**4. iPSC for clinical use**

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

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

validation of such systems is critical for iPSC-derived therapies. To circumvent and manage the safety risk of the iPSC for regenerative medicine, several groups worked at the early stage on the development of standardized clinical grade iPSC banks from allogeneic donors. Indeed, the use of highly defined iPSC as starting cells presents many advantages as overcoming the genetic variations inducing different immunogenicity, genetic instability, tumorigenicity, and differentiation outcomes. Moreover, generation of iPSC from each patient is costly and time-consuming. In this regard, several groups in the world have developed banking of allogeneic iPSC lines for clinical use with validated and standardized protocols. The possibility of creating off-the-shelf iPSC-based therapies has attracted not only academics but also industrial groups as Lonzo and Cellular Dynamics International, a Fujifilm company.

iPSC banks can provide a cost-effective mass-production strategy. Several groups have developed iPSC banks from selected HLA donors trying to cover the majority of the population [41, 42]. The Center for iPSC Research and Application (CiRA), in Kyoto University, started the iPS Cell Stock for Regenerative Medicine in 2013. Initially, based on the limited diversity of the Japanese population, CiRA wanted to generate clinical-grade iPSCs from samples of peripheral blood and umbilical cord blood from healthy selected donors that would cover 90% of Japanese population with only 50 iPSC lines [43]. This strategy is valuable for countries such as Japan, but could be difficult to expand to the worldwide population. It has been evaluated that a multiethnic iPSC bank of the 100 most common HLA types in each population would cover only 78% of European individuals, 63% of Asians, 52% of Hispanics and 45% of African Americans [44]. This probabilistic model highlights the necessity of a large-scale international collaboration for the constitution of haplobank of iPSC lines. Using HLA-homozygous donors limits the numbers of iPSC lines needed to cover a given population, but identification of the potential donors would need large screenings or the use of established data from cord blood banks. The potential development of "universal" iPSCs made of genetically modified cells offering an off-the-shelf product that is readily available could be an alternative to the iPSC bank using materials from HLA-homozygous donors. The "universal" iPSC could solve the problem of immune rejection profile of iPSC-derived cells by artificially expressing, for example, HLA molecule as HLA-E allowing iPSC-derived cells to escape T cell-mediated rejection and to be resistant to NK-cell lysis [37, 45].

Nevertheless, stochastic events potentially occurring during reprogramming, colony expansion, iPSC selection, differentiation, iPSC-derived cell expansion and purification, storage and transport could complicate efforts toward a standardized product. Consequently, it has to be taken into consideration that variation may exist within any iPSC bank, between iPSC and final product composed of iPSC-derived cells in the clinic. Such variability requires continual extensive genotypic, phenotypic and functional assessment and highlights the need of a global quality control confirming the iPSC and the iPSC-derived cells' quality whatever the manufacturer, the reprogramming method or the cell donors.

#### **4.2 Quality control of clinical-based iPSC**

Given the high variability across iPSC lines and their differentiated derivatives in terms of their epigenetic status, tumorigenic and immunogenic potential, differentiation capacity, batch variability and existence of heterogeneous populations and/or non-relevant cells such as contaminating cell, the clinical outcome of the cell replacement therapy, in terms of efficacy and safety with these iPSC-based products, highly relies on the acceptable quality and safety standards of these products. Because of dissimilarities between institutions on these criteria, agreement on the critical quality attributes (CQAs) of such lines and the assays that should be used

**21**

product without any contaminating other lineage cell types.

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

is required. The CQAs correspond to the chemical, physical and biological properties of the product. As well as the type of assay, they have to be defined within an appropriate limit, range or distribution to ensure quality and safety of the product. For cell therapy product and for clinical-grade iPSC, the CQAs include identity, microbiological sterility, genetic fidelity and stability, viability, characterization and potency. In the last few years, there was a common effort made on the banking and the quality control of the iPSC lines. After a series of workshop, adaptation to iPSC of the established recommendations and guidance realized by the International Stem Cell Banking Initiative (ISCBI) for human embryonic stem cell banking, has generated initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade [46]. During these workshops, the researchers, industrial and regulation agencies pointed out the requirement of standardization and validation of process and quality and safety controls. For each criterion, one or several tests are required with regard to the recommended analytical methods. Global consensus recommends the performance of assays by accredited and licensed laboratories. When it is not available, in-house tests should be undertaken after validation and qualification, and comparability with other laboratories should be performed if possible.

The first mandatory test is to validate the identity of the iPSC line with the short tandem repeat (STR) analysis to genotype the original cells, the iPSC seeds and the master cell bank to ascertain the absence of switch or cross contamination of several iPSC lines during generation or maintenance process. Due to the nature of the stem cell-based products, they cannot be sterilized. The assessment of the microbiological sterility is of the highest importance and should be performed not only on the final product. This should include the mycoplasma, bacteriology and viral testing supplemented by endotoxins detection assay and should have a negative result. The genetic stability and fidelity of the iPSC lines should be evaluated by residual vector testing and karyotype. To eliminate the risk of potential cell transformation and the risk of malignancy development in patients, residual vector testing has to be ≤1 plasmid copy per 100 cells in seed and master cell banks and the karyotype should be normal on more than 20 metaphases. So far, techniques with high precision such as single nucleotide polymorphism (SNP) and whole genome analysis or other genetic markers are not required but could be performed for information. To give an appropriate dosage of cells, viability should be >60%. Calculation of doubling time and detection of cell debris are not required but could provide useful information. To manage the risk associated with the presence of non-desired or spontaneously differentiated cells, iPSCs have to be characterized by the expression of a minimum of two markers from the standard human pluripotent stem cells panel (positive for Oct4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, Sox2, Nanog). A combination of one intracellular and one extracellular marker should be used and should be >70%. Finally, for the potency assay, reflecting the biological activity of the cells, embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers is mandatory. The teratoma formation in severe combined immune-deficient (SCiD) mouse injection assay is not mandatory for the iPSC due to a reproducibility problem, high cost and non-ethical procedure. Molecular pluripotency assays such as mRNA array- and RNA-Seq-based gene expression assays could be kept for information if they are performed molecular pluripotency assays such as mRNA array- and RNA-Seqbased gene expression assays could be for information but are not required. For the iPS-derived differentiated therapeutic products, the minimal criteria are mostly identical except for the phenotypical characterization, which should validate the absence of pluripotent stem cell markers, the expression of differentiation markers unique to the therapeutic product and assess 100% purity of the therapeutic cellular

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

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

validation of such systems is critical for iPSC-derived therapies. To circumvent and manage the safety risk of the iPSC for regenerative medicine, several groups worked at the early stage on the development of standardized clinical grade iPSC banks from allogeneic donors. Indeed, the use of highly defined iPSC as starting cells presents many advantages as overcoming the genetic variations inducing different immunogenicity, genetic instability, tumorigenicity, and differentiation outcomes. Moreover, generation of iPSC from each patient is costly and time-consuming. In this regard, several groups in the world have developed banking of allogeneic iPSC lines for clinical use with validated and standardized protocols. The possibility of creating off-the-shelf iPSC-based therapies has attracted not only academics but also industrial groups as Lonzo and Cellular Dynamics International, a Fujifilm company.

iPSC banks can provide a cost-effective mass-production strategy. Several groups have developed iPSC banks from selected HLA donors trying to cover the majority of the population [41, 42]. The Center for iPSC Research and Application (CiRA), in Kyoto University, started the iPS Cell Stock for Regenerative Medicine in 2013. Initially, based on the limited diversity of the Japanese population, CiRA wanted to generate clinical-grade iPSCs from samples of peripheral blood and umbilical cord blood from healthy selected donors that would cover 90% of Japanese population with only 50 iPSC lines [43]. This strategy is valuable for countries such as Japan, but could be difficult to expand to the worldwide population. It has been evaluated that a multiethnic iPSC bank of the 100 most common HLA types in each population would cover only 78% of European individuals, 63% of Asians, 52% of Hispanics and 45% of African Americans [44]. This probabilistic model highlights the necessity of a large-scale international collaboration for the constitution of haplobank of iPSC lines. Using HLA-homozygous donors limits the numbers of iPSC lines needed to cover a given population, but identification of the potential donors would need large screenings or the use of established data from cord blood banks. The potential development of "universal" iPSCs made of genetically modified cells offering an off-the-shelf product that is readily available could be an alternative to the iPSC bank using materials from HLA-homozygous donors. The "universal" iPSC could solve the problem of immune rejection profile of iPSC-derived cells by artificially expressing, for example, HLA molecule as HLA-E allowing iPSC-derived cells to escape T cell-mediated rejection and to be resistant to NK-cell lysis [37, 45].

Nevertheless, stochastic events potentially occurring during reprogramming, colony expansion, iPSC selection, differentiation, iPSC-derived cell expansion and purification, storage and transport could complicate efforts toward a standardized product. Consequently, it has to be taken into consideration that variation may exist within any iPSC bank, between iPSC and final product composed of iPSC-derived cells in the clinic. Such variability requires continual extensive genotypic, phenotypic and functional assessment and highlights the need of a global quality control confirming the iPSC and the iPSC-derived cells' quality whatever the manufacturer,

Given the high variability across iPSC lines and their differentiated derivatives in terms of their epigenetic status, tumorigenic and immunogenic potential, differentiation capacity, batch variability and existence of heterogeneous populations and/or non-relevant cells such as contaminating cell, the clinical outcome of the cell replacement therapy, in terms of efficacy and safety with these iPSC-based products, highly relies on the acceptable quality and safety standards of these products. Because of dissimilarities between institutions on these criteria, agreement on the critical quality attributes (CQAs) of such lines and the assays that should be used

the reprogramming method or the cell donors.

**4.2 Quality control of clinical-based iPSC**

**20**

is required. The CQAs correspond to the chemical, physical and biological properties of the product. As well as the type of assay, they have to be defined within an appropriate limit, range or distribution to ensure quality and safety of the product. For cell therapy product and for clinical-grade iPSC, the CQAs include identity, microbiological sterility, genetic fidelity and stability, viability, characterization and potency. In the last few years, there was a common effort made on the banking and the quality control of the iPSC lines. After a series of workshop, adaptation to iPSC of the established recommendations and guidance realized by the International Stem Cell Banking Initiative (ISCBI) for human embryonic stem cell banking, has generated initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade [46]. During these workshops, the researchers, industrial and regulation agencies pointed out the requirement of standardization and validation of process and quality and safety controls. For each criterion, one or several tests are required with regard to the recommended analytical methods. Global consensus recommends the performance of assays by accredited and licensed laboratories. When it is not available, in-house tests should be undertaken after validation and qualification, and comparability with other laboratories should be performed if possible.

The first mandatory test is to validate the identity of the iPSC line with the short tandem repeat (STR) analysis to genotype the original cells, the iPSC seeds and the master cell bank to ascertain the absence of switch or cross contamination of several iPSC lines during generation or maintenance process. Due to the nature of the stem cell-based products, they cannot be sterilized. The assessment of the microbiological sterility is of the highest importance and should be performed not only on the final product. This should include the mycoplasma, bacteriology and viral testing supplemented by endotoxins detection assay and should have a negative result. The genetic stability and fidelity of the iPSC lines should be evaluated by residual vector testing and karyotype. To eliminate the risk of potential cell transformation and the risk of malignancy development in patients, residual vector testing has to be ≤1 plasmid copy per 100 cells in seed and master cell banks and the karyotype should be normal on more than 20 metaphases. So far, techniques with high precision such as single nucleotide polymorphism (SNP) and whole genome analysis or other genetic markers are not required but could be performed for information. To give an appropriate dosage of cells, viability should be >60%. Calculation of doubling time and detection of cell debris are not required but could provide useful information. To manage the risk associated with the presence of non-desired or spontaneously differentiated cells, iPSCs have to be characterized by the expression of a minimum of two markers from the standard human pluripotent stem cells panel (positive for Oct4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, Sox2, Nanog). A combination of one intracellular and one extracellular marker should be used and should be >70%. Finally, for the potency assay, reflecting the biological activity of the cells, embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers is mandatory. The teratoma formation in severe combined immune-deficient (SCiD) mouse injection assay is not mandatory for the iPSC due to a reproducibility problem, high cost and non-ethical procedure. Molecular pluripotency assays such as mRNA array- and RNA-Seq-based gene expression assays could be kept for information if they are performed molecular pluripotency assays such as mRNA array- and RNA-Seqbased gene expression assays could be for information but are not required. For the iPS-derived differentiated therapeutic products, the minimal criteria are mostly identical except for the phenotypical characterization, which should validate the absence of pluripotent stem cell markers, the expression of differentiation markers unique to the therapeutic product and assess 100% purity of the therapeutic cellular product without any contaminating other lineage cell types.

This consensus on CQA and minimum testing requirements for clinical-grade iPSC lines will evolve with the advances in scientific understanding and development in technology and best practices. The Global Alliance for iPSC Therapies (GAiT), which facilitates the development of general clinical-grade iPSC standards by community engagement and consensus building to support the global application of iPSC-derived cellular therapeutics, is in charge of the future evolution of the consensus on quality and safety standards required for a clinical-grade iPSC. Moreover, GAiT presents objectives to achieve consensus on donor selection and screening criteria and consent standards, which with future commercialization and global distribution also require ethical review.

#### **5. Conclusion**

It is quite remarkable that in just over 10 years, research using iPSC has led to several clinical studies, with many more applications expected to follow. In few years, the iPSC-based therapies induced a switch to a mass production of clinical-grade iPSC for the benefit of a large population at affordable costs, with the generation of clinicalgrade iPSC banks, and with a stronger involvement of biopharmaceutical companies. This shift led to many efforts for the standardization of generation, maintenance and differentiation procedures, and for the establishment of quality and safety standards for the clinical-grade iPSC and their derivatives prior to transplantation to patients.

There are still a number of challenges that must be overcome for iPSCs to reach their full potential. The improvement of manufacturing procedures for a large-scale production would provide higher quality cells for clinical iPSC-based therapies. Quality and safety controls are also challenging. Predicting cancer risk based on sequence information is a formidable task, and failure to detect oncogenic mutations is not necessarily a warrantor of the non-tumorigenicity of iPSC-based products, suggesting that recommendations should still evolve with scientific advances.

Due to their large potential in regenerative medicine, such as the generation of complex 3D structures, tissues or organs, more challenges in differentiation protocols in 3D structures have to be overcome for the up-coming year, without compromising quality and safety of iPSCs.

#### **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Valérie Vanneaux Department of Biotherapies, Saint-Louis Hospital, Paris, France

\*Address all correspondence to: valerie.vanneaux@aphp.fr

© 2019 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.

**23**

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

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