As the final chapter of this series of discussions, this article will focus on discussing the strategies of this type of iPSC-MSC in the commercial development process of products, including the main research ideas and matters needing attention in the development of functionally enhanced products and derived cells and biologics, and discuss issues that need to be focused on during the development of advanced therapeutic drugs (ATMP). Whether ATMP R & D ideas are developing MSC or iMSC cells into an advanced therapeutic product, the core issue in the drug process is how to carry out the research and development of drug manufacturing processes and how to achieve good quality control of stem cell drugs.

Pharmaceutical companies need to be based on the concept of Quality by Design (QbD), based on reasonable science and quality risk management, start from predetermined quality goals, and focus on the understanding of MSC/iMSC products and their processes. Research and development methods for production process control systems (ICHQ8). Enterprise R & D personnel also need to identify Critical Quality Attributes (CQAs) that directly affect product safety and effectiveness based on risk assessment, find Critical Process Parameters (CPP), develop a design space to quantify the impact of parameter variability on quality attributes, and then develop control strategies to maintain process parameters to ensure product quality, and scale up process verification.

Difficulties in the MSC/iMSC product development process include: how to establish an experimental protocol that conforms to GMP standards, factors affecting the selection of raw materials, cell expansion into formulation formulations, establishing quality control (QC) parameters, and quality standards that comply with GMP requirements [1]. The critical quality parameters (CQAs) of MSC products include four dimensions: identification, purity, biological efficacy and safety of MSC products.

In addition, more research work is still needed on the comparison of iMSCs and tissue-derived MSCs in terms of regeneration, differentiation and immunoregulation. When developing iMSC products, additional attention needs to be paid to the heterogeneity of the product, and different subpopulations of iMSC cells can be further analyzed through single cell transcriptome sequencing methods. In addition, because iPSC seed cells upstream of the process have potential tumorigenic risks, it is necessary to develop stable and highly sensitive methods for detecting iPSC residues and unexpectedly differentiated cell residues to prove that the final product is not tumorigenic [2].

Compared with primary MSCs, iMSC products will undergo a longer in vitro culture and passage process. Whole genome sequencing is required to pay attention to the genetic safety of iMSC products. By standardizing standardized quality control procedures and establishing manufacturing standards, we ensure that mesenchymal stem cells from different sources or iMSC cells from different differentiation paths have consistent quality and efficacy, thereby improving product consistency, reliability and safety and promoting its transformation into GMP level clinical application products. Development of genetically modified iMSC second-generation products The current main areas for commercial use of iPSC and its derivatives include cell therapy, disease modeling, drug development and discovery, personalized medicine and toxicology screening. Combined with the research and development and market performance of this type of product, we found that in addition to the advantages of simple preparation process, consistent cell source, good uniformity, and suitable for large-scale production, iMSC products are also more convenient to genetically modify from seed cells, thereby creating iPSC-derived cells. Second generation product. Studies have reported that CAR-iPSC formed by specifically editing iPSC cells can be used as seed cells for stable CAR-NK cell production [3].

CRISPR/Cas9 gene editing technology can also be widely used for genetic modification of MSCs [4]. Genetically modifying MSC cells can be freed from the stimulation of exogenous factors and enhance their therapeutic potential. Sustained enhancement of expression of multiple genes can significantly improve the efficacy of MSC. There are significant differences in the functions played by different genes in different indications. The therapeutic effect of MSC is highly dependent on the ability to migrate and homing in vivo, which is related to chemokine receptors expressed on MSC and chemokines in tissues. One of the functionally enhanced genetic modifications is to overexpress specific chemokine receptors on MSCs (such as CCR1 and CXCR2).

There are also reports that IL-4, IL-10, TGF-β1, GATA-4 and CXCR4 genes have the effect of improving stem cell viability and enhancing curative effect [5]. Overexpression of VEGF in BM-MSCs promotes angiogenesis and improves cerebral infarction, increases cell viability and enhances paracrine effects [6]. To achieve the effect of hypoxia pretreatment, transfection of HIF-1α into BM-MSCs can simulate its stronger therapeutic effect under hypoxia exposure [7]. However, the application of genetic modification to primary mesenchymal stem cells requires long-term culture and screening, which will inevitably cause replicative aging of cells and reduce the therapeutic effect of MSC. It is not suitable for practical clinical applications and development of cell therapy products.

Therefore, selecting suitable genes for specific indications, functionally editing the iPSC as the seed for differentiation, and further inducing differentiation to form iMSC can help researchers obtain iMSC cells that are stably modified by specific genes and have low heterogeneity. This operation will help develop functionally enhanced iPSC-derived second-generation products. However, sustained activation or knockdown of a certain gene still requires long-term and complex preliminary demonstration to determine the safety of the product. iMSC-derived products-exosomes (iMSC-EVs) have broad application prospects in immune regulation and tissue regeneration as exosomes released by mesenchymal stem cells (MSC-Exos) with advanced therapeutic possibilities. The main functions of exosomes can be divided into tissue repair and anti-fibrosis, and have therapeutic potential for various types of indications (such as knee osteoarthritis, spinal cord injury, skin injury, liver, kidney and lung tissue fibrosis, neurologic related diseases, etc.)[8].

A series of preclinical studies have also proved that, similar to the biological functions of MSC-EVs, iMSC-derived EVs also have good therapeutic effects on multiple indications [9]. At present, EVs as a core difficulty in drug development are mainly reflected in two aspects: the batch-to-batch difference caused by the heterogeneity of exosome-derived cells and the operability of large-scale production and purification. The iMSCs are derived from a uniform iPSC seed. Appropriate iPSC cell lines can be screened in the early stage of the process and iMSCs with high homogeneity can be obtained through a stable differentiation process. Exosomes can be prepared from these cells, which directly overcomes the heterogeneity of primary cells and ensures a small batch-to-batch difference of the product.

In addition, iMSC products obtained through appropriate preparation processes can ensure sustained and strong in vitro expansion capabilities of cells, thereby expanding the production scale of EVs by directly expanding the scale of upstream seed cells. In addition, the efficacy of iMSC-EVs can also be improved in terms of improving the concentration, targeting and loading of functional molecules. iMSCs can be cultured in perfusion or batch at reactor scale through 3D process to accelerate the cell growth rate and accelerate the secretion of exosomes; or iMSCs can be pretreated with small molecule drugs to regulate and increase the secretion of exosomes; iMSCs can also be genetically modified to allow specific target molecules to be fused and expressed with proteins on the surface of exosomes to enhance the load of exosomes on functional molecules [10].

For exosome products, it is necessary to strictly refer to the exosome-related research specifications in the international standard (ISO 24603) to standardize the upstream steps of exosome production. Two group standards have also been recently released in China,"Small extracellular vesicles derived from human pluripotent stem cells"(T/CRHA002 -2021) and "Small extracellular vesicles derived from human mesenchymal stem cells"(T/CRHA001 -2021). Key quality attributes of exosomes should be paid attention to in product development include:

1. The key quality attributes of human mesenchymal stem cells used to produce small extracellular vesicles should comply with regulations;

2. Key quality parameters such as shape, particle size, marker protein, purity, immunomodulatory ability, and microbial safety of small extracellular vesicles should meet the standards;

3. Detailed process control: Record the isolation method of small extracellular vesicles in detail. In the "Summary of the Second Classification and Definition Results of Medical Device Products in 2024" released by the State Food and Drug Administration on October 31 this year, it was clarified that stem cell exosomes are products that are recommended not to be managed as medical devices. The "identity" in which exosomes can be put on the market and used in patients still requires continuous in-depth research on exosomes, gradual improvement of exosomes production specifications and quality standards and other regulations, and EVs derived from iMSC will gradually show potential advantages and gradually realize the transformation and application into GMP-level products.

Summary: As summarized in this series of articles, iPSC-MSC has superior biological characteristics and good therapeutic effects. However, we must realize that the core determinants of iMSC preparation are similar to MSC. In addition to pharmacy, it also includes clinical efficacy. The clinical efficacy of patients after MSC infusion varies greatly. The key factor is that the in vivo treatment mechanism of MSC is very complex and unclear. Although a large number of in vitro studies have recognized that MSC has multi-lineage differentiation potential, can play immune regulation and anti-inflammatory functions, produce cytokines, exosomes, and exosicles through paracrine effects, or play anti-fibrosis by affecting the transfer of mitochondria and other cell organelles [11], the changes in the human body are unclear. In clinical practice, R & D personnel still need to establish a correlation between efficacy and biological activity indicators.

If key effector cells and key acting molecules can be found, pathways related to the mechanism of action can be detected, and analytical methods that correlate with product characterization and clinical outcomes can be developed, the probability of MSC being overdosed will be significantly increased. At this stage, my country has issued the "Technical Guidelines for Pharmaceutical Research and Evaluation of Human-derived Stem Cell Products"(Trial Implementation), which stipulates the product quality standards and clinical application standards. Therefore, before iPSC and its derived cell products are truly used in clinical practice, it is necessary to establish a reliable, systematic safety assessment system and release that runs through the entire product production process, including residual testing of pluripotent stem cells in the final product, clone formation experiments, nude mouse tumorigenesis experiments, Long-term tumorigenicity experiments in animals are used to strictly control the production process and product quality. In terms of the selection of testing methods, testing costs, experimental cycles, data analysis workload and complexity of result analysis should all be comprehensively considered and improved and improved based on practice.