**3.1 Tumorigenicity**

The potential risk of tumorigenicity to patients from both teratomas and malignant tumors could arise if transplanted cells are contaminated with undifferentiated iPSC, or if transplanted cells have been genetically modified and become unstable during the *in vitro* production steps.

The major concern related to iPSC-based tumorigenicity is the reprogramming method. In the original cocktail of transcription factors developed by Yamanaka, somatic cells are transduced by retroviral vectors that become integrated into the genome of the host cells. Two of these factors—*c-Myc* and *klf4*—are potent oncogenes [6]. Subsequently, reports of tumorigenicity after transplantation of iPSC or iPSC-derived cells are not surprising. Thereby, teratoma formation could be induced by the undesired activation/suppression of essential host genes proximal to integration sites or by residual expression of reprogramming factors in the derived cells in animal model [7, 8]. With hindsight, there is evidence for the necessity to select a non-integrative method for reprogramming, a higher rate of genomic alterations occurring when human iPSCs are generated with viral vectors, compared to mRNA [7, 9]. Numerous studies, focused on the choice of reprogramming factors and methods of delivery, have developed various novel strategies to enhance the efficiency of reprogramming and reduce the potential risk of tumorigenicity. To circumvent this risk, human iPSCs have been generated by several "integration-free" methods, based on the use of viral vectors (adenoviral vectors and Sendai virus-based vectors) or non-viral vectors (piggyBac system, minicircle vector, and episomal vectors). Originally, the four transcription factors needed for complete cell reprograming were *c-myc*, *klf4*, *oct4* and *sox2* [1]. The protumorigenic transcription factor *c-myc* has been found to be unnecessary for the reprogramming process, but the overall efficiency is decreased without it. Several strategies have been developed with the use of different transcription factors and/ or replacement of *c-myc*, or the use of direct protein delivery and synthesized mRNA [10–12].

Furthermore, the tumorigenicity risk is often linked to the genetic instability of iPSC. Random genomic alterations are frequently observed in human iPSCs showing their intrinsic instability, essentially due to the massive genome remodeling, and probably also resulting from various mechanisms such as replicative stress, reactivation of the telomerase and metabolism modification from the oxidative to the glycolytic state. Epigenetic modifications may also contribute to iPSC variation due to residual epigenetic memories of the starting cell type [13]. The incomplete resetting of the non-CpG methylation patterns during reprogramming leads to a biased differential potential in certain cell types depending on the donor cell source [14, 15]. However, it has been shown that their residual epigenetic memory diminishes with the *in vitro* expansion over a period of time [16, 17]. As just mentioned, the selection of the donor cell type is of importance. Many human somatic cell types have been successfully reprogrammed. However, even if the use of different transcription factors, delivery methods and culture conditions does not facilitate any comparison, it is well known that reprogramming efficiencies, kinetics and tumorigenicity vary between somatic cell types. Firstly, cell sources have to be permissive to avoid to turn to integrative methods and to the use of oncogenes. Some human, adult somatic cells, such as melanocytes, are known to naturally express endogenously reprogramming factors, for instance Sox 2, at sufficiently high levels [18, 19]. Moreover, some types of donor cells such as dermal fibroblasts and blood cells are easily accessible, but they might carry more mutational burdens

and chromosomal abnormalities, due to their frequent exposure to environmental stress factors, like ultraviolet rays, or due to the donor's age, thereby leading to increased tumorigenicity, and significant safety problems [20, 21]. With all these considerations of cell variability and tumorigenic potential in mind, reflection on the generation of homogeneous cell source and banking emerged.

Many approaches have been evaluated to address the tumorigenicity challenge by eliminating the pluripotent cells of the final product such as small molecule, genetic approach to introduce a suicide gene; miRNA switch; antibodies targeting a surface-specific antigen; phototoxic approach; live detection and quantification of the residual human iPSC [22]. For the suicide gene approach, the most widely used gene is herpes simplex virus thymidine kinase (HSV-TK) that phosphorylates ganciclovir (GCV) and induces apoptosis by inhibiting DNA synthesis. Many studies demonstrated its efficacy as safeguard to eliminate tumoral cells [23]. Until then, this genetic approach with an inducible suicide system may remain not necessary enough to induce tumor elimination because of potential acquired resistance to GCV due to variability of insertion location sites and to the uncontrolled number of inserted transgene [24]. Another study demonstrated the same mechanism of inducing apoptosis in 95% of iPSCs and iPSC-derived cells by transducing an inducible Caspase 9 [25]. Recently, with development of targeted genetic strategies such as gene-editing, researchers try to identify the location of "genomic safe harbors" (GSH), corresponding to the safest permissive loci for transgenes' insertion [26]. The already known GSH candidates could be AAVS1 (adeno-associated virus integration site 1), CCR5 (chemokine CC motif receptor 5), human ROSA26 and some extragenic loci. Recently, to predict the influence of gene integration on nearby genes, it has been suggested that the combination of several distinct approaches such as the analysis of the topologically associated domains of GSH candidates of chromosomes could reduce the risks associated with cell therapy [27]. Another targeted alternative, eliminating selectively residual pluripotent cells sparing precursors and differentiated cells, involves PluriSIns, pluripotent cellsspecific inhibitors [28]. Alternatively, antibody, lectin or miRNA-mediated removal undesired cells were developed to suppress the pluripotent stem cells from the final product [29]. Lastly, a novel methodology using synthetic microRNA switch is developed to improve the purity of the final product even if the cell surface markers are not available to tag the relevant cells [30, 31].

#### **3.2 iPSC immunogenicity**

The immunogenicity of differentiated cells derived from iPSC is of clinical significance. At the beginning, because of the use of the patient's own cells, theoretically there is no risk of rejection after their transplantation. Some studies demonstrated no immune rejection of autologous iPSC-derived cells, but an activated immune response after the use of allogeneic iPS derived cells. Contrarily, immune rejection has been observed after autologous transplantation of iPSC-derived cells, suggesting that *in vitro* operations could also impact on the immunogenicity of the iPSC [32]. Moreover, the immune response to undifferentiated iPSC is different from their derivatives, emphasizing the need to perform similar comparative analyses in starting cell populations in order to predict immune tolerance after transplantation. Whereas autologous hiPSC-derived smooth muscle cells were highly immunogenic, autologous hiPSC-derived retinal pigment epithelial (RPE) cells were immune tolerated, suggesting a potential abnormal expression of some immunogenic antigens in smooth muscle cells [33]. These results demonstrated that the nature of the differentiated cells could trigger an immune response suggesting the importance of the differentiation protocol.

**19**

*Induced Pluripotent Stem Cells for Clinical Use DOI: http://dx.doi.org/10.5772/intechopen.88878*

preferred for clinical settings [35, 36].

**4. iPSC for clinical use**

control of the "Universal" iPSC quality for clinical settings.

**4.1 Clinical-grade allogeneic iPSC line bank**

As mentioned earlier, because of their genomic instability, generation, amplification and differentiation of iPSC could induce a modified immune response of the iPSC *in vivo*. Concerning reprogramming, the RNA-based methods are relatively efficient and do not integrate in the genome, but they are also known to be highly immunogenic. Concerning cell type, it has been widely shown that iPSCs could be generated from a patient's own cells including fat cells, nerve cells, skin fibroblasts, cuticle cells, fetal foreskin cells, B cells, T cells, peripheral blood mononuclear cells, umbilical cord mesenchymal cells, chorionic mesenchymal cells and amniotic mesenchymal cells. But, some studies showed that the genetic memory of the cellular immunogenicity is conserved after reprogramming and differentiation. So, the selection of donor cell type/origin is crucial. As an example, iPSCs derived from less immunogenic cells, such as umbilical cord mesenchymal cells, generated less immunogenic neural derivatives than those from skin fibroblasts-derived iPSCs [34]. Recently, several researchers showed the less immunogenic potential of some iPSC-derived cells as cartilage and retinal pigment epithelium cells when they are implanted *in vivo*, arguing that some cell types are less immunogenic and should be

Recently, a novel approach of "Universal" iPSC was developed to address the difficulty of immunogenicity of allogeneic iPSCs. Hypoimmunogenicity of iPSC was induced by inactivation of major histocompatibility complex class I and II genes and overexpression of CD47 enabled them to escape to immune rejection in fully HLA-mismatched allogeneic recipients. This strategy allowed the long-term survival of the transplanted cells without the use of immunosuppression. However, overexpression of CD47 is associated with malignant transformation, leading to include some suicide strategies as a safety concern [37]. These immune escape approaches open the door to the clinical use of allogeneic iPSC-derived cell products without immune rejection concerns and complications. However, their complex production process including a combination of several transduction and geneediting operations could add many safety issues. Even though other vectors and gene-editing techniques [38, 39] could also be used to reduce the risks, the multiple genetic manipulations and additional expansions in culture require a reinforced

The use of human iPSCs in medicinal applications requires the establishment of standardized and validated protocols that will allow large-scale, cost-effective cultivation procedure, while maintaining their quality. Implementation of good manufacturing practice (GMP)-compliant protocols for the generation and maintenance of human iPSC lines is crucial to increase the application safety and to fulfill the regulatory requirements to obtain clinical trials' approval. Many efforts to increase the overall iPSC stability, reproducibility and quality have been performed by (1) selecting the cell type that is easily accessible, less immunogenic, and permissive for reprogramming and presents the ability to be stored for longer periods of time; (2) improving reprogramming efficiency, which should be as high as possible without genomic integration-based delivery method and without using oncogene and (3) improving cultivation methods with xeno- and feeder-free products, with defined and scalable conditions for maintenance and differentiation of human iPSC such as automation, closed cell systems and validated protocols [40]. Moreover, selection of cell source is of importance. Demonstration of comparability, standardization and

#### *Induced Pluripotent Stem Cells for Clinical Use DOI: http://dx.doi.org/10.5772/intechopen.88878*

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

are not available to tag the relevant cells [30, 31].

the importance of the differentiation protocol.

The immunogenicity of differentiated cells derived from iPSC is of clinical significance. At the beginning, because of the use of the patient's own cells, theoretically there is no risk of rejection after their transplantation. Some studies demonstrated no immune rejection of autologous iPSC-derived cells, but an activated immune response after the use of allogeneic iPS derived cells. Contrarily, immune rejection has been observed after autologous transplantation of iPSC-derived cells, suggesting that *in vitro* operations could also impact on the immunogenicity of the iPSC [32]. Moreover, the immune response to undifferentiated iPSC is different from their derivatives, emphasizing the need to perform similar comparative analyses in starting cell populations in order to predict immune tolerance after transplantation. Whereas autologous hiPSC-derived smooth muscle cells were highly immunogenic, autologous hiPSC-derived retinal pigment epithelial (RPE) cells were immune tolerated, suggesting a potential abnormal expression of some immunogenic antigens in smooth muscle cells [33]. These results demonstrated that the nature of the differentiated cells could trigger an immune response suggesting

**3.2 iPSC immunogenicity**

and chromosomal abnormalities, due to their frequent exposure to environmental stress factors, like ultraviolet rays, or due to the donor's age, thereby leading to increased tumorigenicity, and significant safety problems [20, 21]. With all these considerations of cell variability and tumorigenic potential in mind, reflection on

Many approaches have been evaluated to address the tumorigenicity challenge by eliminating the pluripotent cells of the final product such as small molecule, genetic approach to introduce a suicide gene; miRNA switch; antibodies targeting a surface-specific antigen; phototoxic approach; live detection and quantification of the residual human iPSC [22]. For the suicide gene approach, the most widely used gene is herpes simplex virus thymidine kinase (HSV-TK) that phosphorylates ganciclovir (GCV) and induces apoptosis by inhibiting DNA synthesis. Many studies demonstrated its efficacy as safeguard to eliminate tumoral cells [23]. Until then, this genetic approach with an inducible suicide system may remain not necessary enough to induce tumor elimination because of potential acquired resistance to GCV due to variability of insertion location sites and to the uncontrolled number of inserted transgene [24]. Another study demonstrated the same mechanism of inducing apoptosis in 95% of iPSCs and iPSC-derived cells by transducing an inducible Caspase 9 [25]. Recently, with development of targeted genetic strategies such as gene-editing, researchers try to identify the location of "genomic safe harbors" (GSH), corresponding to the safest permissive loci for transgenes' insertion [26]. The already known GSH candidates could be AAVS1 (adeno-associated virus integration site 1), CCR5 (chemokine CC motif receptor 5), human ROSA26 and some extragenic loci. Recently, to predict the influence of gene integration on nearby genes, it has been suggested that the combination of several distinct approaches such as the analysis of the topologically associated domains of GSH candidates of chromosomes could reduce the risks associated with cell therapy [27]. Another targeted alternative, eliminating selectively residual pluripotent cells sparing precursors and differentiated cells, involves PluriSIns, pluripotent cellsspecific inhibitors [28]. Alternatively, antibody, lectin or miRNA-mediated removal undesired cells were developed to suppress the pluripotent stem cells from the final product [29]. Lastly, a novel methodology using synthetic microRNA switch is developed to improve the purity of the final product even if the cell surface markers

the generation of homogeneous cell source and banking emerged.

**18**

As mentioned earlier, because of their genomic instability, generation, amplification and differentiation of iPSC could induce a modified immune response of the iPSC *in vivo*. Concerning reprogramming, the RNA-based methods are relatively efficient and do not integrate in the genome, but they are also known to be highly immunogenic. Concerning cell type, it has been widely shown that iPSCs could be generated from a patient's own cells including fat cells, nerve cells, skin fibroblasts, cuticle cells, fetal foreskin cells, B cells, T cells, peripheral blood mononuclear cells, umbilical cord mesenchymal cells, chorionic mesenchymal cells and amniotic mesenchymal cells. But, some studies showed that the genetic memory of the cellular immunogenicity is conserved after reprogramming and differentiation. So, the selection of donor cell type/origin is crucial. As an example, iPSCs derived from less immunogenic cells, such as umbilical cord mesenchymal cells, generated less immunogenic neural derivatives than those from skin fibroblasts-derived iPSCs [34]. Recently, several researchers showed the less immunogenic potential of some iPSC-derived cells as cartilage and retinal pigment epithelium cells when they are implanted *in vivo*, arguing that some cell types are less immunogenic and should be preferred for clinical settings [35, 36].

Recently, a novel approach of "Universal" iPSC was developed to address the difficulty of immunogenicity of allogeneic iPSCs. Hypoimmunogenicity of iPSC was induced by inactivation of major histocompatibility complex class I and II genes and overexpression of CD47 enabled them to escape to immune rejection in fully HLA-mismatched allogeneic recipients. This strategy allowed the long-term survival of the transplanted cells without the use of immunosuppression. However, overexpression of CD47 is associated with malignant transformation, leading to include some suicide strategies as a safety concern [37]. These immune escape approaches open the door to the clinical use of allogeneic iPSC-derived cell products without immune rejection concerns and complications. However, their complex production process including a combination of several transduction and geneediting operations could add many safety issues. Even though other vectors and gene-editing techniques [38, 39] could also be used to reduce the risks, the multiple genetic manipulations and additional expansions in culture require a reinforced control of the "Universal" iPSC quality for clinical settings.
