Antibodies were visualized with Alexa Fluor 488 goat antimouse (1:1,000; Invitrogen), Alexa Fluor 594 rabbit anti-mouse (1:1,000; Invitrogen), and Alexa Fluor 594 goat anti-rabbit (1:1,000; Invitrogen). Nuclei were stained with DAPI (1:1,000; Sigma) as shown in Figure 11.

**Figure 11.** *In vivo* differentiation potential of established clones. Phase contrast images of neuron-like (top left) and retinal pigmented epithelium (RPE) differentiation (top middle) of established clone PFX#7. Cells were fixed and stained with antibodies against AFP, βIII-tubulin, GFAP and vimentin to identify specific cell lineages [18].

allows complete control over the culture environment, resulting in more consistent cell

**Figure 12.** Teratoma with cystic structure. It was derived from iPSCs (PFX #9) implanted in the testicular capsule of a

Generation and Maintenance of iPSCs From CD34+Cord Blood Cells on Artificial Cell Attachment Substrate

http://dx.doi.org/10.5772/58591

131

**Figure 13.** Maintenance of iPS cells (PFX#9) on recombinant vitronectin (VTN-N, Life Technology) in ReproFF2 medi‐

populations and reproducible results in clinical applications.

NOD-SCID mouse. It was stained with hematoxylin and eosin for histological observation.

um.

#### *3.3.7. In vivo differentiation potential of reprogrammed cells by Teratoma formation assay*

Reprogrammed cell lines should demonstrate differentiation potential reflecting three germ layers, *in vivo* as well as *in vitro*. To this end, one million iPSCs were injected beneath the testicular capsule of NOD-SCID mice (SLC Japan) to determine the ability of the transplanted cells to form teratomas containing cells of all three germ layers. Tumor formation was observed approximately four weeks after cell transplantation. Tumor tissues were fixed with 4% formalin, sectioned, and stained with hematoxylin and eosin (Figure 12).

#### *3.3.8. Preservation of Feeder-free iPS cells*

Human ES/iPS clones generated and maintained in a feeder-free system could be frozen in cell clumps using DMSO-free, chemically defined and serum-free freezing medium, CryoStemTM Freezing Medium (Stemgent), and could be cultured again on a Pronectin F-coated dish after thawing. Approximately 10-20% of the colony number scored before cryopreservation in CryoStemTM emerged after thawing.

#### *3.3.9. Long-term, Low-cost and Stable maintenance of undifferentiated human induced pluripotent stem cells in feeder-free condition*

Vitronectin provides a completely defined culture system for the maintenance of hiPSC under feeder-free conditions such as ReproFF2 medium (Figure 13, Figure 14, Table 5). This system Generation and Maintenance of iPSCs From CD34+Cord Blood Cells on Artificial Cell Attachment Substrate http://dx.doi.org/10.5772/58591 131

**Figure 12.** Teratoma with cystic structure. It was derived from iPSCs (PFX #9) implanted in the testicular capsule of a NOD-SCID mouse. It was stained with hematoxylin and eosin for histological observation.

**Figure 11.** *In vivo* differentiation potential of established clones. Phase contrast images of neuron-like (top left) and retinal pigmented epithelium (RPE) differentiation (top middle) of established clone PFX#7. Cells were fixed and

Reprogrammed cell lines should demonstrate differentiation potential reflecting three germ layers, *in vivo* as well as *in vitro*. To this end, one million iPSCs were injected beneath the testicular capsule of NOD-SCID mice (SLC Japan) to determine the ability of the transplanted cells to form teratomas containing cells of all three germ layers. Tumor formation was observed approximately four weeks after cell transplantation. Tumor tissues were fixed with 4%

Human ES/iPS clones generated and maintained in a feeder-free system could be frozen in cell clumps using DMSO-free, chemically defined and serum-free freezing medium, CryoStemTM Freezing Medium (Stemgent), and could be cultured again on a Pronectin F-coated dish after thawing. Approximately 10-20% of the colony number scored before cryopreservation in

*3.3.9. Long-term, Low-cost and Stable maintenance of undifferentiated human induced pluripotent stem*

Vitronectin provides a completely defined culture system for the maintenance of hiPSC under feeder-free conditions such as ReproFF2 medium (Figure 13, Figure 14, Table 5). This system

stained with antibodies against AFP, βIII-tubulin, GFAP and vimentin to identify specific cell lineages [18].

130 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

*3.3.7. In vivo differentiation potential of reprogrammed cells by Teratoma formation assay*

formalin, sectioned, and stained with hematoxylin and eosin (Figure 12).

*3.3.8. Preservation of Feeder-free iPS cells*

CryoStemTM emerged after thawing.

*cells in feeder-free condition*

allows complete control over the culture environment, resulting in more consistent cell populations and reproducible results in clinical applications.

**Figure 13.** Maintenance of iPS cells (PFX#9) on recombinant vitronectin (VTN-N, Life Technology) in ReproFF2 medi‐ um.

**Primers Size(bp)**

REX1-R TCA TAG CAC ACA TAG CCA TCA CAT

NANOG-R TCC CTG GTG GTA GGA AGA GTA AA

**Table 5.** List of primers used for quantitative real-time PCR (qRT-PCR)

hGAPDH-F CCA CTC CTC CAC CTT TGA CG

hGAPDH-R ATG AGG TCC ACC ACC CTG TT

**4. Conclusion**

**Acknowledgements**

CD34+

Japan (2013).

**Author details**

Shin Kawamata

REX1-F TGC AGG CGG AAA TAG AAC CT undifferentiated

Generation and Maintenance of iPSCs From CD34+Cord Blood Cells on Artificial Cell Attachment Substrate

NANOG-F CTC AGC TAC AAA CAG GTG AAG AC undifferentiated

In this chapter we have shown the method for generating iPSC from non-cultured CD34+

blood cells using feeder-free conditions. The established cell clones were characterized at a single cell level. This robust iPSC generation method will solve some of the safety concerns related to tumorigenicity ariseing from chromosomal integration of exogenous genes and/or infection hazards associated with the use of by xenogeneic biological products in the culture system. These methods will contribute to future application of iPSCs-derived cell therapy.

We thank SI Nishikawa for useful discussions, N Fusaki for samples gift, and S Sawada for gene chip analysis. We thank Riken Bio Resource Center (Tukuba, Japan) for fresh

Naoki Nishishita, Takako Yamamoto, Chiemi Takenaka, Marie Muramatsu and

Foundation for Biomedical Research and Innovation, Kobe, Japan

CBCs. This work was partly supported by the Regulatory Science Study for Safety issue for pluripotent stem cells of JST Tokyo Japan (2010-2014) and Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) project of JST Tokyo

ES cell

ES cell

control 114

64

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cord

**Figure 14.** Expression of endogenous pluripotency related genes in iPSC (PFX#9) on VTN-N determined by qRT-PCR.

Following, the PFX#9 cells cultured with VTN-N was the gene expression of pluripotency markers comparable iPS cells cultured on Matrigel or on SNL in Figure 12. It was found that only a recombinant vitronectin (VTN-N) can be maintained in culture for long-term feeder free conditions.



**Table 5.** List of primers used for quantitative real-time PCR (qRT-PCR)
