The extracts of osteoblast developed from adipose-derived stem cell and its role in osteogenesis

Abstract Cell-based therapy has become an achievable choice in regenerative medicines, particularly for musculoskeletal disorders. Adipose-derived stem cells (ASCs) are an outstanding resource because of their ability and functions. Nevertheless, the use of cells for treatment comes with difficulties in operation and safety. The immunological barrier is also a major limitation of cell therapy, which can lead to unexpected results. Cell-derived products, such as cell extracts, have gained a lot of attention to overcome these limitations. The goal of this study was to optimize the production of ASC-osteoblast extracts as well as their involvement in osteogenesis. The extracts were prepared using a freeze–thaw method with varying temperatures and durations. Overall, osteogenic-associated proteins and osteoinductive potential of the extracts prepared from the osteogenic-induced ASCs were assessed. Our results demonstrated that the freeze–thaw approach is practicable for cell extracts production, with minor differences in temperature and duration having no effect on protein concentration. The ASC-osteoblast extracts contain a significant level of essential specialized proteins that promote osteogenicity. Hence, the freeze–thaw method is applicable for extract preparation and ASC-osteoblast extracts may be beneficial as an optional facilitating biologics in bone anabolic treatment and bone regeneration

Computational identification of miRNAs that modulate the differentiation of mesenchymal stem cells to osteoblasts

MicroRNAs (miRNAs) are small endogenous noncoding RNAs that play an instrumental role in post-transcriptional modulation of gene expression. Genes related to osteogenesis (i.e., RUNX2, COL1A1 and OSX) is important in controlling the differentiation of mesenchymal stem cells (MSCs) to bone tissues. The regulated expression level of miRNAs is critically important for the differentiation of MSCs to preosteoblasts. The understanding of miRNA regulation in osteogenesis could be applied for future applications in bone defects. Therefore, this study aims to shed light on the mechanistic pathway underlying osteogenesis by predicting miRNAs that may modulate this pathway. This study investigates RUNX2, which is a major transcription factor for osteogenesis that drives MSCs into preosteoblasts. Three different prediction tools were employed for identifying miRNAs related to osteogenesis using the 3’UTR of RUNX2 as the target gene. Of the 1,023 miRNAs, 70 miRNAs were found by at least two of the tools. Candidate miRNAs were then selected based on their free energy values, followed by assessing the probability of target accessibility. The results showed that miRNAs 23b, 23a, 30b, 143, 203, 217, and 221 could regulate the RUNX2 gene during the differentiation of MSCs to preosteoblasts

The Use of Human Platelet Lysate as a Coating Substance for Adipose-Derived Stem Cell Expansion

Background: Large-scale production of mesenchymal stromal cells is essential for sufficient therapeutic doses in regenerative medicine. However, long-term cultivation encounters limited cell growth and cellular aging. Therefore, an alternative cell culture approach that promotes proliferation and attenuates cell senescence is required. Human platelet lysate (HPL) is a potent supplement for in vitro cell expansion. Applying HPL as a coating material can potentially improve mesenchymal stromal cell cultures. Method: To examine the capacity of HPL, it was used to pre-coat a tissue culture plate for in vitro adipose-derived mesenchymal stromal cell expansion. Alterations in biological features of adipose-derived stem cells (ADSCs) were investigated, including cell adhesion assays, cell proliferation, population doubling time, and cellular senescence. Results: ADSCs cultured on HPL-coated plates significantly increased cell adhesion rate, shortened population doubling time, and stimulated cell growth. The senescent cells were significantly decreased in ADSCs cultured in an HPL-coated plate, and the expression levels of senescence-associated genes, including p16, p21, and p53, were downregulated. Furthermore, Western blotting analysis revealed that HPL was enriched with fibronectin and vitronectin, essential cell adhesive proteins. Conclusions: HPL was effectively used as a coating material for ADSC expansions. Cellular cultivation on the HPL coating is an alternative approach for producing mesenchymal stromal cells

miR-130a and miR-27b Enhance Osteogenesis in Human Bone Marrow Mesenchymal Stem Cells via Specific Down-Regulation of Peroxisome Proliferator-Activated Receptor γ

<p>Mesenchymal stem cell (MSC) is a type of stem cell that is capable of differentiating into osteoblasts and adipocytes. The pathological perturbation of MSC fate determination is well demonstrated by the replacement of bone tissues with fat in those with osteoporosis and osteopenia. Cell fate determination can be regulated by epigenetic and post-transcriptional mechanisms. MicroRNAs (miRNAs) are small endogenous non-coding RNA molecules that mediates the post-transcriptional regulation of genes expression. We hypothesized that miRNA specified to PPARγ, a major transcription factor of adipogenesis, is responsible for the differentiation of MSCs into osteoblasts. Candidate miRNA that is responsible for target gene inhibition was identified from the miRNA database via bioinformatic analyses. In this study, miR-130a and miR-27b were selected for investigation on their role in specifically binding to peroxisome proliferator-activated receptor γ (PPARγ) via in vitro osteogenesis of human MSCs. During osteogenic differentiation of human MSCs, the expression level of miR-130a and miR-27b were found to be upregulated. In the meanwhile, adipogenic marker genes (PPARγ and C/EBPβ) were found to decrease, which is in contrary to the increased expression of osteogenic marker genes (RUNX2 and Osterix). MSCs were transfected with mimics and inhibitors of miR-130a and miR-27b during in vitro osteogenesis followed by evaluation for the presence of osteogenic markers via quantitative gene expression, Western blot analysis and alkaline phosphatase activity assay. The overexpression of miR-130a and miR-27b is shown to enhance osteogenesis by increasing the gene expression of RUNX2 and Osterix, the protein expression of RUNX2, COL1A1, and Osterix as well as the alkaline phosphatase activity. Taken altogether, these results suggested that miR-130a and miR-27b could promote osteogenesis in human MSCs by targeting the PPARγ.</p

Endothelial Progenitor Cell Migration-Enhancing Factors in the Secretome of Placental-Derived Mesenchymal Stem Cells

Therapeutic potentials of mesenchymal stem cells (MSCs) depend largely on their ability to secrete cytokines or factors that modulate immune response, enhance cell survival, and induce neovascularization in the target tissues. We studied the secretome profile of gestational tissue-derived MSCs and their effects on functions of endothelial progenitor cells (EPCs), another angiogenic cell type that plays an important role during the neovascularization. MSCs derived from placental tissues (PL-MSCs) significantly enhanced EPC migration while BM-MSCs, which are the standard source of MSCs for various clinical applications, did not. By using protein fractionation and mass spectrometry analysis, we identified several novel candidates for EPC migration enhancing factor in PL-MSCs secretome that could be used to enhance neovascularization in the injured/ischemic tissues. We recommend that the strategy developed in our study could be used to systematically identify therapeutically useful molecules in the secretomes of other MSC sources for the clinical applications