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Special Column for Quality Research and Evaluation of Stem Cell Products
Editor: Invited Column Editor-in-chief: RAO Chun-ming, PANG Lin

[Editor’s Note] 

Stem cells are a class of primitive, undifferentiated cells characterized by their capacity for self-renewal, intrinsic homing capability, low immunogenicity, multipotent differentiation potential, and the ability to regenerate various tissues and organs. These unique biological properties make them a promising therapeutic tool for numerous intractable and severe human diseases. Over the years, the significant global attention has been directed toward advancing stem cells research and clinical translation, with the publication of a series of guidelines to facilitate their clinical application. In 2008, the International Society for Stem Cell Research (ISSCR) published the Guidelines for the Clinical Translation of Stem Cells. In 2009, the International Stem Cell Forum (ISCF) released the Guidelines for the Derivation and Distribution of Human Embryonic Stem Cell Lines, which provided foundational standards for the translational development of stem cell-based therapies. Subsequently, regulatory agencies, including the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), the World Health Organization (WHO), the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), ISSCR, and ISCF, have developed technical specification and updated technical guidelines. These guidelines encompass quality control from the procurement of raw materials to the final product, as well as preclinical and clinical studies. As of October 23, 2024, a keyword search for “stem cell” on ClinicalTrials.gov identified 13 021 globally registered clinical trials focused on stem cell-based therapies, including 1 942 studies with published results. To date, over 20 stem cell-based pharmaceutical products have been approved for market worldwide. Stem cell therapy has thus emerged as a critical focus in international biomedical research, driving innovation and advancing the frontiers of regenerative medicine. 

In our country, the National Health and Family Planning Commission and the China Food and Drug Administration jointly issued the Administrative Measures for Stem Cell Clinical Research (Trial) on July 20, 2015. Shortly thereafter, on July 31, 2015, the two agencies collaboratively formulated the Guidelines for Quality Control of Stem Cell Preparations and Preclinical Research (Trial) to ensure the safety and efficacy of stem cell therapies. In recent years, the Center for Drug Evaluation (CDE) of the National Medical Products Administration (NMPA) has released a series of relevant technical guidelines, including: Technical Guidelines for Research and Evaluation of Cell Therapy Products (Trial) (December 2017), Technical Guidelines for Pharmaceutical Research and Evaluation of Human Stem Cell Products (Trial) (April 2023), and Technical Guidelines for Nonclinical Research of Human Stem Cell Products (Trial) (January 2024). As of the end of December 2024, a total of 159 stem cell pharmaceutical products have submitted IND applications to the NMPA. Among these, 72 products have been granted clinical trial approval by default. The indications for these therapies include pulmonary fibrosis, knee osteoarthritis, bone fractures, liver cirrhosis, acute-on-chronic liver failure, stroke, rheumatoid arthritis, diabetic foot ulcers, Crohn’s disease, and others.Crohn's disease, and others. On January 2nd, 2025, China's first stem cell therapy drug, Amy Metotrexate Injection (trade name: Ruibo Sheng), was approved for marketing, marking a new stage in China's stem cell therapy field. 

Compared with other pharmaceuticals, stem cells possess self-renewal and multipotent differentiation capabilities. As a result, stem cell products exhibit diverse cell types, with the cells themselves capable of in vivo survival, proliferation, differentiation, and intercellular interactions. As living, sterile injectable medicines, stem cell products are exposed to significant variability in aspects such as cell source, type, expansion, genetic modification, and induced differentiation during production. This inherent heterogeneity and instability result in relatively short usage windows and present challenges in ensuring consistent quality. Consequently, quality control for stem cell products is more complicated, necessitating the development of novel, rapid, and effective testing methodologies and technologies. To tackle these challenges, we applied for and undertook part of the CGT (Cell and Gene Therapy) research project funded by the Beijing Municipal Science and Technology Commission. The project, titled Key Technologies for Quality Control and Service Platform Construction for Genetically Modified Immune Cells and Gene Therapy Drugs, includes the development of innovative testing methods for stem cell products. These advancements lay a foundation for enterprises to conduct testing required for clinical research applications or new drug approvals for stem cell products. 

To embrace the forthcoming boom in stem cell product research, we have organized a team of stem cell product quality analysis experts to author several specialized articles. These contributions form a three-part series titled Special Column on Quality Research and Evaluation of Stem Cell Products. The series covers novel techniques and methods for quality control and testing of stem cell products, as well as the establishment of quality systems for stem cell testing laboratories. This issue features four articles, including: “Research Progress on Quality Control Analysis Methods for Stem Cell Products” “Biological Activity Methods Development of Mesenchymal Stem Cell Products” “Establishment of RT-qPCR Method for Telomerase Catalytic Subunits of Mesenchymal Stem Cells” and “The Construction of AAV Vector-HPV16-HPV18-HPyV Pseudoviruses and Application in the Examination of Human-derived Viruses in Stem Cells”. We hope the insights shared in this column will serve valuable opinions for relevant enterprises and research institutions, accelerating the clinical research and industrialization of stem cell products.

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  • Special Column for Quality Research and Evaluation of Stem Cell Products
    Research progress on quality control analysis methods for stem cell products*
    YANG Ying, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 4-11. https://doi.org/10.16155/j.0254-1793.2024-1299
    Abstract (71) PDF (110)   Knowledge map   Save
    Stem cells have varying degrees of proliferation, self-renewal, and differentiation potential, and can be applied in regenerative medicine and the treatment of various diseases. To ensure safety and effectiveness of stem cell products, it is important to establish quality control methods and standards. Herein, we review the regulations and guidelines for stem cell products, and provide an overview of the detection assays on the basic biological characteristics, microbiological safety, biological safety, biological effectiveness and other conventional testing methods and quality research methods for stem cells. We further describe the quality research methods for genetically modified stem cell products, functional cells derived from stem cells, and extracellular vesicles, etc.
  • Special Column for Quality Research and Evaluation of Stem Cell Products
    Research on biological activity methods of mesenchymal stem cell products*
    WANG Yao, FANG Ji-qing, YUAN Zi-wei, LI Yao-ling, YANG Ying, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 12-19. https://doi.org/10.16155/j.0254-1793.2024-1325
    Abstract (133) PDF (110)   Knowledge map   Save
    Objective: To explore the biological function methods of mesenchymal stem cells(MSCs) for quality analysis. Methods: The surface markers of MSCs were detected by flow cytometry. MSCs osteogenic differentiation was induced by ascorbic acid and β-glycerophosphate sodium, etc., followed by Alizarin Red S staining. MSCs adipogenic differentiation was induced by IBMX, Rosiglitazone, etc., followed by Oil Red O staining. MSCs could differentiate into chondrocytes with treatment of ITS and TGFβ3, etc., followed by Alcian Blue staining. Cell co-culture of THP-1-macrophage with MSCs and ELISA assay were applied to detect the effects of MSCs on macrophage polarization. The expression levels of IL-10 and TNF α in the cell co-culture supernatant were detected by ELISA. To observe the effects of MSCs on lymphocyte proliferation, MSCs cultured with PBMCs, which were labeled with CFSE and activated by CD3/CD28, followed by flow cytometry. Results: The expression of MSCs surface markers, CD105, CD73, and CD90, was more than 95% respectively, while the expression of CD45, CD34, CD14, CD19, and HLA-DR expression was less than 2%. MSCs osteogenic differentiation assay showed red calcium nodules. Red lipid vacuoles were observed in MSCs adipogenic induction differentiation. Furthermore, MSCs have the differentiation potential to chondrocyte spheroids, and typical cartilage pits were observed. Co-culture of MSCs with THP-1 macrophages, an increase in IL-10 expression and downregulate TNFα secretion were observed. MSCs played inhibitory effects on the proliferation of PBMC activated by CD3/CD28, with an inhibition rate of 76.4%. Conclusions: This study established some of biological activity detection methods for MSCs, including MSCs surface markers, differentiation abilities, promotion of macrophage polarization, and inhibitory effects on lymphocyte proliferation. It provides a potential application for MSCs products quality control.
  • Special Column for Quality Research and Evaluation of Stem Cell Products
    Establishment of RT-qPCR method for telomerase catalytic subunits of mesenchymal stem cells*
    CHEN Xiao-fei, LI Hui-ting, DONG Ying-ying, CAO Yi-dan, FU Xin-yue, LIU Ming-yue, ZHANG Rui-rui, WANG Yan-hui, WANG Xin-yue, CUI Meng-shan, ZHANG Tong, PANG Lin, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 20-29. https://doi.org/10.16155/j.0254-1793.2024-1333
    Abstract (124) PDF (84)   Knowledge map   Save
    Objective: To establish a simple and efficient method for detecting telomerase activity for evaluating tumorigenic risk of cell products. Methods: Specific primers and probes were designed for the conserved domain of telomerase catalytic subunit (TERT), and the primers and probes of TERT gene were optimized and screened, and the primers and probes of internal reference genes were set up. Multiple quantitative PCR reaction was performed using two-color fluorescent probes in a reaction system, and RT-qPCR probe method was established. The expression of TERT gene in human mesenchymal stem cells HMSC was used to determine whether telomerase activity existed in the cells indirectly. Results: TERT gene of 293T/17 positive control cells could be stably and specifically detected by this method, with a mean Ct value of 23.96 and a RSD of 1.5. The internal reference gene GAPDH could be detected successfully, the mean Ct value was 14.13, the RSD was 1.3%. The reference gene GAPDH in MRC-5 could be detected in negative control cells, the mean Ct value was 12.81, the RSD was 0.46%, the TERT gene was not detected, and the telomerase activity was negative. The reference gene GAPDH in HMSC could be detected, the Ct mean value was 13.01, and the RSD was 3.8%, whereas, the telomerase activity of HMSC was negative. Conclusion: The real-time fluorescent quantitative RT-qPCR probe method established in this study can accurately detect the expression of catalytic subunit mRNA in telomerase positive cells with good repeatability and high specificity. It can be used to analyze telomerase activity of stem cells and indirectly evaluate tumorigenic risk of cell products derived from stem cells.
  • Special Column for Quality Research and Evaluation of Stem Cell Products
    The construction of AAV vector-HPV16-HPV18-HPyV pseudoviruses and application in the examination of human-derived viruses in stem cells*
    ZHANG Tong, CHEN Xiao-fei, DONG Ying-ying, CAO Yi-dan, WANG Yan-hui, LI Hui-ting, LIU Ming-yue, WANG Xin-yue, CUI Meng-shan, FU Xin-yue, ZHANG Rui-rui, PANG Lin, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 30-38. https://doi.org/10.16155/j.0254-1793.2024-1301
    Abstract (41) PDF (33)   Knowledge map   Save
    Objective: To employ adeno-associated viruses as vectors to design and prepare pseudoviruses comprising multiple DNA viral gene fragments. These were used as positive controls for the detection of human viruses in the field of molecular testing, thereby compensating for the absence of wild-type viral controls in viral molecular testing methods. Methods: The adeno-associated viral vectors served as the backbone of the pseudoviruses, with a total of four target gene fragments from three viruses incorporated: human papillomavirus type 16 (HPV16), type 18 (including HPV18-1 and HPV18-2) and human polyomavirus (HPyV) were, respectively, inserted into one viral vector, and the adeno-associated viral pseudoviruses were packaged by multiple plasmid cotransfection. The infectious activity of the pseudoviruses was confirmed by a cell infection assay, and a quantitative analysis of the pseudoviruses genome was conducted using fluorescence quantitative PCR. Results: The titers of HPV16, HPV18-1, HPV18-2, and HPyV genomes in the pseudoviruses were 7.77×108 copies · mL-1, 6.77×108 copies · mL-1, 7.04×108 copies · mL-1, and 1.24×109copies · mL-1, respectively, as determined by fluorescence quantitative PCR. Following a 48 h period of infection in 293T/17 cells, the viral gene sequences of HPV16, HPV18-1, HPV18-2, and HPyV were successfully identified using fluorescence quantitative PCR with the intracellular pseudoviruses. The copy numbers were 1.30×106 copies · mL-1, 6.59×105 copies · mL-1, 6.27×105 copies · mL-1, and 4.17×106 copies · mL-1. Following the infection of 293T/17 cells with the reporter gene pseudoviruses for a period of 48 hours, a distinct green fluorescence was evident under a fluorescence microscope, thereby confirming the infectious activity of the pseudoviruses. The pseudoviruses was employed as a positive control for the verification of applicability in human mesenchymal stem cells, and the recoveries of the four viral gene fragments by fluorescence quantitative PCR for nucleic acid extraction were 94.4%, 70.7%, 83.1% and 90.9%, respectively. Conclusion: The present study demonstrates that the adeno-associated virus packaging method can be employed to produce a multiviral gene pseudoviruses with infectious activity. The pseudoviruses can be employed as a positive control for the replacement of multiple wild-type viruses in the viral fluorescence quantitative polymerase chain reaction (PCR) of stem cell samples.
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