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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

Clinical-grade hiPSCs can randomly differentiate into all three germ layers and directly differentiate into functional neural cells and cardiomyocytes in Xeno-free media. a Morphology of EBs at day 8 derived from one clinical-grade hiPSC line. Scale bar, 200 μm. b Gene expression profile for germ-layer marker genes of the day 8 EBs, ectoderm (Gad1 and Pax6), mesoderm (Enolase and Osteonectin), endoderm (Nicastrin and Alpha-fetoprotein), and pluripotency (Oct4, Sox2 and Nanog). c Hematoxylin and eosin staining of teratoma derived from one clinical-grade hiPSC line. Scale bar, 100 μm. The teratomas contain tissues of all three germ layers. d Immunofluorescence results of neural cells differentiated from one clinical-grade hiPSC line. The neural cells express TH1 and TUJ1 at protein level. Scale bar, 100 μm. e Immunofluorescence results of cardiomyocytes differentiated from one clinical-grade hiPSC line. The cardiomyocytes express NKX2.5 and CTNT at protein level. Scale bar, 100 μm. EB embryoid body, GMP good manufacturing practice, iPSC induced pluripotent stem cell
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Fig3: Clinical-grade hiPSCs can randomly differentiate into all three germ layers and directly differentiate into functional neural cells and cardiomyocytes in Xeno-free media. a Morphology of EBs at day 8 derived from one clinical-grade hiPSC line. Scale bar, 200 μm. b Gene expression profile for germ-layer marker genes of the day 8 EBs, ectoderm (Gad1 and Pax6), mesoderm (Enolase and Osteonectin), endoderm (Nicastrin and Alpha-fetoprotein), and pluripotency (Oct4, Sox2 and Nanog). c Hematoxylin and eosin staining of teratoma derived from one clinical-grade hiPSC line. Scale bar, 100 μm. The teratomas contain tissues of all three germ layers. d Immunofluorescence results of neural cells differentiated from one clinical-grade hiPSC line. The neural cells express TH1 and TUJ1 at protein level. Scale bar, 100 μm. e Immunofluorescence results of cardiomyocytes differentiated from one clinical-grade hiPSC line. The cardiomyocytes express NKX2.5 and CTNT at protein level. Scale bar, 100 μm. EB embryoid body, GMP good manufacturing practice, iPSC induced pluripotent stem cell

Mentions: The EB and teratoma formation assays are commonly used to evaluate the differentiation ability of hPSCs. Both of the clinical-grade hiPSC lines were grown in suspension in clinical-grade hESC basic medium. Eight days later, they all formed round EBs (Fig. 3a; and Figure S1E in Additional file 3). RT-PCR analysis showed that the EBs expressed markers of all three germ layers, including ectoderm (Gad1 and Pax6), mesoderm (Enolase and Ostenectin), and endoderm (Nicastrin and Alpha-fetoprotein) (Fig. 3b). The teratoma formation assay was performed by injection of the two hiPSC lines to the testis of SCID mice separately. Two months later, teratomas were visualized in all injected testes. Histological analysis revealed that the teratomas comprised tissues of all three germ layers (Fig. 3c; and Figure S1F in Additional file 3).Fig 3


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)

Clinical-grade hiPSCs can randomly differentiate into all three germ layers and directly differentiate into functional neural cells and cardiomyocytes in Xeno-free media. a Morphology of EBs at day 8 derived from one clinical-grade hiPSC line. Scale bar, 200 μm. b Gene expression profile for germ-layer marker genes of the day 8 EBs, ectoderm (Gad1 and Pax6), mesoderm (Enolase and Osteonectin), endoderm (Nicastrin and Alpha-fetoprotein), and pluripotency (Oct4, Sox2 and Nanog). c Hematoxylin and eosin staining of teratoma derived from one clinical-grade hiPSC line. Scale bar, 100 μm. The teratomas contain tissues of all three germ layers. d Immunofluorescence results of neural cells differentiated from one clinical-grade hiPSC line. The neural cells express TH1 and TUJ1 at protein level. Scale bar, 100 μm. e Immunofluorescence results of cardiomyocytes differentiated from one clinical-grade hiPSC line. The cardiomyocytes express NKX2.5 and CTNT at protein level. Scale bar, 100 μm. EB embryoid body, GMP good manufacturing practice, iPSC induced pluripotent stem cell
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4643509&req=5

Fig3: Clinical-grade hiPSCs can randomly differentiate into all three germ layers and directly differentiate into functional neural cells and cardiomyocytes in Xeno-free media. a Morphology of EBs at day 8 derived from one clinical-grade hiPSC line. Scale bar, 200 μm. b Gene expression profile for germ-layer marker genes of the day 8 EBs, ectoderm (Gad1 and Pax6), mesoderm (Enolase and Osteonectin), endoderm (Nicastrin and Alpha-fetoprotein), and pluripotency (Oct4, Sox2 and Nanog). c Hematoxylin and eosin staining of teratoma derived from one clinical-grade hiPSC line. Scale bar, 100 μm. The teratomas contain tissues of all three germ layers. d Immunofluorescence results of neural cells differentiated from one clinical-grade hiPSC line. The neural cells express TH1 and TUJ1 at protein level. Scale bar, 100 μm. e Immunofluorescence results of cardiomyocytes differentiated from one clinical-grade hiPSC line. The cardiomyocytes express NKX2.5 and CTNT at protein level. Scale bar, 100 μm. EB embryoid body, GMP good manufacturing practice, iPSC induced pluripotent stem cell
Mentions: The EB and teratoma formation assays are commonly used to evaluate the differentiation ability of hPSCs. Both of the clinical-grade hiPSC lines were grown in suspension in clinical-grade hESC basic medium. Eight days later, they all formed round EBs (Fig. 3a; and Figure S1E in Additional file 3). RT-PCR analysis showed that the EBs expressed markers of all three germ layers, including ectoderm (Gad1 and Pax6), mesoderm (Enolase and Ostenectin), and endoderm (Nicastrin and Alpha-fetoprotein) (Fig. 3b). The teratoma formation assay was performed by injection of the two hiPSC lines to the testis of SCID mice separately. Two months later, teratomas were visualized in all injected testes. Histological analysis revealed that the teratomas comprised tissues of all three germ layers (Fig. 3c; and Figure S1F in Additional file 3).Fig 3

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