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Journal of Neurorestoratology  2021, Vol. 9 Issue (1): 13-30    doi: 10.26599/JNR.2021.9040005
Special Reports     
Standards of induced pluripotent stem cells derived clinical-grade neural stem cells preparation and quality control (2021 China version)
Meng Cai1,Fabin Han2,Nanxiang Xiong3,(✉)(),Yihao Wang4,Shiqing Feng5,Jiajing Wang4,Xiang Li3,Jun Wei1,Changkai Sun6,On behalf of Chinese Association of Neurorestoratology (Preparatory), China Committee of International Association of Neurorestoratology and Special Committee of Neurorestoratology,Chinese Medical Doctor Association
1iRegene Therapeutics Ltd., Wuhan 430000, Hubei, China
2The Institute for Tissue Engineering & Regenerative Medicine, Liaocheng University / Liaocheng People’s Hospital, Liaocheng 252000, Shandong, China
3Department of Neurosurgery of Zhongnan Hospital, Wuhan University, Wuhan 43071, Hubei, China
4Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China
5Department of Orthopedics, Tianjin Medical University, Tianjin 300070, China
6The Research and Educational Center for the Control Engineering of Translational Precision Medicine, School of Biomedical Engineering, and the Key Laboratory of Integrated Circuit and Biomedical Electronic System of Liaoning Province, Dalian University of Technology, Dalian 116024, Liaoning, China
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Abstract  

Induced pluripotent stem cells (iPSCs) have become the leading research object in the clinical application of restorative medicine. They are easily generated from diverse cell sources and functionally indistinguishable from embryonic stem cells without the accompanying ethical issues. To date, the use of iPSC-derived neural stem cells and their progeny in the treatment of neurodegenerative and injurious diseases has achieved good results, with great potential in cell drug development. However, because of some unique biological properties and differences from traditional drug production processes, cell drug research and development has many problems that can hinder clinical applications. Given this situation, the Chinese Association of Neurorestoratology (Preparatory) and China Committee of the International Association of Neurorestoratology have organized relevant professional experts to formulate the standard presented here. Overall, the aim was to promote the clinical application of neural stem cells (NSCs) and their further derived neural cells from iPSC sources and promote cell drugs’ production and development. This standard refers to the latest research results, quality evaluation criteria for traditional medicines, and the regulatory framework for cellular treatments. The standard considers general biological properties of cells, including cell morphology, cell cycle, karyotype, and cell viability. The specific biological properties of NSCs, such as cell surface markers and differentiation ability, general drug standards, such as aseptic testing, endotoxins, human virus detection, and cell-related drug standards, such as telomerase activity and tumorigenicity, are also considered. This standard will serve as a reference for physicians and scientists who focus on clinical nervous cell applications and studies related to iPSCs.



Key wordsinduced pluripotent stem cells      preparation      quality control      standard      clinical-grade      neural stem cells     
Received: 03 November 2020      Published: 17 June 2021
Corresponding Authors: Nanxiang Xiong     E-mail: mozhuoxiong@163.com
Cite this article:

Meng Cai, Fabin Han, Nanxiang Xiong, Yihao Wang, Shiqing Feng, Jiajing Wang, Xiang Li, Jun Wei, Changkai Sun. Standards of induced pluripotent stem cells derived clinical-grade neural stem cells preparation and quality control (2021 China version). Journal of Neurorestoratology, 2021, 9: 13-30.

URL:

http://jnr.tsinghuajournals.com/10.26599/JNR.2021.9040005     OR     http://jnr.tsinghuajournals.com/Y2021/V9/I1/13

Test itemSpecificationTest method
Cell morphologyConsistent with the morphology of iPSCMicroscopy
Cell viability≥ 85% (live cells) or ≥ 70% (frozen cells)Annexin V and PI staining by flow cytometry
BacteriaNegativeChinese Pharmacopeia 2020, IV, 1101
FungusNegativeChinese Pharmacopeia 2020, IV, 1101
Endotoxin< 2.0 EU/mLChromogenic substrate Limulus amebocyte lysate (LAL) assay; Chinese Pharmacopeia 2020, IV, 1143
MycoplasmaNegativeChinese Pharmacopeia 2020, IV, 3301
HCV, HBV, EBV, CMV, HIV, and TPNegativeELISA or quantitative PCR
Cell cycleIn the period of G0\S\G2\M and no sub-G1Flow cytometry
Cell doubling timeReport resultCell culture
STR (short tandem repeat) analysisSingle sourcePCR
Chromosome karyotype analysis46, XX or 46, XYFluorescence in situ hybridization
OCT4-positive rate≥ 70%Flow cytometry
NANOG-positive rate≥ 70%Flow cytometry
alkaline phosphatase (AP)-positive rate≥ 95%AP colorimetric assay
Telomerase activityReport resultQuantitative PCR or immunoassay
Differentiation ability (in vitro)Differentiated NSCsCell culture
Differentiation ability (in vivo)Form teratomas with three germ layers: endodermal, mesodermal, and ectodermalTeratoma formation assay
Genomic mutationsReport resultNext-generation sequencing
Table 1Quality requirements and testing method for iPSCs.
Test itemSpecificationTesting method
Cell morphologyConsistent with the morphology of NSCMicroscopy
Cell viability≥ 85% (live cells) or ≥ 70% (frozen cells)Annexin V and PI staining by flow cytometry
BacteriaNegativeChinese Pharmacopeia 2020, IV, 1101
FungusNegativeChinese Pharmacopeia 2020, IV, 1101
MycoplasmaNegativeChinese Pharmacopeia 2020, IV, 3301
Endotoxin< 2.0EU/mLChromogenic substrate LAL assay; Chinese Pharmacopeia 2020, IV, 1143
HCV, HBV, EBV, CMV, HIV, and TPNegativeELISA or quantitative PCR
Cell cycleIn the period of G0\S\G2\M and no sub-G1Flow cytometry
Cell doubling timeReport resultCell culture
STR analysisSingle sourcePCR
Chromosome karyotype analysisExpected 46, XX or 46, XYFluorescence in situ hybridization
PAX6-positive rate≥ 80%Flow cytometry
NURR1-positive rate≥ 80%Flow cytometry
Differentiation abilityDifferentiate into dopamine neuronsCell culture
Immunological reactionReport resultPBMC coculture proliferation assay
Telomerase activityReport resultQuantitative PCR or immunoassay
TumorigenecitySoft agar colony formation test and in vivo detection show no clone or tumor formedChinese Pharmacopeia 2020, III
Genomic mutationsReport resultNext-generation sequencing
Table 2Quality testing items and standards for clinical-grade NSCs
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