Stem Cells Properties Shifting and Reprogramming

#### **Chapter 1**

## Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM-Free Reprogramming to Pluripotency

*Zorica A Becker-Kojić, José Manuel García-Verdugo, Anne-Kathrin Schott, Vicente Herranz-Pérez, Ivan Zipančić and Vicente Hernández-Rabaza*

#### **Abstract**

The generation of induced pluripotent stem cells (iPSCs) by forced expression of defined transcription factors has revolutionized regenerative medicine. These cells have similar features to embryonic stem cells (ESCs) regarding self-renewal and their ability to differentiate into any cell type in the body. In spite of many improvements, in using nonviral delivery reprogramming methods, there are still challenges to overcome regarding safety before patient-made iPSCs can be used in regular clinical practice. We have recently reported about a gene manipulation-free method of generating human pluripotent stem cells (PSCs), based on activation of the novel human GPI-linked glycoprotein ACA. The process of dedifferentiation of blood progenitor cells that leads to the generation of blood-derived pluripotent stem cells (BD-PSCs) is initiated upon cross-linking of this protein *via* activation of PLCγ/PI3K/Akt pathway. These cells are mortal, express pluripotent markers, and redifferentiate *in vitro* into cells of all three germ layers. The ultrastructural analysis of BD-PSCs, by means of electron microscopy, revealed them similar to human ESCs with large dense nucleolus and scarce cytoplasm. BD-PSCs are autologous stem cells and while nonteratogenic offer a new alternative that overcomes immunological, ethical, and safety concerns and opens up a new avenue in treating contemporarily intractable diseases and generally in human therapeutics.

**Keywords:** blood progenitor cells, membrane signaling, dedifferentiation, pluripotency, redifferentiation

#### **1. Introduction**

Human induced pluripotent stem cell (iPSC) technology has paved the way for new possibilities to investigate and potentially cure diseases. The iPSCs derived from patients can be used in at least two ways: regenerative medicine and drug discovery,

for example, screening chemicals, natural compounds, and derivatives to identify drug candidates. This new technology promises to provide a powerful tool for modeling human pathology that allows for investigation and understanding of the underlying mechanisms and causes of various human diseases. Particularly, disease-specific iPSCs are of great potential for disease modeling and therapeutic benefits [1, 2].

iPSCs have characteristics of human embryonic stem cells (ESCs), including pluripotency and potentially unlimited self-renewal. During the last decade, patientmade iPSCs have been differentiated into a variety of functional cell types *in vitro* and are expected to reconstruct disease phenotypes, as already demonstrated in several animal disease models [3].

Originally, iPSCs were generated by retroviral transduction of four specific transcription factors, Oct3/4, Sox2, Klf4, and cMyc or Oct3/4 Sox2/Nanog/LIN, using retroviral or lentiviral vectors [4, 5]. Later, lentivirus was the preferred delivery method, since, unlike retrovirus can infect proliferating and nondividing cells. Viral vectors for iPSC generation are very effective for integrating exogenous genes into the genome of somatic cells; however, they could be permanently integrated into the cell's genome, which generates serious concerns about changes in cell behavior and therefore, limiting their use in patients [6].

Despite the fact that iPSCs, as well as ESCs, are being proclaimed to have a great advantage as a source of stem cells that can be used in regenerative medicine, the ultimate goal to use them in clinical practice has not been achieved.

Cells convert one kind of signal into another through a process called signal transduction. This mechanism comprises the coupling of a ligand-receptor interaction to many intracellular events. These events include phosphorylation by tyrosine kinases and/or serine/threonine kinases. Differential localization of protein that participates in signaling pathways is essential for cells to respond efficiently to changes in their environment. In the plasma membrane, such compartmentalization is performed through lipid rafts [7] that are enriched in cholesterol, sphingolipids, and GPI-anchored proteins. During the signaling processes, various lipids are phosphorylated, recruited, and activated by different signaling components, which are essential for the regulation of cell survival and growth.

PI3Ks are a family of intracellular signal transducer enzymes involved in a variety of cellular functions like growth proliferation and differentiation. These enzymes are capable of phosphorylating position 3 of the inositol ring of phosphatidylinositol (PtdIns), and this lipid modification initiates a set of events that leads to cell activation and growth [8].

We have recently shown that human GPI-linked glycoprotein ACA, expressed in all adult stem cells, including hESCs, is involved in developmental process, which shapes the human embryo and controls adult stem cell compartments. Activation by this protein on the membrane of human blood progenitor cells drives membrane-tonucleus signaling pathways, thus regulating pluripotency [9].

We investigate here the signaling machinery behind the antibody cross-linking activation of GPI-linked membrane glycoprotein ACA that drives the immature blood progenitor cells to pluripotency and the capacity of these cells to redifferentiate into cells belonging to different germ layers.

The newly generated human blood-derived (BD-PSCs) as well as their redifferentiated progeny was assessed by the methods of immunocytochemistry (ICC), flow cytometry, and electron microscopy (EM). Signaling competence of ACA receptor was analyzed by studying the phosphorylation pattern and real-time analysis of developmentally relevant genes, such as *NOTCH, WNT, CTNNB, C-KIT,* and others. *Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

#### **2. Materials and methods**

#### **2.1 Cell cultures**

Human mononuclear cells (MNCs) were isolated from peripheral blood (PB) samples obtained from healthy donors. All of the human blood samples were used after obtaining written informed consent from the donors. PBMNCs were isolated by density gradient centrifugation using Ficoll and activated by specific antibody cross-linking as described elsewhere. Briefly, 6106 MNCs in a 15 ml polystyrol tube were incubated for 30 min with antibody (30 μg/mL in 1% PBS/BSA) to GPI-linked membrane protein ACA and further cultured and maintained in suspension in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FBS (Gibco Life Technology, Grand Island, NY), 0.1 mM nonessential amino acids (NEAA), 1 mM L-glutamine (all from Invitrogen, Carlsbad CA). The cells were taken at different time points for immunophenotyping (IP) by flow cytometry, ICC, and western blot (WB) analysis.

For growing of human ESCs line H9 (Wi Cell Inc., Medison, Wi) on feeder cells, mouse embryonic fibroblasts (MEFs), obtained from mouse strain CF-1 (American Type Culture Collection Manassas, VA, USA) mitotically inactivated by radiation were prepared according to standard protocol and approved (07-WO25) by the ethics committee established at Príncipe Felipe Centro de Investigación (CIPF) in Valencia (Spain), where these experiments were conducted. Human ES cells were placed on a freshly prepared MEF layer and further cultivated in ESC medium consisting of Knockout-DMEM (Invitrogen, Carlsbad CA), 100 μM β-mercaptoethanol (Sigma, St Lois), 1 mM L-glutamine (Invitrogen), 1% penicillin/streptomycin (Invitrogen) and 8 ng/ml bFGF (Invitrogen), or in condition media (see below). The medium was changed every other day. Recovery of pluripotent phenotype of the differentiated H9 cells line was done by cross-linking the membrane of these cells with a GPI proteinspecific antibody. Cells were maintained in IMDM supplemented with 10% FBS.

The culture of BD-PSCs on feeder-free culture dishes was performed to assess the expression of hESC markers by means of immunofluorescence. For that purpose, BD-PSCs were grown in MEF-conditioned media supplemented with 8 ng/ml bFGF on culture dishes coated with Matrigel 1:30 (BD Bioscience, Franklin Lakes, NJ).

MNCs isolated from PB were cultured in Iscove's media supplemented with 10% FBS, preincubated with the inhibitors Et-18-CH3 (ET) at 50 μM, LY 294002 (LY) at 20 μM or ET + LY purchased from Calbiochem (USA) for 1 h, and after activation in the presence of inhibitors cultured at 37°C, as specified. After labeling with CD34APC, CD45FITC (BD Pharmingen), and CD14PE (eBioscience), the generation of CD34 cells was daily assessed by multiple flow cytometry analyses. The nonviable cells were excluded after performing a propidium iodide (PI) assay. Conjugated isotypematched irrelevant antibodies were used as controls.

Antibodies to SSEA-4 and TRA-1-81 purchased by Chemicon (Temecula, CA) labeled with phycoerythrin (PE) were used for phenotypic analysis of pluripotent markers expressed on BD-stem cells by means of flow cytometry. Gating was done with matched isotype control monoclonal antibodies. Conjugated isotype-matched irrelevant mAbs were used as controls.

#### **2.2 BD-PSCs differentiation toward neuronal and hepatic cells**

Differentiation to neuronal lineages was adapted from previously described protocol [10]. Briefly, BD-PSCs were grown on glass coverslips coated with 1:5 diluted poly-L-ornithin/laminin for 2 days in neuronal initiating medium N2, followed by neuronal differentiation medium (Neurobasal medium, L-glutamin, B27 supplement, nonessential amino acids (NEAA), recombinant human glial-derived neurotrophic factor (GDNF), recombinant human brain-derived neurotrophic factor (BDNF), and ascorbic acid solution. The cells were grown at 37°C, 5% CO2 for 130 days. Cells were taken at D8, D20, and D30 for ICC analysis.

Differentiation of BD-PSCs toward endoderm/hepatocytes was performed on Biolaminin 111 (Biolamina Sundbyberg, Sweden) treated glass coverslips in KSR/DMSO media consisting of 80% of knockout DMEM media (KO-DMEM), 20% knockout serum replacement, 0.5% NEAA (all from Invitrogen), 0.1% β-mercaptoethanol, 1% DMSO (Sigma), and 1% penicillin–streptomycin for 7 days, followed by culturing cells in HepatoZYME maturation medium, 1% glutamax, 10 μM hydrocortisone 21-hemisuccinate sodium salt (HCC) (Biomol, Hamburg, Germany), supplemented with 10 ng/mL hepatocyte growth factor (HGF) (Life Technology) and 2 ng oncostatin M (OSM) (Biotechne), for additional 2 weeks. The cells were taken at D7 and D21 for ICC analysis.

#### **2.3 Cell culture, inhibition, and western blot analysis**

MNCs isolated from PB were cultured in Iscove's media supplemented with 10% FBS, preincubated with the inhibitors ET at 50 μM, LY at 20 μM, and PD098059 (PD) at 10 μM, or (ET + LY + PD) purchased from Calbiochem (USA) for 1 h, and after activation in the presence of inhibitors cultured at 37°C, 5% CO2. Cell-free extracts of these cells were subjected to western blot analysis. Nonactivated MNCs were used as controls.

#### **2.4 Western immunoblotting**

Cell lysis, protein extraction, and western blot analysis of ACA-activated PBMNCs vs. nontreated samples were performed as described elsewhere [9, 11]. Briefly, cells were lysed in Triplex buffer (50 mM Tris HCl pH 8, 120 mM NaCl, 0.1% SDS, 1% Nonidet P-40, and 0.54% deoxycholate), 300 μg of protein extracts were submitted to electrophoresis by using 10% SDS-PAGE. Immunodetection was performed by using appropriate primary antibodies followed by incubation with HPR-conjugated secondary antibodies (all purchased by cell Signaling technology, GAPDH antibody by Santa Cruz biotechnology). ECL Western blotting substrate (Pierce) was used for the detection of proteins on PVDF.

#### **2.5 Quantitative PCR analysis**

Total RNA was extracted from cells using TRIzol (Invitrogen) and transcribed into cDNA using oligo (dT) 16 and ReverTra Ace reverse transcriptase. PCR reactions were carried out by mixing 1 μL of cDNA template, 250 nM of each primer, 200 μM dNTP mixture, and 1 U of Taq DNA polymerase in a total volume of 20 μL. Samples were amplified in a thermocycler. For qPCR, each sample was analyzed in triplicate with GADH as the internal control. Amplification data were collected using ABI PRISM 7900 and analyzed using the sequence detection system 2.0 software. The primers and TaqMan probes used in this experiment are presented in **Table 1**.

*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*


#### **Table 1.**

*List of primers and TaqMan probes used in Figure 1d.*

#### **2.6 Immunocytochemistry (ICC)**

Immunofluorescence analysis of cells growing on Matrigel (Corning CA) was first performed to evaluate the presence of pluripotent stem cell markers, such as SSEA-4, TRA-1-60, and TRA-1-81. All antibodies were purchased by chemicon. Secondary PE or Alexa Fluor 488 labeled antibody (Life Technology, Carlsbad CA) was used to reveal the expression of pluripotent markers on BD-PSCs.

Activated PBMNCs growing in suspension were taken at different time points during culture time period from D1D14, transferred on glass coverslips, coated with Poly-L-lysine (Sigma-Aldrich St. Louis, USA), and the acquisition of pluripotent markers, TRA-1-60, SOX2, NANOG, and OCT3/4 monitored by means of ICC. Cells were fixed in 4% paraformaldehyde (PFA) for 15 min at RT followed by permeabilization with 0.1% Triton X-100 in PBS for 30 min in phosphate buffer saline (PBS) containing 3% BSA. All directly labeled primary antibodies were diluted in the same blocking buffer and incubated with samples overnight at 4°C. The nuclei were stained with DAPI (Sigma-Aldrich) for 3 min at RT. All images were acquired with an inverted Olympus IX71 Microscope. All antibodies used in this experiment are presented in **Table 2**.

ICC of human neuronal cells generated from BD-PSCs was performed using antibodies to Nestin, GFAP, MAP2, Neun, and Tuj1.

Cells were fixed with prewarmed fixative (PBS, PFA, MgCL2, EGTA, and sucrose) for 15 min, then treated with 0.3% Triton X-100 in PBS containing 3% BSA for 5 min as previously described [10]. Appropriate dilution of antibodies was prepared in PBS containing 1% BSA and incubated for 1.5 h, at RT, washed three times with PBS, and anti-chicken, anti-rabbit, and anti-mouse fluorochrome-conjugated antibodies were used to reveal the expression of specific neuronal markers (antibodies used in this experiment are presented in **Tables 3** and **4**). DAPI was used for staining the nuclei of cells. All images were acquired with an inverted Olympus IX71 microscope.

BD-PSC Differentiation to human endoderm/hepatocytes was assessed by means of ICC using antibodies to alpha-fetoprotein (AFP), anti-transthyretin (TTR) (endoderm), and anti-Albumin (ALB), anti-Hepatocyte Nuclear Factor 4 alpha (HNF4 α) (hepatocytes) and their relevant fluorescent-labeled secondary antibodies (all antibodies used are presented in **Tables 5** and **6**). Cells were fixed with 4% PFA for

#### *Possibilities and Limitations in Current Translational Stem Cell Research*

#### **Figure 1.**

*Relative protein expression and phosphorylation status of mediators of ACA-signaling and its inhibition with pharmacological inhibitors. The PBMNCs activated cells were cultured either in presence or absence of inhibitors ET, LY, PD, or ET + LY. Relative protein expression and phosphorylation status of mediators of this signaling were determined by western blot analysis. Nonactivated mononuclear cells isolated from PB were used as controls. (a): Western blotting of cell lysate derived from activated PBMNCs expression and phosphorylation status of proteins involved in the signaling mechanisms. (b): The PB-generated progenitor/HSCs were cultured either in presence or absence of inhibitors ET, LY, PD, or ET + LY. Nonactivated MNCs isolated from PB were used as controls. ET, LY, and ET + LY inhibited membrane protein antibody cross-linking induced generation of CD34 cells. Flow cytometry shows the generation of CD34 cells in the presence or absence of ET, LY, or ET + LY respectively in a 15 days' culture time period. (c): The total number of viable CD34 cells in cultures stimulated with or without ET, LY or ET + LY was estimated by flow cytometry. Data are presented as the mean SEM for three independent experiments. (d): Regulation of gene expression after GPI-anchored protein-induced generation of BD-PSCs. Living cells were isolated before (D1) and after activation at D6 and D12. The total RNA of these cells was reverse transcribed, and the expression of the following genes was studied by real-time analysis: CTNNB1 HOXB4, C-KIT, NOTCH1, WNT10, BMI1,TGFB1, and BCL2. Represented is the average of triplicate gene-expression changes measured by TaqMan as described in Methods.*

*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*


#### **Table 2.**

*Directly labeled antibodies from BD Pharmingen (California, US) are used in Figure 2.*

#### **Figure 2.**

*Inducing pluripotency in BD-progenitor/stem cells throughout cell culture time. (a-d): PBMNCs were activated by antibody-specific cross-linking and cultured in Iscove's medium for 14 days. Aliquots were taken at D3, 8, and 14, incubated with fluorescent-labeled antibodies to pluripotent markers, and subjected to immunofluorescence microscopy. Immunofluorescence microscopy of cell cultures for 14 days showing the expression* in vitro *of the pluripotency markers, (a) TRA-1-60, (b) Nanog, (c) Oct3/4, and (d) Sox2, throughout the cell culture time. Nuclei were stained with DAPI. Scale bars 50 μm.*


#### **Table 3.**

*Primary antibodies used in Figure 3.*

10 min and permeabilized with 0.1% Triton-X-100 for 3 min. DAPI was used for nuclear staining. Expressions of these markers were visualized with an inverted Olympus Microscope CKX53.

#### **2.7 Electron microscopy**

Cells were seeded at 6.25<sup>10</sup><sup>5</sup> cells/cm<sup>2</sup> in 8-well Lab-Tek chamber slides (Nalgene Nunc International, Naperville, IL). Cells were fixed in 3.5% glutaraldehyde for 1 h at 37°C, postfixed in 2% OsO4 for an additional 1 h at RT, and stained in 2% uranyl acetate in the dark at 4°C for 2 h. Finally, cells were rinsed with distilled water, dehydrated in ethanol, and embedded in Durcupan (Fluka) epoxy resin overnight. Following resin hardening, embedded cultures were detached from the chamber slide and glued to araldite blocks. Serial semi-thin sections (1.5 μm) were cut with an

#### **Figure 3.**

*Ectodermal/neural differentiation of BD-PSCs in adherent monolayer from N8-N30. Immunocytochemical and immunophenotypic profiling of BD-neuronal cells throughout the culture time period from N8 to N30. Scale bars 100 μm (N8-N20) and 50 μm (N30).*

*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*


#### **Table 4.**

*Secondary antibodies used in Figure 3.*

Ultracut UC-6 (Leica, Heidelberg, Germany), mounted onto glass slides, and lightly stained with 1% toluidine blue. Selected semi-thin sections were glued with Super-Glue-3 Loctite (Henkel, Düsseldorf, Germany) to resin blocks and detached from the glass-slide by repeated freezing (in liquid nitrogen) and thawing. Ultrathin sections (6080 nm) were prepared with an ultramicrotome and contrasted with lead citrate. Finally, photomicrographs were obtained at 80 kV using an FEI Tecnai G2 Spirit transmission electron microscope (FEI Europe, Eindhoven, and Netherlands) equipped with a Morada CCD digital camera (Olympus Soft Image Solutions GmbH, Münster, Germany).

#### **3. Results**

#### **3.1 Membrane-to-nucleus signaling induced by ACA activation**

To investigate a phosphorylation pattern across the plasma membrane, we activated GPI-linked protein by means of ACA-antibody cross-linking. The cell-free extracts were prepared from peripheral blood (PB) cells after Ficoll gradient centrifugation before and 6 days after activation. The evaluation of the expression and phosphorylation status of the proteins, presumably involved in this signaling network, was assessed by western blot analysis.

As shown in **Figure 1**(**a**), antibody cross-linking induces PI3K activation that phosphorylates and activates the known members of this pathway described in [12].

The phosphoinositol-phospholipase γ (PLCγ) is a member of family of PLC enzymes consisting of various isoforms with different cellular functions. PLCγ is linked to tyrosine kinase signaling pathways with its primary function to catalyze the hydrolysis of phosphatidylinositol-4,5-bisphosphat (PIP2) to generate inositol (1,4,5) triphosphate (PIP3) and 1,2-diacylglycerol (DAG). PIP3 initiates an increase in intracellular, whereas DAG activates protein kinase C, and the control over this important


**Table 5.** *Primary antibodies used in Figure 4.*

**Figure 4.**

*Endodermal/hepatocytes differentiation of BD-PSC in culture time period from L7 to L20. Shown is immunocytochemical and immunophenotypic analysis of endoderm/hepatocyte-specific markers through culture time period from L7 to L20. Scale bars 50 and 20 μm (L20 ALB/DAPI).*

second messenger pathway is a key to changes in cellular activity and function [13]. Phosphorylation of PLCγ and its activation seemed to be the earliest event in ACAinitiated specific signaling network, leaving the hydrolytic products of this protein, like inositol phosphate and a diacyl-myristate, the latter known as the most powerful activator of PI3K.

To evaluate the function and role of PLC in the signaling pathway, we used the specific inhibitors of PLCγ, ET-18-0-CH3 (ET), as well as the inhibitors of various kinases involved in this signaling like LY 294002 (LY) inhibitor of PI3K, and the MAP kinase inhibitor, PD098059 (PD). Peripheral blood mononuclear cells (PBMNCs) were preincubated with these inhibitors and cultured for 16 days after activation. Relative protein expression and phosphorylation status of various mediators of the initiated signaling were assessed by western blot analysis. Nonactivated PBMNCs were used as controls. The inhibitor of PLCγ caused a partial suppression of expression as well as reduced phosphorylation extent of various kinases involved in this signaling, whereas specific inhibitors of PI3K or MAP kinase alone had no effect on the initialization of the signaling (data not shown). Conversely, preincubation with all three inhibitors caused significant suppression of protein expression and phosphorylation extent of all participants (**Figure 1**(**a**)).


**Table 6.**

*Secondary antibodies used in Figure 4.*

*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

We further assessed the role of PLCγ and PI3K in membrane-to-nucleus-induced signaling, which first leads to generation of hematopoietic progenitor cells. This is an inevitable intermediate step on the described route to pluripotency [11]. Cells expressing CD34 protein are normally found in umbilical cord blood as well as in bone marrow cells, and antibodies to this protein are often used clinically to quantify the number of HSCs used in HSC transplantations [14]. CD34+ cells were generated in the presence or absence of their specific inhibitors, such as ET, LY, and ET + LY. The culture conditions were used to follow the effect of inhibitors on generation of CD34+ cells for the culture time period of 5 days.

PBMNCs were preincubated with ET, LY, and ET + LY and upon activation; newly generated cells were assessed by multiple flow cytometry analyses using antibodies to CD34, CD45, and CD14. The growing population of CD34<sup>+</sup> during culture time, from D1D5, was monitored. Upon inhibition with ET, we observed a slight decrease in the number of newly generated CD34+ cells, whereas LY alone had no effect on induced hematopoiesis. Most significantly, a dramatic decrease in fluorescence intensity occurred when both inhibitors were used together. Inhibition of PLCγ alone as well as inhibition of PLCγ and PI3K with their specific inhibitors ET + LY induced significant changes regarding *de novo* generation of CD34<sup>+</sup> cells, whereas no changes were observed when IP3K was inhibited by using LY alone see **Figure 1**(**b**).

The same experiment was performed with PBMNCs from various donors confirming the previous findings (**Figure 1**(**c**)).

Herewith, we conclude that phosphorylation and activation of PLCγ are indispensable for initiation of the signaling cascade. The changes induced by ET during the generation of CD34<sup>+</sup> cells are highly likely to be responsible for the initial events started by GPI-linked protein stimulation. This implies that phosphorylation and activation of PLCγ is a crucial event in this mediated signaling, while PI3K and AKT represent downstream activators, essential for induced route to pluripotency *via* generation of human hematopoietic progenitor cells.

#### **3.2 Antibody activation upregulates the expression of developmentally relevant genes**

In order to study these specific signal transduction networks, we used quantitative RT-PCR (TaqMan) analysis to determine the gene-expression pattern of the molecules potentially involved in this signaling.

We activated the GPI-linked protein at the surface of blood progenitor cells as described above and analyzed the way in which the initiated signaling machinery regulates the expression of genes known to play a role in human embryonic development *via* specific protein phosphorylation as an important regulatory mechanism in cellular processes.

We compared the gene expression profile of the PBMNCs before and after activation to assess a transcript level for candidate molecules, the most important among them NOTCH and WNT/CTNNB1 (**Figure 1**(**d**)). The signaling pathways linked to these genes are developmentally conserved and play a significant role in embryonic development as well as in the regulation of adult cell compartments [15].

Dysregulation of Wnt and Notch pathways due to their involvement in the key functions of human cells is a reason for their implication in many human diseases [16].

As shown in **Figure 1**(**d**), we demonstrated that GPI-linked glycoprotein upregulates both Notch and Wnt signaling pathways, thus acting in a hierarchical manner in the relationship to both signaling pathways. In consistency with the previous report about β-catenin as a downstream regulator of the canonical Wnt pathway [17], our results clearly show the upregulation of β-catenin as a consequence of this activation.

The involvement and significance of this GPI-linked protein in the signaling process regarding development and dedifferentiation of the human (ES) cell line H9 is shown in **Figure 5**, in which a spontaneously differentiated colony of ESCs was restored (dedifferentiated) to its primordial state, upon specific antibody cross-linking and culturing for 1 day in Iscove's medium supplemented with 10% FBS.

#### **3.3 BD-PSCs are generated from unmanipulated steady-state PB**

A PB sample (30 mL) of healthy donors was collected after obtaining informed consent. Mononuclear cells (MNCs) were isolated after Ficoll centrifugation and activated at the membrane by antibody cross-linking using specific antibodies. **Figure 6** shows the steady growing new population of cells in time course modus from day 5 to 14, while the nonactivated PB cells, under identical culture conditions, show gradual deterioration of cell structure and function, which leads to disappearance and death of the majority of the cells from day 5 to 14 of culture time period.

#### **3.4 Expression of ESC markers on BD-PSCs**

As shown, membrane-to-nucleus signaling network initiates *via* PLCγ/PI3K/Akt/ mTor/PTEN a process of de-differentiation of blood cells that leads *via* generation of HSCs to PSCs. By means of immunofluorescence, we analyzed the expression of pluripotent markers that BD-PSCs share with ESCs.

Human PSCs are characterized by specific cell surface markers, such as the glycolipid antigens SSEA-3 and SSEA-4, as well as the glycoprotein antigens TRA-1-60 and TRA-1-81. Stage-specific embryonic antigen (SSEA-4) is a glycosphingolipid expressed in early human embryonic development and PSCs and acts as a mediator of cell adhesion as well as a modulator of signal transduction. The expression of human SSEA-4 decreases following differentiation of ESCs [18]. Glycoprotein antigens TRA-1-60 and TRA-1-81 are expressed in early human embryonic development and PSCs; they also mark cells of the inner cell mass of preimplantation embryos [19].

Activated PBMNCs were grown in MEF-conditioned media in Matrigel-coated cell culture dishes for 2 weeks and immunocytochemistry (ICC) analysis was performed using antibodies to SSEA-4, TRA-1-60, and TRA-1-81. As shown in **Figure 7**(**ae**), cell surface protein marker analysis demonstrated that BD-PSCs express the pluripotent markers SSEA-4 as well as TRA-1-60 and TRA-1-81.

Unlike ESCs and PSCs, BD-PSCs can grow in suspension in Iscove's medium supplemented with 10% FBS without any addition of cytokines or growth factors. Flow cytometry expression analysis of BD-PSCs grown in suspension revealed the expression of ESCs specific markers SSEA-4 and TRA-1-81 on BD-PSCs (**Figure 7**(**f**–**m**)).

#### **3.5 Expression of the factors that maintain pluripotency**

To further investigate the expression of pluripotency markers on BD-PSCs at the protein level, we extended the immunofluorescence analysis to the transcription

*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

#### **Figure 5.**

*Immunofluorescence analysis during redifferentiation process of spontaneously differentiated human ESCs initiated after membrane antibody cross-linking activation of human GPI-linked protein. A spontaneously differentiated ESCs colony was activated by antibody cross-linking incubated with TRA-1-81 and DAPI and micrographs were taken by confocal microscopy. (a): Brightfield micrograph of spontaneously differentiated ESC colony. (b): Nuclei stained with DAPI. (c): Brightfield/DAPI (merged). (d): Spontaneously differentiated ESC colony stained with DAPI. (e): Spontaneously differentiated ESC colony stained with antibody to TRA-1-81. (f): Spontaneously differentiated ESC colony stained with TRA-1-81/DAPI (merged). Scale bars 200 μm. Fluorescence images of restored (activated) pluripotency in spontaneously differentiated ESC colony. (g): Activated ESC colony stained with DAPI. (h): Activated ESC colony stained with antibody to TRA-1-81. (i): Activated ESC colony stained with TRA-1-81/DAPI (merged). Scale bars 100 μm.*

factors Nanog, Sox2, and Oct3/4 that reside at the core of pluripotency network, where they can regulate their own expression and interact with a number of other pluripotency factors. Nanog, also called a pluripotency master molecule, is a

#### **Figure 6.**

*Reprogramming of PBMNCs after activation. PBMNCs isolated by Ficoll gradient centrifugation were activated by specific antibody cross-linking and cultured in Iscove's medium supplemented with 10% FBS. The brightfield images were taken at D5, 8, and 14. Nonactivated PBMNCs were used as controls. Scale bars 100 μm.*

unique homeobox transcription factor that is critical in regulating the cell fate of the pluripotent inner cell mass during embryonic development maintaining the pluripotency and blocking the differentiation of PSCs [20]. Activated preparations of PBMNCs were grown in suspension in a time-course manner from day 3 to 14. The newly generated cells were plated on Poly-L-lysine coated glass coverslips and ICC was performed using appropriate antibodies for specific pluripotency markers on BD-PSCs.

Expression of TRA-1-60, Sox2, Nanog, and Oct3/4 was induced following day 3, showing a rising trend from day 8 and had been completed at day 14.

Immunofluorescence analysis of the newly generated cells at different time points showed the gradual enhancement of the expression of the pluripotency markers on activated PBMNCs cultures. In contrast, nonactivated PBMNCs cultures showed no expression of these markers. These data are presented in **Figure 2**(**ad**).

Karyotype analysis was conducted on BD-PSCs by a qualified service provider (CELL Line Service Heidelberg, Germany) using standard G-banding methods as described elsewhere [21]. Analyses showed that BD-PSCs maintained a normal karyotype (data not shown).

*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

#### **Figure 7.**

*BD-PSCs express PSC markers. (ae): Activated PBMNCs were grown in MEF-conditioned media on Matrigelcoated culture dishes. Immunofluorescence analysis was performed with characteristic pluripotent markers, including SSEA-4,TRA-1-81, and TRA-1-60. Immunofluorescence images were taken by confocal microscopy. DAPI was used to stain nuclei. Generation and culture of BD-PSCs in suspension. (f-m): Activated PBMNCs cultures were grown in suspension in Iscove's medium supplemented with 10% FBS for 16 days, and flow cytometry analysis for pluripotency marker was performed using antibodies to SSEA-4 and TRA-1-8. Percentages were determined relative to appropriate isotype control.*

#### **3.6 Ultrastructural studies**

Various blood donors provided blood samples for this study showed no differences among them in terms of cell morphology or ultrastructure. *In vitro* cell culture studies, after 1 day of dedifferentiation (D1), upon activation, resulted in the observation of a large cell population comprised of approximately 60% of agranular mononuclear cells and 40% of granular mononuclear cells (**Figure 8AC**). Granular cells displaying deeply invaginated nuclei, scarce cytoplasm, and abundant primary and secondary granules disappeared from the culture after 5 days. Furthermore, red blood cells and platelets were detected. The population of interest for this study are agranular cells, morphologically characterized by small size and a nucleus with condensed chromatin (**Figure 8BC**). These cells showed rounded shapes with slender filipodia-like cytoplasmic expansions. Their cytoplasm was electron-dense and contained few organelles, highlighting the presence of small dictiosomes, some mitochondria, and rough endoplasmic reticulum cisterns. The nucleus occasionally showed deep invaginations with the emphasis on large nucleoli and condensed chromatin, preferentially associated with the nuclear membrane. On day 5 (D5), most cells in the culture were classified as agranulocytes, which exhibited a clear decrease in the number of cytoplasmic organelles (**Figure 8D**). The nuclear/cytoplasmic ratio of these cells was high,

#### **Figure 8.**

*Blood cells progressively showed morphological de-differentiation features upon activation. A, B: One day (D1) after activation in appropriate culture medium, typical blood cell types could be detected in the samples. Red blood cells, platelets, granulocytes, and agranulocytes could be found in the sample. CE: After 5 days (D5), most cells could be classified as agranulocytes, showing some nondifferentiation characteristics, such as scarce cytoplasm, low number of organelles, and the presence of annulate lamellae (E, arrowhead). F-H: The homogeneity of the culture peaked 8 days upon activation (D8), showing similar ultrastructural characteristics as defined in D5. This state was maintained in D12 (not shown). IK: After 16 days (D16), cells slowly started to show some differentiation signs, such as lipid drops (K, arrow). A small population of cells started to change their morphology and appeared as bigger elongated cells (L), with increased cytoplasm and more organelles. Photomicrographs in the first column correspond to toluidine blue-stained semithin (1.5 μm) sections. The center and right columns are transmission electron microscopy (TEM) images. Scale bars: A, C, F, I, K = 10 μm; B, D, G, H, J = 2 μm; E = 200 nm.*

similar to ESCs (**Figure 8DE**). We could observe small dictiosomes, some mitochondria, and rough endoplasmic reticulum cisterns**.** On the other side, polyribosomes and filamentous structures were abundant. Moreover, some of these cells occasionally presented annulate lamellae (**Figure 8EF**). Regarding the nucleus, we observed nuclear invaginations with abundant condensed chromatin (heterochromatin). On the cell surface, some short cytoplasmic expansions were appreciated (**Figure 8E**). At this stage of the cellular culture, we could occasionally see another subpopulation of large cells as well, containing a wide heterogeneity of cellular structures and highlighting the presence of lysosomes (**Figure 8D**). On day 16 (D16), reprogrammed cells underwent morphological changes as the culture medium was gradually changed to neuronal medium, showing signs of differentiation with the presence of lipid drops, bigger cytoplasm, and more organelles. Their shape changes to larger cells with elongated appearance resembling cells of neuroectodermal origin.

*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

#### **3.7 Redifferentiation of BD-PSCs to the cells belonging to different germ layers**

The capability of BD-PSCs to redifferentiate in neuroectodermal layer was demonstrated by growing these cells on laminin-ornithin coated plates in N2 medium to initiate the differentiation toward neuronal cells and further cultivation in neuronal differentiation media containing B27 supplement BDNF and GDNF as described in Material and Methods. The conditions described above enable for redifferentiation of BD-PSCs toward various neuronal lineages. Depicted are different populations of cells expressing the specific markers (**Figure 3**). Specific neuronal lineages from BD-PSCs can be generated by slightly modifying time and culture conditions. In neuronal differentiation culture time period from D8 to D30, we observed a clear decrease in the expression of neuroepithelial stem cell protein Nestin, which is a major intermediate filament (IF) protein of embryonic central nervous system also known as neuronal progenitor marker, while the expression of MAP2, a member of neuron-specific microtubule-associated protein family, neuronal nuclear antigen NeuN, a common neuron marker, and class III β-tubulin element of tubulin family, Tuj 1 a specific marker for human neurons, significantly increases during neuronal differentiation.

Glial fibrillary acidic protein GFAP is a type of IF expressed in various cells belonging to central nervous system, such as glial cells and astrocytes. These cells are mainly expressed in the central nervous system, such as brain and spinal cord, contributing to astrocytes-neuron interactions as well as cell–cell communication. Using antibodies to GFAP, we confirmed that such structures are recognized in newly generated BD-neuronal cells confirming the feature of BD-PSCs to redifferentiate to neuroectoderm [22].

Capacity BD-PSCs to redifferentiate into endoderm/hepatocytes was assessed by growing the cells in appropriate medium as described in Material and Methods. Following initial differentiation into endoderm in KSR/DMSO medium, as confirmed by ICC using antibodies to AFP and TTR, in the second phase by using hepatocytes maturation medium cells turned to mature hepatocytes like cells expressing their specific marker ALB and HNF4α, (**Figure 4**), recapitulating liver development *in vivo* [23].

The membrane activation of human glycoprotein ACA initiates a dedifferentiation process, consecutively generating more primitive cells until the final stage of this process is reached. BD-PSCs capable of redifferentiation into all three germ layers are the final product of this dedifferentiation process initiated by the membrane glycoprotein ACA [9–11] depicted in **Figure 9**.

#### **4. Discussion**

#### **4.1 Signaling**

Antibody cross-linking of a GPI-linked protein ACA initiates, *via* PLCγ/PI3K/Akt mTor/PTEN up-regulation of Wnt, Notch, c-Kit, and/or HoxB4 genes, among others. Signaling network linked to these genes induces dedifferentiation of blood progenitor cells leading to generation of BD-PSCs [9, 11]. Briefly, PI3K activation phosphorylates and activates Akt localizing it at the plasma membrane. Akt is a serine/threoninespecific protein kinase that plays a key role in multiple cellular processes like cell proliferation, transcription, and apoptosis [25]. The components of the PI3K/Akt Pathway, such as α, β, and γ p110 catalytic subunits, as well as subunits of 3-

#### **Figure 9.**

*Schematic presentation of dedifferentiation process that starts with human blood progenitor cells expressing CD34* via *HSCs, following side population (SP) cells mainly not expressing CD34, (low HLA) and ending up with the generation of BD-PSCs expressing pluripotency marker (SSEA-4) [24].*

phosphoinositide dependent kinase 1 (PDK1), which is the major transducer of IP3 Kinase, likewise the downstream effector proteins like glycogen kinase-3 (GSK-3), which plays a central role in the regulation of the stability and synthesis of proteins involved in the cell cycle entry; C-Raf, a serine/threonine kinase, whose main role in cells is the phosphorylation and activation of the MAP kinases MEK1 and MEK2, and the mTOR complex that controls translation and acts as a critical regulator of protein synthesis, are regulated in ACA-dependent manner.

Most importantly, a lipid-protein phosphatase PTEN, also called an anti-tumor agent, which is the natural inhibitor of PI3K/Akt signaling pathway that regulates p53 protein level and activity as well. PTEN works by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to phosphatidylinositol-4, 5-bisphosphat (PIP2), which limits Akt's ability to bind to the membrane, decreasing its activity. Deletion of the PTEN tumor-suppressor gene in adult hematopoietic stem cells (HSCs) leads to myeloproliferative diseases, and recent studies showed the inactivation of PTEN in human T-ALL cell lines as well as primary cells [26–28].

PTEN is a downstream target of phosphorylation at this specific route to pluripotency. Upregulation of PTEN indicates that a proliferative control of the

#### *Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

process that leads to generation of self-renewable BD-PSCs is tightly regulated in a GPI-linked protein-dependent manner. Our results indicate that this protein promotes the generation of self-renewing cells by activating PI3K/AKT pathway, but one of the best-conserved functions of AKT in promoting growth in this induced signaling cascade appears to be under the control of activated PTEN by preventing oncogenic outgrow.

Canonical Wnt/ß-catenin pathway is involved in the regulation of various functions like embryonic development, proliferation, survival, cell polarity, migration, and maintenance of somatic stem cells in many tissues, modulating a delicate balance between stemness and differentiation. Binding of Wnt proteins to their receptors inhibits the phosphorylation of β-catenin resulting in stabilization and accumulation of β-catenin in the cytosol and its nuclear translocation followed by transcriptional regulation of target genes [29]. Notch protein is a hetero-oligomer type of transmembrane receptor and the pathway linked to his protein is highly conserved and functionally involved in various processes from development to cell growth and cell death. Among them, the most important, together with other signaling pathways, such as Wnt, are the regulation of stem cell self-renewal, maintenance of homeostasis, cell– cell communication, regulation of cell-fate decision, neuronal function and development, and expansion of HSCs [30].

Tyrosine-protein kinase (c-Kit) is a receptor tyrosine kinase that belongs to the type III family of kinases. When the ligand binds to this receptor, the dimer is formed, which activates intrinsic kinase activity that initiates the phosphorylation of various signal-transducing molecules and propagates the signals within the cells.

C-Kit signaling is involved in various mechanisms, such as differentiation, cell survival, and proliferation. It is expressed in various cell types, most importantly in HSCs, and binding on its ligand SCF causes blood progenitor cells to grow [31].

Further downstream partner genes in the initiated signaling pathways, such as HOXB4 and BMI1, belong to the Homeobox and/or Polycomb group (PcG) family of genes involved in the development. HOXB4 gene encodes a nuclear protein with a homeobox DNA-binding domain. Ectopic expression of this protein expands HSCs and progenitor cells *in vivo* and *in vitro,* making it a potential candidate for therapeutic stem cell expansion [32]. BMI1, as a member of the PcG family of transcriptional repressors, is involved in the control of development by regulating cell growth and differentiation. It is also expressed in HSCs proven to be essential for generation of self-renewing HSCs [33].

Transforming growth factor beta (TGF-β) is a multifunctional cytokine that belongs to the transforming growth superfamily that includes endogenous growthinhibiting proteins [34]. Activation by ACA down-regulates TGF-β, which is one of the most potent inhibitors of HSC growth *in vitro.* One of the features of HSCs is their relative quiescence and given the strong inhibitory properties of TGF-β, it has been proposed to be the main regulator of quiescence *in vivo* [35]. Anti-apoptotic BCL-2 family proteins represent a family of evolutionary conserved cytoplasmic proteins that are known for their regulation of programmed cell death and survival. In response to intracellular damage, signals initiate the proteolytic cascade that disintegrates the cells. In our findings, these genes are down-regulated compared to unmanipulated PBMNCs, indicating that apoptosis represents an important regulatory factor in the maintenance of stem cells and is a part of the molecular mechanisms regulated by GPI-anchored membrane protein.

ACA signaling network *via* PLCγ/IP3K/Akt/mTOR/PTEN up-regulates the critical genes that are involved in the signaling pathways that regulate human development, such as NOTCH and WNT. Moreover, due to hierarchy among them, upregulation of these genes remains under the control of tumor suppressor gene PTEN, which is also upregulated in an ACA-specific manner. Apoptosis through downregulation of BCL-2 is an additional mechanism that regulates growth and proliferation. Finally, tumor suppressor gene P53 remains constant during reprogramming by ACA (data not shown).

Notably, the highest extent of upregulation of target genes is reached with c-Kit receptor tyrosine kinase [36], which is the gene critical for proliferation and survival of HSCs, indicative of a direct link that exists between these two proteins. C-Kit, a receptor tyrosine kinase type II, activates signaling through second messengers, such as cyclic adenosine monophosphate (cAMP), which are membrane-associated and diffuse from the plasma membrane into intermembrane space, where they can reach and regulate other membrane proteins. This reaction is probably the key to molecular mechanisms regulated by GPI-anchored membrane glycoprotein.

#### **4.2 Reprogramming by dedifferentiation**

IPSCs appear to represent the greatest promise for regenerative medicine without the ethical and immunological concerns incurred by the use of ESCs. They are pluripotent and have high replicative capability. Furthermore, iPSCs have the potential to generate all the tissues of the human body and provide researchers with patient- and disease-specific cells, which can recapitulate the disease *in vitro,* allowing for specific drug discovery. The iPSC technology provides an opportunity to generate cells with characteristics of ESCs, including pluripotency and potentially unlimited self-renewal.

Although methods have been improved from viral integration to integration-free, there are still challenges down the road to achieving their clinical application in humans.

The use of iPSCs in autologous cell-based therapy represents an ideal approach for regenerative medicine since the patients do not require long-term immunosuppressive drugs. The derivation of iPSCs over a decade ago has been raising high expectations and enthusiasm that iPSC technology can deliver autologous cell-based therapeutics to treat a high number of degenerative diseases, but actually, autologous therapy is related to the high cost and long period of time which should be spent in the manufacturing process that includes generation, characterization, differentiation into relevant cell types, scale up, and careful validation of the generated cell product. In order to reduce production time and costs, iPSCs therapies are moving toward allogeneic approaches by establishing clinical-grade iPSC banking [37, 38].

Banking of iPSCs from healthy donors would throw the iPSC reprogramming strategy once claimed as advantageous when compared to hESCs, while autologous at its beginning background. Therefore, the reprogramming strategies entirely free of DNA-based vectors could lead to solving the problems regarding genetic induced pluripotency.

#### **4.3 Blood-derived pluripotent stem cells**

Stem cell therapy is the ultimate goal of personalized medicine and individual care for many degenerative diseases, such as Alzheimer's disease, Parkinson, diabetes, and others. It has already been shown that human PB cells can be successfully reprogrammed into blood cells using the Yamanaka factors [39]. Blood is one of the most easily accessible sources of patient cells for reprogramming because there is no need to maintain cell cultures extensively prior to reprogramming experiments. Therefore, it is a potentially unlimited and safe source of cells.

#### *Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

Our own work showed recently that blood cells can be reprogrammed to PSCs without any genetic manipulation [9–11]. The present study shows that the signaling network activated by human GPI-linked protein ACA is sufficient to generate cells from circulating blood that is pluripotent, according to their morphology, pluripotent marker proteins, and differentiation potential. In fact, it is possible to reprogram adult progenitor cells that can be obtained from PB through protein activation, by means of antibody cross-linking, making them return to a similar state to that of ESCs.

Immunophenotyping of BD-PSCs by using antibodies to pluripotency markers by means of flow cytometry and immunofluorescence analysis, revealed the expression of SSEA-4, TRA-1-60, TRA-1-81, NANOG, SOX2, and OCT3/4, indicating that newly generated cells possess the properties of ES cells. Electron microscopy analysis showed the morphological changes during the culture time period from D1-D16. A scarce cytoplasm and decreased number of organelles indicate that undifferentiated characteristics appeared through culture time. Most importantly, the appearance of annulate lamellae, stacked sheets of membranes embedded with pore complexes which are frequently found in cells with high proliferative activity, such as oocytes, embryonic, and tumor cells [40], suggest that BD-PCSs correspond to an actively dividing cell population. In addition, when neuronal differentiation medium was added to the cell culture system the morphology of these cells changed to larger elongated cells with more organelles and increased cytoplasm, supporting the notion that they are able to redifferentiate [10].

Activation of a membrane protein ACA at the surface of blood progenitor cells by cross-linking at the membrane of blood cells with its specific antibody and analysis of the mode of how this signaling machinery regulates the expression of genes known to play a role in human development *via* specific protein phosphorylation as an important regulatory mechanism in the cellular processes related to its signaling competence showed its involvement in the processes that determine the cell type, its fate, and identity.

Our results confirm the previously published data that the initiation of GPI-linked protein ACA upon activation of PBMNCs described here is sufficient to induce signaling machinery that leads to generation of self-renewing PSCs. Moreover, it ensures the maintenance of pluripotency in ESCs as well, indicating the involvement of this protein in pluripotency signaling network in humans.

Dedifferentiation process initiated upon membrane activation follows exactly the opposite way that is known for differentiation of PB progenitor cells. It is generally accepted that the proliferation capacity is higher by hematopoietic progenitor cells and declined by more primitive cells like HSCs and SP cells. The process initiated by ACA is due to activation of tumor suppressor genes, under strict proliferative control, leading to generation of BD-PSCs and explaining the lack of teratogenicity of these cells resulting in advantage for their application in cell tissue replacement.

BD-PSCs generated through dedifferentiation process are capable of redifferentiate into cells belonging to all three germ layers.

Most importantly, an antibody that acts at the surface of PB progenitor cells initiating membrane-to-nucleus signaling pathways may have numerous potential advantages regarding clinical safety for application of these cell products in regenerative medicine.

#### **5. Conclusions**

So far today, no iPSCs-based therapy is implemented into routine clinical use [41]. The hurdles regarding use of iPSCs in clinical practice are related primarily to genetic

instability of these cells that may cause mutations leading to tumor formation and cancer. Another safety concern, when it comes to their application in humans, is the presence of residual undifferentiated iPSCs, also linked to tumorigenicity [42]*.*

Allogenic approaches by establishing clinical-grade iPSC banking must consider the populations with heterogeneous genetic background, which might be very challenging [43]. Therefore, it is very important to further improve the current iPSCs technology to minimize the possible side effects and genetic and epigenetic differences between reprogrammed cells and donors. Human ES cells derived from blastocyst imply its destruction that cause serious ethical concern. In addition, these cells are by their nature nonautologous and may cause graft-versus-host disease. They are mostly used for studying early human development using currently available hES cell lines, but they also have limited potential in medicine due to restrictions related to ethical and immunological issues [44].

Great effort is made to assure safe clinical applications using stem cell therapies. The international stem cell banking initiative (ISCB) published guidelines for the development of pluripotent stem cell stocks for clinical applications [45]. Due to complex nature of the cells to be used for therapies in regenerative medicine compared to drug therapies, standard regulations must include purity of the cells, sterility, viability, genomic stability, specific gene expression profile, functional evaluation of reprogrammed, and differentiated cells, absence of infectious pathogens and tumorigenicity [46]. The greatest attention is taken to ensure the safety of transgenic cells when compared to genetically unmodified cells, even more so when it comes to the use of more modern technology like CRISPR/Cas9 for removing randomly inserted foreign genes into human genome during reprogramming process because there is the possibility of off target mutagenesis [47, 48].

Our results reveal insight into the molecular events regulating cellular reprogramming and indicate that pluripotency may be controlled *in vivo* through the binding of soluble ligand(s) to ACA-protein and initiating the cascade of already known and partly characterized signaling pathways. The process of reprogramming is short (1012 days), the source is easily accessible unmanipulated peripheral blood and no use of growth factors is necessary. BD-PSCs are autologous, capable of generating *in vitro* cell types of all three layers exhibiting neuronal, liver, or hematopoietic characteristics [9–11]. Due to their differentiation capacity, they could be potentially utilized to regenerate any type of tissue, and thus treat neurological and immune disorders, as well as injuries to critical organs, such as the heart and brain. Moreover, due to lack of teratogenicity, BD-PSCs can be used *in situ* without the necessity to be differentiated before their application [11] as is also shown in the wound healing experiment currently ongoing in our laboratory. Due to tight proliferation control, BD-PSCs do not form cell lines and therefore must be freshly prepared.

It can be expected that the standard regulations for the use of BD-PSCs would be similar to that of genetically unmodified cells implying fewer hurdles compared to iPSCs ensuring a fast and safe application of these cells in routine clinical practice.

The potential application of BD-PSCs in regenerative stem cell therapies is innovative and promising. Additional studies are underway in order to determine *in vivo* therapeutic potential and to ensure a safe platform for translation of basic research to new clinical therapies.

Our report provides a practical and efficient way to generate patient-specific PSCs. This will also be valuable for the generation of clinical-grade PSCs for future therapeutic applications so that the possibility to develop a truly personalized medicine becomes more realistic.

*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

#### **Acknowledgements**

Dedicated to the memory of Dr. Rainer Saffrich.

The authors express unending gratitude to Professor Gennady T. Sukhikh for his encouragement and permanent support. We gratefully acknowledge the technical assistance provided by Patricia García-Tárraga in Valencia and by John and Oksana Greenacre in Heidelberg.

This work was supported by the Prometeo Grant for Excellence Research Groups [PROMETEO/2019/075] given to José Manuel García-Verdugo; and private funding from ACA CELL Biotech GmbH Heidelberg, Germany.

#### **Conflict of interest**

The corresponding author declares that she is a patent holder related to Novel Human GPI-linked Protein ACA, she also cofounded and works in ACA CELL Biotech GmbH. The other authors declare that there is no conflict of interest.

#### **Abbreviations**



*Membrane-to-Nucleus Signaling in Human Blood Progenitor Cells Reveals an Efficient GM… DOI: http://dx.doi.org/10.5772/intechopen.108950*

### **Author details**

Zorica A Becker-Kojić<sup>1</sup> \*, José Manuel García-Verdugo<sup>2</sup> , Anne-Kathrin Schott<sup>1</sup> , Vicente Herranz-Pérez<sup>2</sup> , Ivan Zipančić<sup>3</sup> and Vicente Hernández-Rabaza<sup>3</sup>

1 ACA CELL Biotech GmbH, Heidelberg, Germany

2 Laboratory of Comparative Neurobiology, Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, Paterna, Spain

3 Department of Biomedical Sciences, Cardenal Herrera-CEU University, Valencia, Spain

\*Address all correspondence to: z.becker-kojic@aca-cell.de

© 2022 The Author(s). Licensee IntechOpen. 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.

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#### **Chapter 2**

## Activation and Metabolic Shifting: An Essential Process to Mesenchymal Stromal Cells Function

*Patricia Semedo-Kuriki, Gabriel Pereira, Danilo Cândido de Almeida and Niels Olsen Saraiva Camara*

#### **Abstract**

To elucidate the basal metabolism of Mesenchymal Stromal Cells (MSCs), as well as knowing how they are activated, can bring important clues to a successful cellbased therapy. Naive MSCs, in their niche, mainly keep the local homeostasis and the pool of tissue stem cells. Once activated, by an injury, MSCs' response leads to a lot of physiological differences in its metabolism that are responsible for its healing process. Since endogenous MSC seems to be ineffective in pathologic and aging conditions, cell-based therapy using MSC is focused on administration of exogenous MSC in patients to exert its healing functions. From quiescent to activated state, this "Metabolic Shifting" of MSC interferes directly in its secretion and cellular-derived particle generation. We will address here the differences between the MSCs activation phases and how they can modify the MSCs metabolism and its function. Moreover, understanding MSC in their niche and its damped function in pathologic and aging processes can improve stem cell-based therapies.

**Keywords:** mesenchymal stromal cell, metabolism, MSC activation, MSC niche, cell therapy

#### **1. Introduction**

Stem cells research has brought great insight in regenerative medicine. Currently, over 1700 clinical trials are registered at Clinicaltrials.gov (clinicaltrials.gov "mesenchymal stem cell OR mesenchymal stromal cell", August 2022), with ten approved MSC therapies worldwide [1].

Besides efforts to promote standardization of procedures and classifications for MSCs, the translation of promising preclinical results to human clinical trials has not matched full desired effects. Such variability may come from differences among species or source-tissues of MSCs in both *in vivo* or *in vitro* preclinical studies [1].

Initially, MSCs therapeutic potential was associated with engraftment of MSCs into tissues and to a contact-dependent cell communication. Advances in the field

now confirm that paracrine mechanisms are the primary effector of MSCs for tissue regeneration, angiogenesis and modulatory effects on inflammation, apoptosis and fibrosis. These effects may be achieved by the secretion of biologically active molecules by MSC, such as cytokines and chemokines, growth factors, extracellular matrix and extracellular vesicles. Indeed, the use of secreted factors in the medicine and research fields lead to a cell-free approach, which can overcome major adversities found in the use of allogeneic or even autologous MSCs therapy [2, 3].

In fact, for some regenerative approaches, no additional cell is necessary, and nowadays, beyond adult stem cells; there are other stem cells-based products such as: (i) conditioned medium, (ii) concentrated supernatant, (iii) lyophilized secretome, (iv) cellular particles (i.e. exosomes, microvesicles, small body particles), and (v) small regulatory molecules (i.e. lncRNAs, microRNAs, ceRNAs, circRNAs). All together, these approaches are new fields to be explored in stem cell technologies and cellular-based therapies [4, 5].

Several questions can be formulated regarding the MSC paracrine mechanism of repair: How did MSC become so secretive? How is MSC activated to secrete those molecules responsible for its regenerative mechanism? How is MSC in its quiescent state?

Currently, the metabolism and cell activation of MSC has been the focus of study of many researchers worldwide. Recent reports have provided evidence that stem cells have a metabolic/activation signature which is distinct and specific to each tissue to maintain the homeostasis. Regarding therapy, the choice of the MSC origin and thus how it is MSC activated directly regulates therapy performance, since MSC metabolism is crucial to the paracrine effect. MSC activation is also controlled by the microenvironment, for instance, a metabolically activated MSC can interact with other cells in their niches and they are able to sense and to adapt to dietary changes, exercise, aging, epigenetics changes etc. [5, 6].

Thus, in this chapter, we will attempt to elucidate the importance of MSCs activation/metabolism in its therapeutic function. More specifically, we will describe here the impact of MSCs activation in its metabolism and function. In addition, we will discuss how this "Metabolic/activation Shifting" can interfere directly in the MSC secretory function and in its cellular-derived particle generation. Moreover, stem cell dysfunction and disabilities will also be discussed. Hence, understanding these basic steps about naïve and activated MSCs should improve the establishment of new stem cell-based therapies and other associated approaches around MSC technologies, expanding its use and resources for future implementation as a translational and effective therapy.

#### **2. Mesenchymal stromal cells definitions**

Isolated from a huge number of tissues, MSC has been used in several clinicals trials, despite its basic studies are still ongoing. Since MSC therapy leads to amelioration of pathologic state, it causes a frenesi in clinicals trials and cell-based therapies. This frenesi creates inconsistencies, for instance at MSC's characterization, nomenclature, culture parameters, etc. Lacking the principle of reproductibile and quality control, several works and clinical trials have still been done improperly [7, 8]. How are the MSCs defined? A brief historic event of its discovery may help to elucidate it.

Described in the early 1970 by Friedenstein and colleagues [9], they observed that bone marrow cells, in a cell culture condition, generated attached cells in culture *Activation and Metabolic Shifting: An Essential Process to Mesenchymal Stromal Cells Function DOI: http://dx.doi.org/10.5772/intechopen.109273*

plates. These cells showed fibroblastic shape that started growing in this condition. Moreover, they observed that these cells induce osteogenesis in an experimental model. In 1990, Caplan first used the name Mesenchymal Stem Cell to describe these cells with differentiation properties, depending on local (niche concept) and genetics factors [10]. In 1999, Pittenger studies had flourished in the MSC area. Pittenger et al. showed the isolation of MSC from human bone marrow, listing some criteria to define them, such as (i) adherent culture cells and (ii) differentiation capabilities under specific stimulation [11].

Then, the mess comes … everybody, everywhere, every tissue could generate MSC. However each culture condition was different from each other, with different techniques of isolation, with different patterns of characterization [12]. And this confusion is used indiscriminately by some clinicals to sell cell therapies treatment [13]. Placing order to it, in 2006, the International Society for Cellular Therapy (ISCT) defined the minimal criteria to MSC [14]. Those criteria were upgraded in 2019 [15], where ISCT defines:


The most cited MSCs are the Bone marrow-MSC (BM-MSC) and adipose tissue-MSC (AT-MSC). Other sites are also well known such as Umbilical cord MSC (UC-MSC) and Wharton's Jelly MSC (WJ-MSC). Each one has different characterization patterns but all have paracrine effects and immunomodulatory properties, however with different amounts of molecules secreted by each one. For instance, AT-MSC shows a higher pro-angiogenic pattern than BM-MSC and WJ-MSC. WJ-MSC shows an increased expression of inflammatory cytokines and chemokines than BM and AT-MSC [16]. MSC secretome not only includes molecules secreted by them but also extracellular vesicles (EV) productions that reflect in its internal content the same pattern of MSC from origin. For clinicals trials, it's a quite exciting way to treat with MSC without MSC *per se*. In this sense, there are several clinicals trials ongoing using EV from MSC.

But again, regarding the use of EV at clinicals trials, the cell culture protocol standardization, as well as detailed description of isolating methods, requires more attention [13, 17]. For immune regulation capabilities of MSC, ISCT describes assays to standardize the protocols for clinicals trials. Several researchers and groups summarize three assays that must be followed by all clinicals trials: real time PCR of selected gene products, immunophenotyping assays by flow cytometry and secretome assays [18]. In addition, clinicals parameters such as time to administer MSC, dosage,

delivery, homing, fresh or frozen MSC, autologous or allogeneic transplantation, etc., all these can generate different responses for patient's' treatment. Thus, more quality control to clinicals trials must be done [19].

#### **3. Healing mechanisms of mesenchymal stromal cells**

The physiological and clinical properties of MSCs include not only differentiation potential but also maintenance of tissue homeostasis, immunomodulation, secretion of particles and molecules, and of course, tissue regeneration/healing [20].

Initially, it was believed that MSCs could act directly in the tissue repair and regeneration through migration and engraftment to the site of injury, differentiating into functional local cells and promoting regeneration to the damaged tissue. However, it is now understood that MSCs major effects are promoted largely through secretion of modulatory factors (paracrine activity) and less due to its tissue replacement [21].

In this sense, the ability of regeneration and healing of tissue depends on multiple factors. In the aspect of wound healing, for example, different cell types are involved, including platelets, macrophages, fibroblasts and MSCs. Thus, the balance among proinflammatory M1 macrophages, transformation to anti-inflammatory M2 macrophages and fibroblast extracellular matrix production are crucial to the process of healing. For instance, Adipose-tissue derived MSCs (AT-MSC), as well as its derived exosomes, have been reported to induce M2 macrophage phenotype, modulating the inflammatory process and to enhance the proliferation and migration of fibroblast, contributing to the wound healing process [22].

MSC paracrine signaling can act as anti-inflammatory, anti-fibrotic and proangiogenic effects leading to tissue healing and regeneration. In this case, MSCs have been shown to promote accelerated peptic ulcer healing leading to higher proliferative cells population over the ulcer margin, by increasing vascularity in the site of lesion with increased expression of interleukin-10, an anti-inflammatory cytokine, resulting in ulcer healing, such as reepithelization, angiogenesis, and reduced inflammation [23].

Furthermore, this triad process of healing of MSCs based on its anti-inflammatory, anti-fibrotic and pro-angiogenic effects was observed in many studies confirming the pleiotropic effect of these cells during therapeutic process. Briefly, the use of MSCs in ischemic diseases have also been explored. In this scenario, transplantation of MSCs induced angiogenesis with reported differentiation of MSCs into endothelial cells to compose new blood vessels in the infarcted cardiac tissue. Classically, MSCs have been used in Graft versus host disease (GVHD) and autoimmune diseases and have presented decreasing of global inflammatory process with modulation of inflammatory cells (lymphocytes, NK cells, macrophages) and expanded survival or reduced the use of corticoids by transplanted patients [24]. MSCs paracrine secretion of extracellular vesicles or soluble factors may also contribute to angiogenic or immunomodulatory activity in the ischemic heart and brain, even leading to activation of endogenous cardiac stem cells responsible for myocardial regeneration [25].

#### **4. Mesenchymal stromal cells at niche**

Cellular turnover varies immensely among the human body tissues. Skin and gut epithelia are replenished every 3–5 days. On the other hand, a neuron's lifespan is

huge [26]. This turnover is regulated by stem cells in adult tissues. How these stem cells are *in vivo* and how they keep the homeostasis of tissue is a challenging subject. Despite this, it is known that the MSC and the stem cells live together in specific areas called niches.

A niche is an area of a tissue that provides a specific microenvironment, in which stem cells are present in an undifferentiated, quiescent and self-renewable state. The niche is composed of: (1) a population of stem cells; (2) a population of stromal cells, mainly MSC; (3) an extracellular matrix in which stem cells, stromal cells and molecular cues are embedded; (4) blood vessels support; and (5) neural inputs [27].

The niche is the place where humoral, neuronal, local (paracrine), positional (physical) and metabolic cues interact with each other to regulate stem cell fate [28]. MSC also lives in this environment and has a crucial role in the niche. The cross-talk between stem cells and MSC is very important to both cells. Cells of the niche, mainly MSC, interact with the stem cells to maintain them or promote their differentiation. And tissue homeostasis depends on this balance [27, 29].

The role of MSC in the niche has been studied in recent years. MSC may be the cell that sustains the niche and the cell that keeps the tissue stem cell in the quiescent state. MSC can secrete soluble factors, produce extracellular matrices due to its sensing of the extracellular signals and thus regulate stem cell fate [30].

MSC can be found in every vascularized tissue. Several studies have demonstrated a population of MSC in different tissues, mainly the ones highly vascularized. Following the minimal criteria defined by ISCT, several studies have demonstrated that MSC are the perivascular cells in tissues. Crisan et al. have isolated cells phenotypically positive for pericytes markers (CD146, NG2 and PDGF-Rβ2) from placenta, adipose tissue, pancreas and skeletal muscle and when cultured these cells shown MSC patterns [31]. Not only microvascular pericytes have been described to be the MSC origin cell but also adventitial perivascular cells [32].

Are the *in vitro* MSC the *in vivo* pericytes? Some authors state that MSCs are cell culture artifacts [33]. They disagreed that MSCs are pericyte because since our body is extremely vascularized thus the MSC population should be huge enough to guarantee efficient repair after injury. However our regeneration is not so efficient. In this sense, it has been shown that pericytes *in vitro* generate a cell similar to MSC, but *in vivo* all the pericyte functions may not release them to act as MSC [17]. In addition, if all MSC should be pericytes *in vivo*, then all MSC *in vitro* should be the same, and they are not. MSC from adipose tissue differs phenotypically (CD markers, secretion of molecules, etc.) from bone marrow-MSC, that differs from Wharton Jelly MSC, that differs from cord blood MSC etc. However, there is a hypothesis of an imprinting of tissue source on MSC properties that make tissue-MSC differs from each other [34]. All these opposite points of view show that the search for MSC *in vivo* continues.

Of note, all the knowledge on the MSC field achieved until now is obtained from cultured cells, expanded ex-vivo. In addition, in a plastic dish, MSC is not a pure population. The isolation methods and expansion in culture conditions did not exclude other cells from rising together. They are a heterogenous population in these conditions. Single cell RNA sequencing studies demonstrate that MSCs are heterogeneous and moreover MSC from different sites differs from each other [34–36].

Since most clinicals trials have been using ex-vivo expanded MSC and showing mild positive results *in vivo*, some researchers claim that the stimulation of the niche and their endogenous MSC should be a better option than administered exogenous MSC [30, 37, 38]. Thus, knowing how a niche works and how to properly stimulate it may result in better clinicals outcomes.

Hypoxic areas in the niches are common. At the bone marrow niche, the concentration of O2 is near 3%. Indeed, tissue O2 concentration may vary from 1 to 5% [39]. Several studies in rodents models as well as with human BM-MSC have demonstrated that a hypoxic condition increased osteogenic capabilities [40], increase the expression of pro-angiogenic factors [41], enhance MSC immunosuppression profile [42], maintain genomic stability [43], etc.

Since hypoxia has a huge effect on MSC metabolism, it is clear that energy metabolism can also be linked to MSC cross-talk to stem cells or its stemness. Several works have been studying the energy metabolic process at MSC. The homeostasis state of MSC can be regulated by metabolic signals leading to its stemness of MSC as described by Sun et al. [44]. They show that low levels of sodium lactate, upregulation of glycolysis, both induced by lysine demethylase 6B (KDM6B), can maintain MSC stemness. Indeed, energy metabolism is extremely important in the activation/ differentiation of MSC [45]. At the pathological stage, glucose, fatty acid, and amino acid metabolism are altered at MSC. If those pathways could be restored, tissular homeostasis can also be restored [46].

Extracellular matrices (ECM) can also be regulated by MSC. Beyond the structural scaffold, ECM is an acellular 3D structure that is in close contact with the cells. ECM is composed of several proteins (mainly collagen and elastin), glycosaminoglycans and proteoglycans. ECM participates in cell adhesion and in signaling through mimicking several receptors. In addition, mechanical patterns of ECM can also interfere in cell response, such as stiffness [47, 48]. During injury, ECM can be remodeled. Stromal cells, including MSC, secrete more ECM to reconstruction, helping other cells to migrate to this injury site. We will exploit it below regarding MSC secretome.

#### **5. Metabolically activated mesenchymal stromal cells**

#### **5.1 The MSC secretome**

The MSC secretome is composed of a soluble fraction of bioactive molecules (cytokines, chemokines and growth factors) and particles (extracellular vesicles and exosomes, responsible for the delivery of microRNAs and proteins) with several regulatory effects such as (1) anti-inflammatory; (2) pro-angiogenic; (3) stimulation of endogenous progenitor cells; (4) anti-apoptotic; (5) anti-fibrotic; and (6) anti-oxidant [49]. In addition, there is secretion of extracellular vesicles (exosomes, microvesicles and apoptotic bodies). Inside these vesicles, there are a pool of active molecules (enzymes, receptors, cytokines, chemokines, miRNA, DNA) that can perform the same function of its mother cells (See **Figure 1**) [50, 51].

The whole MSC secretome, which is composed of proteins, nucleic acids, lipids, carbohydrates and extracellular vesicles can also be obtained from MSC-derived conditioned medium (MSC-CM). The soluble component of the secretome and their extracellular vesicles may be then separated with the use of specific methodologies as centrifugation, filtration and chromatography [2]. MSC-CM and extracellular vesicles are enriched with various regulatory components, including transforming factor-β (TGF- β), hepatic growth factor (HGF), indoleamine 2,3-dioxygenase-1 (IDO-1), prostaglandin E2 (PGE2), interleukin (IL)-10, IL-1 receptor agonist (IL-1Ra) and others. Thus, the exposure of different cells to MSC-CM or extracellular vesicles induces different responses depending on the secreted factor available [52].

*Activation and Metabolic Shifting: An Essential Process to Mesenchymal Stromal Cells Function DOI: http://dx.doi.org/10.5772/intechopen.109273*

#### **Figure 1.**

 *The MSC secretome. Different molecules are secreted by MSCs. Some of them may induce modulation of immune response, by the expression of cytokines and chemokines to act as anti-inflammatory. It mainly occurs by induction of lymphocytes T helper 2 (TH2) and T regulatory cells (Treg). Molecules secreted by MSC can also be proangiogenic, anti-apoptotic, antioxidant and anti-fibrotic. Furthermore, MSCs secretome promotes the stimulation of other stem cells at niche. Extracellular vesicles, also considered part of MSC secretome, contain several molecules and RNA (miRNA, lnRNA) and proteins that may act over other cells through interaction with surface receptors or entering the contact with neighboring cells. IL, interleukin; TGF- β, transforming factor-β; COX-2, Cyclooxygenase-2; GSH, glutathione; GSSG, glutathione disulfide; miRNA, MicroRNA; lnRNA, long noncoding RNA; Th2, T helper 2; Treg, regulatory T cells; TLR2, toll-like receptor 2; Nrf2, nuclear factor erythroid 2-related factor 2; and Mox, oxidized phospholipids-activated macrophages phenotype. Created with BioRender.com .* 

 As appointed by Filidou et al., the anti-inflammatory, anti-fibrotic and tissue regeneration properties of MSC-CM promote *in vivo* and *in vitro* beneficial effects on different disease models that, in general, damages the tissues. Specifically, the use of AdMSC-CM resulted in reduced expression of inflammatory chemokines and cytokines in human pulmonary subepithelial myofibroblasts in response to exposure to IL-1α and Tumor Necrosis Factor-α (TNF-α) and also to TGF-β-induced fibrotic responses in these cells. In addition, the authors showed a reduction in chemotaxis (CCL and CXCL), inflammatory (IL-1α) and fibrotic (collagen Type III) molecules mRNA and protein expression by CM derived from human AT-MSC [ 53 ]. Furthermore, an experimental model of preeclampsia induced by bacterial lipopolysaccharide (LPS) showed that human placenta-derived MSC-CM reduced expression levels of TNF-α and IL-6 in the mice placenta, while also reducing the expression of the anti-angiogenic factor sFlt-1 [ 54 ].

#### **5.2 MSCs extracellular vesicles**

 The use of MSC of extracellular vesicles (MSC-EVs) has attracted attention for its ability to promote beneficial effects even when MSC itself is not present [ 55 ]. MSC biological characteristics may compromise its use as a therapeutic agent. MSCs proliferation decreases over culture passages, studies report concerns about increased tumorigenicity and the uncertainty of MSCs fate after venous injection calls attention to weak points of such therapeutic strategy [ 56 ].

MSC-EVs are classified according to their size, which ranges from apoptotic bodies (> 1000 nm), to microvesicles (100–1000 nm) and exosomes (30–200) [57]. Up to date, 45 MSC-EVs clinical trials are registered in Clinicaltrials.gov [clinicaltrials.gov "(mesenchymal stromal cells OR mesenchymal stem cells) AND (extracellular vesicle OR exosome OR microvesicle)", October 2022] of which 5 studies are currently at phase 3, including therapeutic approaches to rhinitis pigmentosa (NCT05413148), SARS-CoV-2 infection and acute respiratory distress syndrome (NCT05216562, NCT05354141), diabetes mellitus type 1 (NCT02138331) and stroke (NCT01716481).

The MSC-EVs content vary depending on the derived cell, microenvironment and physiological conditions, thus can be modulated by preconditioning methods, but are known to contain molecules such as messenger RNA, microRNAs, others regulatory RNAs (i.e., lncRNAs, microRNAs, ceRNAs, and circRNAs), enzymes, receptors, cytokines, chemokines and growth factors. Once released to the extracellular environment from the donor cell, MSC-EVs can be internalized by another cell via endocytosis or trigger responses through receptor-ligand interaction acting as a paracrine and endocrine agent. Furthermore, these MSC-derived EVs are capable of homing to injured tissue, having immunosuppressive effects or others similar to those promoted by transplanted MSCs [57].

MSC-EVs can be used in almost all therapy conditions that native MSCs are used or predicted to be; for instance, the MSC-derived exosomes were utilized in wound healing and was verified the promotion of collagen synthesis and proliferation and migration of fibroblasts and keratinocytes, important cells in the mechanisms of wound regeneration. Furthermore, it was detected that these effects are, greatly in part, promoted by microRNA in the exosomes. The therapeutic effects of microRNA derived from MSC-exosomes was widely reported in several studies showing benefits in the treatment of chronic skin ulcers, bone repair, promoting the immunomodulation in favor of inflammation resolution, improving angiogenesis, neurogenesis, macrophage polarization and limiting cardiac fibroblast proliferation, and improving tissue function after ischemia-reperfusion injury [55].

Using AT-MSC-derived exosomes Heo and Kim [58] reported a reduction in the gene expression of pro-inflammatory molecules as TNF-α, IL-6 and IL-8 which were induced by LPS in the THP-1 cell line, while the expression levels of antiinflammatory CD163, ARG1, CD206, TGF-β1 and IL-10 were shown to be increased in the LPS + exosomes group. The treatment of human umbilical vein endothelial cells (HUVECs) with AT-MSC-derived exosomes increased the proliferation of HUVECs and gene expression level of pro-angiogenic genes like angiopoietin1 and flk1, while reducing the expression of those with detrimental vascular function as vasohibin-1 and thrombospondin-1. Remarkably, the expression of miR-132 and miR-146a were found increased in exosome-treated HUVECs, and these microRNAs bound to the anti-angiogenic genes thrombospondin-1 and vasohibin-1, respectively [58].

Furthermore, a study aiming to elucidate the role of MSC-EVs in mitochondrial damage showed a reversion of mitochondrial DNA deletion to the treated group that was not observed in injured renal tubular cells. Utilizing an *in vivo* model of acute kidney injury, the authors observed the same effect through up-regulation of mitochondrial factor A pathway activity. These findings suggest that MSC-EVs therapeutic effects can also be related to improvement of mitochondrial function in diverse diseases in addition to its role as anti-inflammatory, antioxidant and anti-apoptotic as observed in many other injury models [59].

Finally, use of MSC-EV are promisor therapies that comprehends the major effects attributed to MSC secretome, promoting desired improvements in regeneration and immunomodulation as that offered by paracrine effects credited to MSCs.

#### **5.3 Activation signaling and pre-conditioning**

The paracrine effect of MSC is highly dependent on the microenvironment around MSCs. The MSCs have some sensors receptors (i.e., TLRs, AhRs, TNFRs, and IFNRs) which act as an "antenna" that captures external signals that drive a special cellular effect. In contrast, in the absence of stimuli the MSCs show little to no expression of molecules responsible for their function, for instance, the immunomodulatory profile, such as the expression of human leukocyte antigen (HLA)-I and intercellular adhesion molecule-1 (ICAM-1).

The production of molecules from MSC secretome can be stimulated by the presence of inflammatory components that induce an immunomodulatory phenotype on MSCs [60]. The MSCs preconditioning with inflammatory factors such as IL-1β and interferon gamma (IFN-γ) result in augmented production of modulatory components by MSCs which can influence and regulate other cell types, such as macrophages, to acquire a regulatory phenotype [61]. Hence, exposure of MSCs to an inflammatory environment, containing for example IFN-γ and TNF-α cytokines, induces MSCs to start the production of specific molecules which will play a role as immunoregulators [62].

TNF-α is one of the first secreted cytokines during an inflammatory event. TNF-α binds to two distinct receptors, TNFR1 and TNFR2. While TNFR1 is expressed ubiquitously, few cellular populations express TNFR2, including immune cells and MSCs. In MSCs, TNFα/TNFR2 interaction promotes the expression or secretion of pro-angiogenic and cytoprotective mediators. Beldi et al. investigated the role of TNFR2 in MSCs and found that in comparison to TNFR2+ wild type MSCs, MSCs lacking TNFR2 were less immunosuppressive to CD4 and CD8 T cells when reducing cellular proliferation and cytokines production in T cells. Furthermore, while TNF-α stimuli did not result in increased expression of early HLA-I, MSC exposure to IFN-γ increased expression of HLA-I, an indicator of MSC activation [63].

Regarding the MSCs-EV, preconditioning may also be expected to happen. In fact, cultures of PBMCs in presence of MSC-derived exosomes preconditioned with TNF-α and IFN-γ, resulted in cytokines shifting: 34 inflammatory cytokines and chemokines were found to be downregulated and several anti-inflammatory, as IL-10, were upregulated. Moreover, preconditioning of MSC-exosomes with atorvastatin enhanced angiogenesis when compared to non-pretreated MSCs in myocardial infarction injury; and also TNF-α preconditioning of adipose tissue MSCs promoted higher osteoblast differentiation upon exosome treatment [64, 65].

Although showing interesting results during preclinical i*n vivo/in vitro* studies, the preconditioning of MSCs is still performed with human and non-human recombinant factors with lack of consistency at human clinical trials. To overcome this, the use of fresh human derived products can be an effective resource when we take in mind the use of preconditioning on the clinical scale. Thus, platelets or platelet-rich plasma have been proposed as a beneficial enhancer to therapeutic properties of MSCs. These platelets or platelet-rich plasma medium stimulates proliferation of MSCs and offer protection against oxidative stress, mainly due to the release of growth factors that exerts beneficial effects on MSCs. Further, the transfer of platelets mitochondria

to MSCs stimulates wound-healing activity. And, the incubation of MSCs with full functional platelets, but not with dysfunctional mitochondria platelets, resulted in increased expression of pro-angiogenic genes [66].

Moreover, other similar approaches aiming to control extrinsic factors in MSCs modulation are available. Considering these aspects, some MSCs variability to its activity is found in response to (i) source or location, that is, Bone marrow-derived MSCs or Adipose tissue-derived MSCs, (ii) passage number in culture, and (iii) oxygen concentration and presence of different compounds in the environment, such as pharmacological agents. These extrinsic factors are useful methods of preconditioning MSCs and can be used to improve its therapeutic potential regulating the secretory MSCs profile. These effects can be reached using the hypoxic environment of cell culture, inflammatory cytokines, pharmacological compounds, and 3D cell culture models [60].

Finally, an interesting cell culture method of 3-dimensional culture can be used as a preconditioning factor as well. In this culture method, the physiological conditions seen as in the *in vivo* cell environment are replicated, as the spheroid 3D culture promotes a physiological-like environment, like those found in MSCs niche. In this spheroid culture, internal cells receive lower oxygen levels than MSCs in the surface of the 3D structure, creating a hypoxic environment. These spheroid cultured MSCs presented an increase in cytoprotective factors and enhanced proliferation, with increased immunomodulatory factors expression, along with elevated angiogenic, anti-fibrotic and anti-apoptotic activity [67].

#### **6. Mesenchymal stromal cell dysfunction**

Our knowledge on MSC is focused on how healthy MSC responds to an injury, by secreting several molecules, trying to rebuild the tissue homeostasis. At cellular therapy, healthy exogenous MSCs are administered to patients and in response to the injury, this exerts its regenerative role and helps heal the damage.

However, there are several conditions that can damp MSC capabilities of healing *in situ*. Autologous transplantations of MSC have mild results in clinical trials despite animal models generated great results [68]. Allogeneic transplantation of MSCs seems to have better outcomes. Moreover, the functional decline of MSCs has been associated with a pathophysiological driver of several diseases and aging [69].

Regenerative properties of endogenous MSC can be decreased *in vivo* and *in vitro.* Aging, metabolic changes due to pathologies and epigenetics changes can interfere at MSC *in vivo*. *In vitro*, MSC can be altered by cell culture passages (senescence), by storage at cryogenic conditions, by culture conditions (such as serum deprivation), by cell contact loss, by normoxia, etc. [69–71]. We will further exploit some of the MSC disabilities below. See **Figure 2**.

#### **6.1 Aging: epigenetic and PMT at MSC disruption**

Aging is a settled multifactorial process. Lopez-Otin has described 9 hallmarks that represent common denominators of aging: (1) genomic instability, (2) telomere attrition, (3) epigenetic alterations, (4) loss of proteostasis, (5) deregulated nutrientsensing, (6) mitochondrial dysfunction, (7) cellular senescence, (8) stem cell exhaustion, and (9) altered intercellular communication [72]. Herein, we will focus on some of these hallmarks and its impact on MSC.

*Activation and Metabolic Shifting: An Essential Process to Mesenchymal Stromal Cells Function DOI: http://dx.doi.org/10.5772/intechopen.109273*

#### **Figure 2.**

 *Senescence In vivo and in vitro : Conditions that lead to MSC senescence. Several factors are correlated with MSC senescence. In vivo , pathologies, metabolic diseases and aging may interfere at epigenetic levels and/or generate chronic inflammation responses that can cause MSC senescence. In vitro , other factors contribute to a senescence culture such as O 2 concentration, culture and cryogenic conditions etc. mainly due to telomerase disruption. Created with BioRender.com .* 

 Aging and age-related diseases have been associated with the higher number of senescent cells in the tissue [ 73 ]. In 1995, Dmiri et al. have described the quantification of the amount of beta-galactosidase as a biomarker of senescence. He demonstrated that a higher amount of beta-galactosidase is present at senescent cultured fibroblasts in human cells [ 74 ]. Since then, several works have been demonstrating that beta-galactosidase is not a reliable marker, so the search for a biomarker for senescence is still ongoing. It has been demonstrated that p16Ink4a-positive senescent cells accumulate with age in multiple tissues [ 75 ]. DNA damage response (DDR) is induced in healthy MSCs leading to the activation of the two main signaling pathways p19 *ARF* and p16 *INK* 4 *A* [ 73 ].

 Evidence suggests that MSC senescence is a dynamic process driven by epigenetic and genetic changes. Moreover, aging can be impacted by both environmental and inherent factors. Genetics factors are associated with long term mutations in DNA that lead to failure of the replicative state of the cell. Environmental factors that do not change DNA, also affect cell cycle. To date, epigenetics refers to the study of heritable phenotypic alterations linked to differential gene expression when the same DNA sequence is maintained [ 76 ]. Epigenetic dysregulation is associated with (1) DNA-based mechanisms: DNA methylation and histone modifications (2) RNA-based mechanisms: noncoding RNAs and RNA modifications [ 69 ].

 These genetics and epigenetic modifications can interfere directly with MSC by inducing the arrest of cell cycle, by producing a defective ECM niche production and by disrupting the MSC differentiation leading to tissue aberrations evidentiated at aging and disease [ 69 , 76 ]. Several articles described differential methylation patterns at MSC isolated from young X olders patients. Moreover, these differential methylation patterns were also observed at long term cultures *in vitro* of MSC [ 77 , 78 ]. Single-cell sequencing analysis of young X elderly BM-MSC have shown that young MSC have higher expression of genes related to tissue regeneration. Moreover, at young BM-MSC there is a cluster of cells that have a lower expression of genes of proliferation, that characterize them as quiescent cells, so stem cells. And these clusters of cells were not observed at elderly BM-MSC [79].

Not only epigenetic modification in DNA but also modifications in protein has huge importance in the differentiation processes. Protein post-transductional modifications (PTM) are protein modifications caused by adding groups of phosphates, acetyl, methyl, etc. in one or multiple amino acids and/or caused by proteolytic cleavage by ubiquitin [80]. These modifications can determine its activity state, localization, turnover, and interactions with other proteins. At MSC, PMT has been associated with differentiation to osteogenic lineage [81] Osteogenic differentiation of BM-MSC has been linked to *O*-GlcNAc cycling to the Runx2-dependent regulation of the early ALP marker [82].

In addition, aging decreases the number of stem cells in the niche, but not only it, aging also affects MSC and stem cell response due to metabolic and epigenetics changes [83]. Muscle stem cells (satellite cells) in aging tend to be converted to a fibroblast lineage instead of myogenic lineage [84, 85]. Several authors have been demonstrating that niche ECM stiffness leads to the aging process, dampening regeneration of the tissue and its homeostasis and moreover, leading to stem cell aging. In central nervous systems (CNS), ECM niche stiffness of oligodendrocyte progenitor cells (OPCs) have been related to aging processes mainly through the mechanoresponsive ion channel Piezo1 [83, 86].

Immunophenotypic profile of MSC can also be affected by senescence. Laschober et al. described that CD295 (leptin receptor or LEPR) have been found to increase during MSC senescence and it correlates with reduced proliferation capacities of MSC [87].

#### **6.2 MSC senescence in culture conditions**

In culture conditions, long term cultures are not welcome to be used in therapy due to its altered therapeutic profile. These cells became large and flattened ("sunny side up egg" morphology), less proliferative and less responsive. Senescence in culture characterized by the arrest of cell cycle. It is a known issue, as described by Hayclifk in fibroblast cultures [88]. Four types of senescence have been distinguished: replicative senescence (RS), oncogene-induced senescence (OIS), stress-induced premature senescence (SIPS), and developmental senescence [29, 89, 90].

Stress conditions at culture, such as adaptations to 2D culture, O2 concentration, confluency condition, the amount of nutrients even though the exposure to light lead to modifications that cause its arrest in the G0 phase of cell cycle of MSC [91, 92].

Cryopreservation is also a concern regarding MSC stability. Dimethyl sulfoxide (DMSO) has been the gold standard agent for cryobiology. However, the use of DMSO has been associated with *in vitro* toxicity. Since it has been associated with DNA methylation processes, DMSO affects many cellular processes and dysregulation of gene expression [93]. Mol et al. described that fresh culture MSC have a trend to have better outcomes for acute graft versus host disease (GvHD) and tissue injury in hemorrhagic cystitis than freeze-thawed MSC. Fresh MSCs have higher mRNA expression of IDO after 24 h IFNg priming, showing higher immunomodulatory properties than cryopreserved MSC [94].

*Activation and Metabolic Shifting: An Essential Process to Mesenchymal Stromal Cells Function DOI: http://dx.doi.org/10.5772/intechopen.109273*

#### **6.3 Inflammation and senescence of MSC**

Cycle arrest occurs due to a persistent DNA damage response (DDR) caused by either intrinsic (oxidative damage, telomere attrition, hyperproliferation) or external insults (ultraviolet, γ-irradiation, chemotherapeutic drugs) [95]. The more DNA damage, the more cell death, senescence and tissue dysfunction contributing to aging. Growing evidence has been describing that inflammation can also lead to DNA damage [96].

DNA damage induces the expression of type I interferons and other inflammatory factors [97]. The connection between DNA damage and inflammation is through the cytoplasmic DNA sensing pathway. Micronuclei formations (formed due to DNA damage during mitosis) can stimulate the cell senescence throughout cyclic GMP– AMP synthase (cGAS), a DNA sensor that stimulates STING (stimulator of interferon genes). To prevent undesired inflammation, besides cGAS-STING pathway, there are also the deoxyribonucleases (DNases) in the cytoplasm, digesting excessive DNA, serving as a negative regulator of cytoplasmic DNA. There are two major DNases in the cytoplasm: DNase2α (encoded by *DNaseII*) and TREX1 (originally designated DNaseIII). Intriguingly, both DNases are downregulated in senescent cells, contributing to aberrant cytoplasmic DNA sensing and inflammation [98].

Inflammaging, a term to define a chronic, low-grade sterile inflammation frequently observed during aging [99]. It is a macrophage centered process, involves several tissues and organs, including the gut microbiota, and is characterized by a complex balance between pro- and anti-inflammatory responses [100]. In elderly, the chronic inflammation observed is due to cells in tissue expressing pro-inflammatory cytokines, such as IL-1α, IL-6, TNF, and NF-κB activity and other inflammatory factors [101]. Chronic inflammation during aging and its negative outcome is supported by clinical data in kidney [102], liver [103], lung [104] etc.

Since MSC are perivascular cells and that they have a close connection with circulant factors in blood, it is possible to consider MSC with a central role in inflammaging, together with macrophages [105]. Rejuvenation strategies, such as culturing MSC with serum from older rats and parabiosis, showed a lower proliferation rate and survival of MSC exposed to serum from elderly subjects [106]. Thus, there are circulant molecules/cytokines that can impair MSC functions in aged individuals. Higher amounts of circulant beta-catenin and SMAD3 have been associated with senescence MSC profile [29]. More basic research must be done in this area.

Senescent cells are functional cells. Senescent cells were shown to secrete a range of inflammatory factors, which was termed the 'senescence-associated secretory phenotype' (SASP) [107]. The SASP mediates many of the cell-extrinsic functions of senescent cells. The SASP has its physiologic role: (1)by maintaining the SASP profile of the senescent cell (maintaining cell cycle arrest and SASP expression), (2) by eliciting immune response to generate a senescent cell clearance and (3) by secreting ECM and angiogenic factors leading to tissue regeneration [90, 108]. However, SASP has also deleterious effects by promoting inflammation (leading to inflammaging) and, potentially, tumor progression in neighboring cells. The correlation of SASP and inflammaging is beginning to be investigated using models to detect and eliminate the senescent cell (the INK-ATTAC model) [90, 108]. SAPS at MSC a is also related to higher secretion of extracellular microvesicles in aged subjects, as well its higher amount of microRNA content [109, 110] .

Interestingly, as it was described early in this chapter, MSCs have potent antiinflammatory functions, whereas senescent MSCs play a pro-inflammatory role

due to SASP, which has been considered a major cause of aged MSCs' detrimental effects [111]. In accordance with this, HMGB1 secreted by senescent fibroblasts is recognized by TLR4, followed by increase in SASP secretion [112]. These findings establish the critical role played by innate immune sensing mechanisms in regulating senescence [91].

#### **6.4 Diseases and MSC**

At MSC therapy, attention must be done regarding the pathological state of the patients at the harvest of MSC, since aging and pathological diseases can interfere at this isolated MSC. Moreover, when treating the patient, the pathogenic milieu where exogenous MSC is administered requires attention, because it may interfere with the MSC mechanism of action.

Obesity can impact BM-MSC. Ulum et al. described BM-MSC from patients with high body mass index (BMI) are more senescent, have disrupted differentiation to osteogenic and adipogenic cells, and highly expressed endoplasmic reticulum genes related to stress [113]. Diabetes can regulate AT-MSC as described by Abu-Shahba et al. They isolated AT-MSC from diabetic and non-diabetic patients and demonstrated that IL-1b is highly expressed in AT-MSC from diabetic patients [114].

#### **7. Conclusion and new perspectives**

The knowledge of MSC still requires much more research to elucidate its regenerative properties. More than 30 years of research and yet there is a lot to understand. The search for a better performance in MSCs cultures, the secretome profile, how to stimulate MSC to secrete higher amounts of such molecules using preconditioning techniques or niche stimulation, how MSC acts *in vivo*: a lot of questions with some clues, but far from the right answer.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Activation and Metabolic Shifting: An Essential Process to Mesenchymal Stromal Cells Function DOI: http://dx.doi.org/10.5772/intechopen.109273*

#### **Author details**

Patricia Semedo-Kuriki1 \*, Gabriel Pereira<sup>2</sup> , Danilo Cândido de Almeida2 and Niels Olsen Saraiva Camara1

1 São Paulo University (USP), São Paulo, Brazil

2 Federal University of São Paulo (UNIFESP), São Paulo, Brazil

\*Address all correspondence to: patricia.semedo@gmail.com

© 2022 The Author(s). Licensee IntechOpen. 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.

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Section 2
