The effectiveness and safety of mesenchymal stem cell therapy have been demonstrated in a variety of diseases. However, problems such as the limited expansion ability of adult-derived mesenchymal stem cells in vitro and the instability of clinical results caused by the high heterogeneity of isolated cells need to be solved urgently. Inducement and differentiation of iPSC into iPSC-derived mesenchymal like MSCs (iPSC-MSC) through a specific experimental route is the most potential product to replace adult MSCs. Many studies have proved that iPSC can obtain iPSC-MSC through multiple differentiation strategies, and studies have shown that compared with adult MSC, iPSC-MSC has better or equivalent characteristics such as proliferation ability, immunoregulatory ability, and biological efficacy than adult MSC.

The author attempted to discuss the development prospects of such cell therapy products with readers from four aspects: methodological research on iPSC-MSC preparation, application of iMSC products in different disease models, latest research progress on clinical grade iPSC-derived cell products, and development strategies for new generation iPSC-derived products, in order to discuss the development prospects of similar pipelines, thereby enriching product development ideas. Regenerative medicine and cell therapy have become the focus of advanced biomedical technologies in various countries around the world today. Its mesenchymal stem cell (MSC) therapy has unlimited potential and has been proven to be safe and effective in clinical trials and research on a variety of diseases. It has attracted widespread attention.

MSC belongs to adult pluripotent stem cells. Due to its high multi-directional differentiation potential, low immunogenicity, few ethical issues and extensive sources, MSC has always been an important direction of stem cell research [1]. MSC has functional properties such as immune regulation, anti-inflammation, supporting hematopoietic function, and tissue repair [2], and can be used in the treatment of autoimmune diseases such as lupus erythematosus, inflammatory diseases such as osteoarthritis, aging-related diseases, and infectious diseases [3]. However, due to different MSC cell sources, different donor conditions, differences in culture systems, cell heterogeneity, limited in vitro expansion capabilities, difficulties and limitations in isolation and extraction, etc.[4], MSC cell therapy has some limitations and unstable effects in clinical application [5].

Progress in methodological research on obtaining iPSC-MSC from iPSC

The author summarized the preparation methods of the mainstream iPSC-derived MSC (iMSC) currently on the market [6]. The direction of differentiation can be roughly classified into three aspects. The mainstream differentiation strategies include MSC medium replacement induction method, signal pathway regulation induction method, and embryoid body method, etc.[7]. In the following, details of the key materials and methods used in each reported research protocol are described in detail. Each study also evaluated the cell surface markers and multi-directional differentiation potential of the derived iMSC. Statistics were also made for the overall differentiation time of each protocol in order to find an efficient and safe differentiation protocol that can be used for industrial production.

(1) The MSC medium replacement induction method can induce the spontaneous differentiation of iPSC by replacing iPSC medium with MSC culture system, and play a role in inducing and selecting cells. MSC basic medium components include high/low sugar DMEM, KO-DMEM (specific component knockout DMEM), DMEM-F12 and α-MEM, etc., and are also supplemented with FBS, KOSR, L-glutamine, P/S, non-essential amino acids and FGF. Kang[8] et al. reported that iMSC cells in the form of fibroblasts could be obtained by directly replacing iPSC medium with DMEM medium containing 10% FBS for two weeks. After passage, the cells were continuously passed in pre-coated culture dishes. Similar induction conditions can also be obtained using α-MEM medium [20]. The overall induction efficiency using this method is low, such as obtaining iMSC with qualified phenotypes (high expression of CD73, CD44, CD105, CD90; low expression of CD19, CD34, CD45, HLA-DR, etc.) and having the ability to differentiate into three lines [10], and the time is as long as 30-50 days.

(2) Signaling pathway modulators induce iMSC to form pathway inhibitors is a common method for iPSCs to induce MSCs. It can quickly and efficiently induce iPSCs to the MSC cell phenotype and have the function of mesenchymal stem cells. Inhibitors used for iPSC-MSCs induction include SB203580 (p38-MAPK inhibitor), SB431542 (TGF-β inhibitor), CHIR (GSK3 inhibitor), β-mercaptoethanol, b-FGF, etc.

-SB203580

SB203580 is a pyrimidazole p38 MAPK inhibitor that also has inhibitory activity on kinases such as GAK, CK1, RIP2, c-Raf and GSK3. The p38-MAPK pathway can promote the differentiation of MSC into epidermal-like cells [11], can also inhibit IL-2-mediated T cell proliferation, participate in various inflammatory reactions or induce autophagy in vivo. It has been reported that SB203580 can promote the transformation of iPSC cells to mesodermal phenotypes [12]. By suspending iPSC to form embryoid bodies (EB), adding a certain concentration of SB203580 to the culture system to induce embryoid bodies, the induced embryoid bodies were cultured and passaged. After 28 days, iMSC cells with qualified surface markers were obtained. After 20 generations, the karyotype was complete and the cells did not age.

-SB431542

SB431542 is an inhibitor of the TGF-β signaling pathway, which is the main signaling pathway that maintains stem cell pluripotency. Inhibition of the TGF pathway can induce the differentiation of ESCs and iPSC into MSC or neuronal cells [13]. Glaeser et al. used 20 μM SB431542 to induce ESCs into neural crest cells step by step over 10-14 days, and then converted to a serum-containing culture system to transform neural crest cells into MSC-like cells [14]. Lee and Sun[15] et al. added 10 μM SB431542 throughout the entire iMSC induction process, and after several passages, they obtained iMSC cell lines with triple-line differentiation capabilities. The effective concentrations of SB431542 used by different researchers ranged from 1 μM to 20 μM [16], and there was no significant difference in the induction efficiency of iMSC.

-CHIR-99021

CHIR-99021 is a highly specific glycogen synthase kinase-3 (GSK-3) inhibitor that has been shown to promote self-renewal and maintain pluripotency in BALB/c mouse ES cells. CHIR-99021 is involved in multiple signaling pathways including Wnt/β-catenin, TGF-β, Nodal and MAPK [17]. Different concentrations of CHIR99021 are often used in combination with SB431542 to induce differentiation of iMSCs. Harada et al.[18] used a compound combination of 10 μM SB431542 +1 μM CHIR99021 to successfully induce mesenchymal phenotypic cells in about 21 days following the iPSC-neural crest cells-MSC-like cell route. Winston et al.[19] obtained iMSC in about 25 days using 4 μM SB431542 +4 μM CHIR99021 +10 ng/mL β-FGF. Compared with direct conversion of MSC medium, the addition of signaling pathway regulators has a higher efficiency in inducing iMSC maturation, effectively shortening the overall differentiation time, and the obtained cells are not significantly different from primary MSC cells in terms of phenotype, triple-line differentiation ability and genomic expression [20].

-β-mercaptoethanol

β-mercaptoethanol is also a commonly used additive during iMSC induction. Liang, McGrath, Hua and Lim et al. added different concentrations of β-mercaptoethanol (final concentrations were 55uM, 0.1mM, and 110mM respectively) to the iPSC culture system. Fibroblast-like iMSC cells were obtained after about 30 days. It was identified that the cell surface markers and differentiation abilities of the three lines were similar to those of BM-MSC.

(3) The embryoid body method can harvest more target cells in a limited culture volume through the embryoid body (EB)-mediated iMSC differentiation path, which is conducive to industrialization and cost control. However, the overall culture process takes a long time and the cells produced have certain heterogeneity. Researchers usually add compound pathway modulators to the culture system during the formation of embryoid bodies and the climbing out of iMSC to improve the efficiency of cell enrichment into iMSC.

Chen YS used DMEM-F12 medium containing 20% KOSR and 0.1 μg/mL bFGF to suspend cultured iPSC to form EB. Then, SB431542 at a concentration of 10 μM was added to the system to promote cell climbing out, and then iMSC was obtained through cell passage [22]. Zhou et al.[23] developed a method for large-scale expansion of embryonic stem cells in a bioreactor. ESC was cultured in suspension to obtain a large amount of EB, and then a KOSR-containing system was used to induce EB differentiation, and further induce EB to climb out to obtain ES-MSC. The researchers also further optimized the induction system and developed a new formula to induce differentiation. Adding a certain concentration of b-FGF and TGFβ-1 to the system to promote the crawling and maturation of ES-MSC [24]. In addition, cytokines including Activin A, VEGF, BMP4, RA[25] are also used in the induction process of differentiation through embryoid bodies, and the number of days of differentiation varies from 20 to 30 days.