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Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

Wang J, Hao J, Bai D, Gu Q, Han W, Wang L, Tan Y, Li X, Xue K, Han P, Liu Z, Jia Y, Wu J, Liu L, Wang L, Li W, Liu Z, Zhou Q - Stem Cell Res Ther (2015)

Bottom Line: Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III".As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC).The hiPSC lines are pluripotent and have passed the safety evaluation.

View Article: PubMed Central - PubMed

Affiliation: State Key of Stem Cells and Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China. juan920846643@163.com.

ABSTRACT

Introduction: Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents.

Methods: Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III".

Results: We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media.

Conclusion: The clinical-grade hiPSC lines therefore could be valuable sources for future hiPSC-based clinical trials or therapies and for drug screening.

No MeSH data available.


Related in: MedlinePlus

Reprogramming of clinical-grade HFF cells into clinical-grade hiPSCs and characterization of their pluripotency. a Time course of clinical-grade hiPSC generation. b Morphology of clinical-grade HFF cells, and clinical-grade hiPSCs grown on HFF feeder cells and in feeder-free conditions. Scale bar, 200 μm. c Clinical-grade hiPSCs express alkaline phosphatase. Scale bar, 200 μm. d Karyotyping results of one of the clinical-grade hiPSC lines. e mRNA expression levels of pluripotency-related genes of the clinical-grade hiPSCs relative to hESCs. f Immunofluorescence results of one of the clinical-grade hiPSC lines. The hiPSCs express pluripotency markers OCT4, SOX2, SSEA-3, SSEA-4, and TRA-1-60 at the protein level and do not express differentiation marker SSEA-1. Scale bar, 100 μm. g PCR analysis results show that the clinical-grade hiPSCs did not express Sendai virus exogenous genes. h PCR analysis results show that the Sendai virus genes did not integrate into the genomic of the clinical-grade hiPSCs. i Flow cytometry analysis of SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 expression of one of the clinical-grade hiPSC lines growing without feeders. D day, ESC embryonic stem cell, GMP good manufacturing practice, HFF human foreskin fibroblast, iPSC induced pluripotent stem cell, SSEA stage-specific embryonic antigen
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Fig2: Reprogramming of clinical-grade HFF cells into clinical-grade hiPSCs and characterization of their pluripotency. a Time course of clinical-grade hiPSC generation. b Morphology of clinical-grade HFF cells, and clinical-grade hiPSCs grown on HFF feeder cells and in feeder-free conditions. Scale bar, 200 μm. c Clinical-grade hiPSCs express alkaline phosphatase. Scale bar, 200 μm. d Karyotyping results of one of the clinical-grade hiPSC lines. e mRNA expression levels of pluripotency-related genes of the clinical-grade hiPSCs relative to hESCs. f Immunofluorescence results of one of the clinical-grade hiPSC lines. The hiPSCs express pluripotency markers OCT4, SOX2, SSEA-3, SSEA-4, and TRA-1-60 at the protein level and do not express differentiation marker SSEA-1. Scale bar, 100 μm. g PCR analysis results show that the clinical-grade hiPSCs did not express Sendai virus exogenous genes. h PCR analysis results show that the Sendai virus genes did not integrate into the genomic of the clinical-grade hiPSCs. i Flow cytometry analysis of SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 expression of one of the clinical-grade hiPSC lines growing without feeders. D day, ESC embryonic stem cell, GMP good manufacturing practice, HFF human foreskin fibroblast, iPSC induced pluripotent stem cell, SSEA stage-specific embryonic antigen

Mentions: The clinical-grade HFF cells of passage 8 were used for integration-free Sendai virus-based reprogramming. The reprogramming procedure was similar to the instructions of the CytoTune®-iPS Sendai Reprogramming Kit except that Xeno-free media were used in our protocol. Pluriton™ reprogramming medium is a type of Xeno-free medium which can efficiently support the generation of hiPSCs in mRNA-based reprogramming. The X medium, which was defined in our laboratory, could greatly improve the induction efficiency of pig and human iPSCs [24, 26] and could be configured with Xeno-free reagents. We proposed that clinical-grade hiPSCs could also be efficiently derived in pluriton™ reprogramming medium and X medium, and thus both media were utilized in the reprogramming process. The hiPSC colonies emerged at day 12 after infection and were picked up 5 days later to generate stable cell lines (Fig. 2a). The reprogramming efficiency of X medium was almost three times that of the pluriton™ reprogramming medium (data not shown).Fig. 2


Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

Wang J, Hao J, Bai D, Gu Q, Han W, Wang L, Tan Y, Li X, Xue K, Han P, Liu Z, Jia Y, Wu J, Liu L, Wang L, Li W, Liu Z, Zhou Q - Stem Cell Res Ther (2015)

Reprogramming of clinical-grade HFF cells into clinical-grade hiPSCs and characterization of their pluripotency. a Time course of clinical-grade hiPSC generation. b Morphology of clinical-grade HFF cells, and clinical-grade hiPSCs grown on HFF feeder cells and in feeder-free conditions. Scale bar, 200 μm. c Clinical-grade hiPSCs express alkaline phosphatase. Scale bar, 200 μm. d Karyotyping results of one of the clinical-grade hiPSC lines. e mRNA expression levels of pluripotency-related genes of the clinical-grade hiPSCs relative to hESCs. f Immunofluorescence results of one of the clinical-grade hiPSC lines. The hiPSCs express pluripotency markers OCT4, SOX2, SSEA-3, SSEA-4, and TRA-1-60 at the protein level and do not express differentiation marker SSEA-1. Scale bar, 100 μm. g PCR analysis results show that the clinical-grade hiPSCs did not express Sendai virus exogenous genes. h PCR analysis results show that the Sendai virus genes did not integrate into the genomic of the clinical-grade hiPSCs. i Flow cytometry analysis of SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 expression of one of the clinical-grade hiPSC lines growing without feeders. D day, ESC embryonic stem cell, GMP good manufacturing practice, HFF human foreskin fibroblast, iPSC induced pluripotent stem cell, SSEA stage-specific embryonic antigen
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643509&req=5

Fig2: Reprogramming of clinical-grade HFF cells into clinical-grade hiPSCs and characterization of their pluripotency. a Time course of clinical-grade hiPSC generation. b Morphology of clinical-grade HFF cells, and clinical-grade hiPSCs grown on HFF feeder cells and in feeder-free conditions. Scale bar, 200 μm. c Clinical-grade hiPSCs express alkaline phosphatase. Scale bar, 200 μm. d Karyotyping results of one of the clinical-grade hiPSC lines. e mRNA expression levels of pluripotency-related genes of the clinical-grade hiPSCs relative to hESCs. f Immunofluorescence results of one of the clinical-grade hiPSC lines. The hiPSCs express pluripotency markers OCT4, SOX2, SSEA-3, SSEA-4, and TRA-1-60 at the protein level and do not express differentiation marker SSEA-1. Scale bar, 100 μm. g PCR analysis results show that the clinical-grade hiPSCs did not express Sendai virus exogenous genes. h PCR analysis results show that the Sendai virus genes did not integrate into the genomic of the clinical-grade hiPSCs. i Flow cytometry analysis of SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 expression of one of the clinical-grade hiPSC lines growing without feeders. D day, ESC embryonic stem cell, GMP good manufacturing practice, HFF human foreskin fibroblast, iPSC induced pluripotent stem cell, SSEA stage-specific embryonic antigen
Mentions: The clinical-grade HFF cells of passage 8 were used for integration-free Sendai virus-based reprogramming. The reprogramming procedure was similar to the instructions of the CytoTune®-iPS Sendai Reprogramming Kit except that Xeno-free media were used in our protocol. Pluriton™ reprogramming medium is a type of Xeno-free medium which can efficiently support the generation of hiPSCs in mRNA-based reprogramming. The X medium, which was defined in our laboratory, could greatly improve the induction efficiency of pig and human iPSCs [24, 26] and could be configured with Xeno-free reagents. We proposed that clinical-grade hiPSCs could also be efficiently derived in pluriton™ reprogramming medium and X medium, and thus both media were utilized in the reprogramming process. The hiPSC colonies emerged at day 12 after infection and were picked up 5 days later to generate stable cell lines (Fig. 2a). The reprogramming efficiency of X medium was almost three times that of the pluriton™ reprogramming medium (data not shown).Fig. 2

Bottom Line: Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III".As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC).The hiPSC lines are pluripotent and have passed the safety evaluation.

View Article: PubMed Central - PubMed

Affiliation: State Key of Stem Cells and Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China. juan920846643@163.com.

ABSTRACT

Introduction: Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents.

Methods: Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III".

Results: We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media.

Conclusion: The clinical-grade hiPSC lines therefore could be valuable sources for future hiPSC-based clinical trials or therapies and for drug screening.

No MeSH data available.


Related in: MedlinePlus