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Background: Placenta-derived human mesenchymal stem cells (PL-MSCs) have gained a lot of attention in the field of regenerative medicine due to their availability and bone-forming capacity. However, the osteogenic differentiation capacity of these cells remains inconsistent and could be improved to achieve greater efficiency. Although metformin, a widely used oral hypoglycemic agent, has been shown to increase bone formation in various cell types, its effect on osteogenic differentiation of PL-MSCs has not yet been investigated. Therefore, the objective of this study was to examine the effect of metformin on the osteogenic differentiation capacity of PL-MSCs and the underlying mechanisms., Methods: The PL-MSCs were treated with 0.5 to 640 µM metformin and their osteogenic differentiation capacity was examined by an alkaline phosphatase (ALP) activity assay, Alizarin red S staining and expression levels of osteogenic genes. The role of adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling in mediating the effect of metformin on the osteogenic differentiation capacity of PL-MSCs was also investigated by determining levels of phosphorylated AMPK (pAMPK)/AMPK ratio and by using compound C, an AMPK inhibitor., Results: The results showed that 10-160 µM metformin significantly increased the viability of PL-MSCs in a dose- and time-dependent manner. Furthermore, 80-320 µM metformin also increased ALP activity, matrix mineralization, and expression levels of osteogenic genes, runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN) and collagen I (COL1), in PL-MSCs. Metformin increases osteogenic differentiation of PL-MSCs, at least in part, through the AMPK signaling pathway, since the administration of compound C inhibited its enhancing effects on ALP activity, matrix mineralization, and osteogenic gene expression of PL-MSCs., Conclusions: This study demonstrated that metformin at concentrations of 80-320 μM significantly enhanced osteogenic differentiation of PL-MSCs in a dose- and time-dependent manner, primarily through activation of the AMPK signaling pathway. This finding suggests that metformin could be used with other conventional drugs to induce bone regeneration in various bone diseases. Additionally, this study provides valuable insights for future osteoporosis treatment by highlighting the potential of modulating the AMPK pathway to improve bone regeneration., Competing Interests: Declarations Ethics approval and consent to participate All experimental procedures were conducted in accordance with the Declaration of Helsinki and the Belmont report. This study was approved by the Human Research Ethics Committee of Thammasat University (Medicine) [Approval title: Effect of metformin on cell viability and osteogenic differentiation potential of human mesenchymal stem cells.] [Approval number: 037/2021] (Date of Approval: February 25, 2021). All samples were obtained from donors with their written informed consent. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Mesenchymal stem cells (MSCs) are important in regenerative medicine because of their potential for multi-differentiation. Bone marrow, chorion and placenta have all been suggested as potential sources for clinical application. However, the osteogenic differentiation potential of MSCs derived from chorion or placenta is not very efficient. Bone morphogenetic protein-2 (BMP-2) plays an important role in bone development. Its effect on osteogenic augmentation has been addressed in several studies. Recent studies have also shown a relationship between miRNAs and osteogenesis. We hypothesized that miRNAs targeted to Runt-related transcription factor 2 (Runx-2), a major transcription factor of osteogenesis, are responsible for regulating the differentiation of MSCs into osteoblasts. This study examines the effect of BMP-2 on the osteogenic differentiation of MSCs isolated from chorion and placenta in comparison to bone marrow-derived MSCs and investigates the role of miRNAs in the osteogenic differentiation of MSCs from these sources. MSCs were isolated from human bone marrow, chorion and placenta. The osteogenic differentiation potential after BMP-2 treatment was examined using ALP staining, ALP activity assay, and osteogenic gene expression. Candidate miRNAs were selected and their expression levels during osteoblastic differentiation were examined using real-time RT-PCR. The role of these miRNAs in osteogenesis was investigated by transfection with specific miRNA inhibitors. The level of osteogenic differentiation was monitored after anti-miRNA treatment. MSCs isolated from chorion and placenta exhibited self-renewal capacity and multi-lineage differentiation potential similar to MSCs isolated from bone marrow. BMP-2 treated MSCs showed higher ALP levels and osteogenic gene expression compared to untreated MSCs. All investigated miRNAs (miR-31, miR-106a and miR148) were consistently downregulated during the process of osteogenic differentiation. After treatment with miRNA inhibitors, ALP activity and osteogenic gene expression increased over the time of osteogenic differentiation. BMP-2 has a positive effect on osteogenic differentiation of chorion- and placenta-derived MSCs. The inhibition of specific miRNAs enhanced the osteogenic differentiation capacity of various MSCs in culture and this strategy might be used to promote bone regeneration. However, further in vivo experiments are required to assess the validity of this approach.

Background: Mesenchymal stromal cells (MSCs) are considered potential candidates that hold great promise in the treatment of immune-related diseases. For therapeutic applications, it is necessary to isolate and expand MSCs with procedures complying with good manufacturing practice (GMP). Recent studies reported the use of human serum (HS) instead of fetal bovine serum (FBS) for the expansion of bone marrow-derived MSCs. Nevertheless, there are only limited data on HS as an alternative to FBS for the isolation and expansion of umbilical (UC-MSCs) and placenta-derived MSCs (PL-MSCs). In this study, we evaluate the effect of HS compared to FBS on the proliferative and immunosuppressive capacities of these MSCs., Methods: PL-MSCs and UC-MSCs were isolated and cultured in HS- or FBS-supplemented media. The MSC characteristics, including morphology, immunophenotype, and differentiation ability, were verified. The proliferative and immunosuppressive capacities were also examined. In addition, the proliferative-enhancing factors in both sera were explored using proteomic analysis., Results: PL-MSCs and UC-MSCs proliferated faster in HS-supplemented medium than in equivalent levels of FBS-supplemented medium. Adipogenic and osteogenic differentiations occurred at nearly identical levels in HS- and FBS-supplemented media. Interestingly, MSCs cultured in HS-supplemented medium had a similar immunosuppressive effect as MSCs cultured in FBS-supplemented medium. Proteomic analysis revealed that Con-A binding glycoproteins with a molecular weight > 100 kDa in FBS could significantly enhance MSC proliferation. In contrast, the proliferative enhancing factors in HS were found in the Con-A non-binding fraction and WGA binding fraction with a molecular weight > 100 kDa., Conclusions: Taken together, our results suggest applications for the use of HS instead of FBS for the isolation and expansion of PL-MSCs and UC-MSCs for cell therapy in the future. Furthermore, this study identifies factors in HS that are responsible for its proliferative and immunosuppressive effects and might thus lead to the establishment of GMPs for the therapeutic use of MSCs.

Objective: Evidence exists indicating that mesenchymal stem cells (MSCs) are promising candidate for therapeutic applications. One major obstacle for their clinical use is the biosafety of fetal bovine serum (FBS), which is a crucial part of all media currently used for culture of MSCs. Although some recent studies recommended substituting FBS with human serum (HS) for the expansion of MSCs for clinical use, the characteristics and functional capacity of the expanded cells has only been partially explored. In addition, limited experience indicates that HS may replace FBS in some but not all culture systems. Currently, relatively little is known about using HS instead of FBS for isolation and expansion of placenta derived MSCs. Therefore, this study aimed to compare the exploit of HS and FBS as a supplement in terms of their impact on biological characteristics of MSCs., Material and Method: MSCs derived from placenta were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum or 10% human serum. The morphology, the expression of MSC markers, the differentiation ability and the proliferation characteristics were examined., Results: The results demonstrated that MSCs cultured in DMEM supplemented with 10% HS had similar characteristics to MSCs cultured in DMEM supplemented with 10% FBS. Interestingly, MSCs cultured in DMEM supplemented with 10% HS had greater expansion potential than that of MSCs cultured in DMEM supplemented with 10% FBS., Conclusion: The results obtained from this study imply some application in the use of HS instead of FBS for expansion of placenta derived MSCs. The HS-expanded MSCs might be useful and safe for use as a therapeutic tool in regenerative medicine.

Background: The role of bone marrow-derived mesenchymal stromal cells (BM-MSC) in preventing the incidence and ameliorating the severity of graft-versus-host disease (GvHD) has recently been reported. However, as the collection of BM-MSC is an invasive procedure, more accessible sources of MSC are desirable., Aim: This study aimed to explore the alternative sources of MSC from amnion, placenta, Wharton's jelly and umbilical cord, which are usually discarded., Methods: MSC from those tissues were isolated using mechanical dissociation and enzymatic digestion. Their capacity for proliferation and differentiation, and ability to suppress alloreactive T-lymphocytes were studied and compared with those of BM-MSC., Results: MSC derived from amnion, placenta, Wharton's jelly and umbilical cord were similar to BM-MSC regarding the cell morphology, the immunophenotype as well as the differentiation ability. These MSC also elicited a similar degree of immunosuppression, as evidenced by the inhibition of alloreactive T-lymphocytes in the mixed lymphocyte reaction, compared with that of BM-MSC. MSC from umbilical cord and Wharton's jelly had a higher proliferative capacity, whereas those from amnion and placenta had a lower proliferative capacity compared with BM-MSC., Conclusion: The results obtained from this study suggest that MSC from amnion, placenta, Wharton's jelly and umbilical cord can therefore be potentially used for substituting BM-MSC in several therapeutic applications, including the treatment of GvHD., (© 2012 The Authors; Internal Medicine Journal © 2012 Royal Australasian College of Physicians.)