Improved osteogenesis and upregulated immunogenicity in human placenta-derived mesenchymal stem cells primed with osteogenic induction medium

MSCs are indispensable in regenerative medicine, specifically in bone tissue engineering. However, MSCs derived from different tissues display undesirable therapeutic effects in various preclinical studies because of low survival and differentiation potential as well as unexpected immunogenicity in vivo [9, 13]. In the present study, we isolated MSCs from human placenta and developed a cell population termed De-MSCs via induced osteogenic differentiation and dedifferentiation [15, 17]. We demonstrated that De-MSCs can regain their multilineage differentiation into osteoblasts, adipocytes, and chondrocytes [23], being morphologically and phenotypically similar to uncommitted MSCs. Therefore, we further explored the osteogenic ability of MSCs and De-MSCs both in vitro and in vivo. Compared to MSCs, De-MSCs exhibited a predisposition to the osteoblastic lineage supported by increased osteogenic gene expression and enhanced ALP production.

It is widely accepted that BMP2 plays an important role not only in adjusting the proliferation and differentiation of stem cells [24], but also in osteogenic differentiation and bone formation [25]. As a major transcription factor, Runx2 is essential in bone development and more effectively when introduced together with BMP2 [26, 27]. Lying downstream of Runx2, Osx directs the differentiation of pre-osteoblasts into mature osteoblasts [26, 28]. Meanwhile, as a result of the early gene introduction of BMP2, ALP activity and matrix mineralization are promoted [29–31]. In our in vitro study, De-MSCs exhibited higher potential of proliferation and differentiation than MSCs, illustrated by the proliferation curves of MSCs and De-MSCs during their osteogenic differentiation. Upon osteo-induction, Re-MSCs exhibited statistically higher level of BMP2, Runx2 and Osx mRNA than Ob-MSCs did, which paralleled a similar induction of ALP activity. In vivo, we implanted collagen scaffolds loaded with MSCs and De-MSCs in SCID mice. Similar to the observation in vitro, the ALP activity of implanted De-MSCs and the mRNA levels of BMP2, Runx2 and Osx in implanted De-MSCs were remarkably higher than those of implanted MSCs. The immunohistochemistry staining of collagen II and HE further evidenced morphologically that De-MSCs had higher osteogenic potential compared to MSCs in situ. These data suggest that De-MSCs have osteogenic superiority to MSCs and may function as a potential cell candidate for bone regeneration.

To date, MSCs have shown great potential in clinical applications, such as for bone regeneration, cardiac repair and treatment of liver diseases [32–35]. However, the engraftment efficiency, migration behaviors, and functionality of transplanted MSCs in a living animal model are poorly understood. Rui et al showed that De-MSCs could survive longer in unfavorable environment, the mechanism of which is associated with microRNA3, resulting in epigenetic memory gained by priming with osteogenic induction medium [17].

MSCs express immunosuppressive molecules and various growth factors which can facilitate tissue repair and maintain immune homeostasis [36]. MSCs have no or low expression of co-stimulatory molecules CD40, CD80, CD86, moderate expression of MHC class I molecules, absent expression of MHC class II molecules, which contribute to low immunogenicity of MSCs [37]. In order to better understand the immunogenic properties of De-MSCs, we compared the immunogenicity and co-stimulatory expression of MSCs and De-MSCs during their osteogenesis. It is well-documented that co-stimulatory molecules play an essential role in modulating immune response through a variety of mechanisms [38]. Specifically, the B7 family members provide a key checkpoint in the regulation of T cell immunity. The upregulation of the expression of CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells can directly influence the activation and proliferation of T cells, initiating immune response [39]. In contrast, PD-1/PD-L1 pathway attributes to damping T cell responses, promoting T cell tolerance and preventing autoimmunity at most [18]. Among the immunoglobulin superfamily, CD83 (HB15) is necessary for effective DC-mediated activation of naive T cells, thymic T cell maturation and the regulation of B cell activation and homeostasis [40]. In the present study, we explored the expression of B7 family members of the immunoglobulin superfamily proteins on different cell populations. As stated, MSCs, as well as De-MSCs, did not express co-stimulatory molecules CD80, CD83, CD86, but highly expressed co-inhibitory molecules PD-L1 and B7-H3. But Ob-MSCs and Re-MSCs expressed higher CD80, CD83, and CD86, lower PD-L1 and B7-H3 than MSCs and De-MSCs did. In addition, MSCs and De-MSCs significantly suppressed T cell proliferation more strongly than Ob-MSCs and Re-MSCs did. Thus, Ob-MSCs and Re-MSCs gained higher immunogenicity upon osteogenic induction than MSCs and De-MSCs in vitro. However, the cell population pattern primed with MSCs or De-MSCs was similar to the one with vehicle delivery. In vivo study, we showed that activated T cells, B cells, monocytes, and macrophages were found in PBMCs and splenocytes of mice immunized with MMC-treated Ob-MSCs and Re-MSCs. Furthermore, the activation of Re-MSCs was higher than Ob-MSCs. These results indicate that the immunogenicity of differentiated MSCs and De-MSCs increase functionally compared with the undifferentiated counterparts, and Re-MSCs elicit more enhanced immunogenicity compared to Ob-MSCs. Given the complexity of immune-modulation in vivo, it is plausible that other partner cell types are involved in the immune-regulated effect of MSCs. During MSCs differentiation, multiple mechanisms are involved in the fate determination process, including genetic and epigenetic regulation [17]. B7-H3 is expressed on antigen-presenting cells and downregulates T cell functions by engaging an unknown counter-receptor on T cells. As well, it is also identified to have a role in the bone-immune interface, playing a positive regulatory role in bone formation [41, 42]. We demonstrated that undifferentiated cells had upregulated immunogenicity, in association with differentiation. Consequently, we speculate that the mechanism of this phenomenon may be related to both epigenetics and signal pathways. In-depth research needs to be conducted to verify all these.

In summary, De-MSCs share similar morphology, cell surface markers, and lower immunogenicity to MSCs. Moreover, they have higher potential for proliferation and differentiation compared to MSCs during osteogenesis. Our study further supports the notion that De-MSCs may serve as an alternative source of cells with enhancing therapeutic efficacy for regenerative medicine and tissue engineering. However, we have demonstrated that De-MSCs had upregulated immunogenicity as MSCs during their osteogenesis. Thus, the immunologic intervention may serve as a beneficial strategy in MSC-based therapy to maximize the potential of MSCs and De-MSCs. Furthermore, more explicit and detailed mechanisms involved in the differentiation potential and immunogenicity of MSCs and De-MSCs need to be elucidated in the future.