Project description:We identified LINC01638 as a lncRNA that is highly expressed in proliferating MSCs and decreases in expression during osteogenesis. We demonstrate that knockdown of LINC01638 in hMSC-hTERT20 cells by CRISPRi during osteogenic differentiation causes an decrease in alkaline phosphatase (ALP) activity and ALPL gene expression. Altered expression of several extracellular matrix proteins was noted. We identified LINC01638 as a nuclear lncRNA that binds to chromatin and where it regulates gene expression. These results reveal that LINC01638 may act as a negative regulator of osteoblast differentiation, thereby providing a novel mechanism of regulatory control of osteogenesis.
Project description:We identified MIR181A1HG as a lncRNA that is ubiquitously expressed in non-mineralized tissues, highly expressed in proliferating MSCs and decreases in expression during osteogenesis. We demonstrate that knockdown of MIR181A1HG in hMSC-hTERT20 cells by CRISPRi during osteogenic differentiation causes an increase in alkaline phosphatase (ALP) activity and ALPL gene expression. Increased expression of several extracellular matrix proteins was noted. We identified MIR181A1HG as a nuclear lncRNA that binds to chromatin and where it regulates gene expression, including the transcription factor SOX5. These results reveal that MIR181AHG may act as a negative regulator of osteoblast differentiation, thereby providing a novel mechanism of regulatory control of osteogenesis.
Project description:Differentiation of human skeletal stem cells (hMSC) into osteoblasts is regulated by a few well described transcription factors. Our study used clustering and gene expression data to identify a novel transcription factor. ZNF25, which we showed is involved in osteoblast differentiation. We used microarrays to study gene expression of hMSC-TERT4 cells after siZNF25 knockdown. hMSC-TERT4 cells were sampled as undifferentiated hMSC and as differentiated osteoblasts.
Project description:Differentiation of human skeletal stem cells (hMSC) into osteoblasts is regulated by a few well described transcription factors. Our study used clustering and gene expression data to identify a novel transcription factor. ZNF25, which we showed is involved in osteoblast differentiation. We used microarrays to study gene expression of hMSC-TERT4 cells during osteoblast differentiation.
Project description:We aimed in this study to identify the differentially regulated genes by Dlk1 in hMSC cells using microarray technology in order to gain a better understanding of Dlk1-mediated signaling pathways during hMSC differentiation. Both control (hMSC-TERT)(not expressing Dlk1) and Dlk1 overexpressing cells (hMSC-Dlk1) were cultured in triplicate at 3×104 cells/cm2 in Petri-dish in standard growth medium. At 90-100% confluence, highly purified total cellular RNA was isolated from each of three independent cultures per cell line using RNeasy Kit (QIAGEN Nordic, West Sussex, UK) according to the manufacturer?s instructions
Project description:The transcriptome of hMSC in late passages was compared to hMSC in early passages. Both hMSC were obtained from the umbilical vein of three donors, two of hMSC have a normal karyotype (MSC/n) and another has a constitutional paracentric chromosomal inversion (hMSC/inv).
Project description:We have employed whole microRNA microarray to identify changes in microRNA expression in human bone marrow MSCs (hMSC-TERT) during adipocytic differentiation in culture on day 7 and day 13
Project description:Differentiation of human skeletal stem cells (hMSC) into osteoblasts is regulated by a few well described transcription factors. Our study used clustering and gene expression data to identify a novel transcription factor. ZNF25, which we showed is involved in osteoblast differentiation.
Project description:Adult human mesenchymal stem cells (hMSCs) have shown promise as a valuable new therapeutic tool in a wide range of diseases. hMSCs from bone marrow stroma are currently isolated by their adherence to tissue culture treated polystyrene (TCP) and passaged multiple times on the same plastics until they are able to produce enough cells to be useful for research or clinical therapeutic trials. However, evidence in the literature has shown that culture on TCP can negatively alter hMSC function. Our aim was to expand hMSCs in an in vitro environment more closely resembling that of the hMSCs' native microenvironment to maximize proliferation while retaining therapeutic potential. We used decellularized hMSC-derived extracellular matrix (hMSC-ECM) to test hMSCs' maintenance of stem cell properties during in vitro expansion. We found that hMSC-ECM was able to increase hMSC proliferation while retaining the stem cells‘ immature state and increasing differentiation potential. In addition, the hMSC-ECM could be covalently cross-linked to polymer substrates and was effective in the isolation and expansion of hMSCs in the presence of fetal bovine serum and human serum. Using proteomics and transcriptomics, we were able to determine the mechanism behind the hMSCs’ increased proliferation was due to their ability to downregulate their otherwise required gene expression for ECM proteins by on hMSC-ECM. The effects of hMSC-ECM were largely hMSC specific and were not found with several other types of human cells. Providing a pre-formed in vitro niche for hMSCs can provide the cells with the critical components required for hMSC function during in vitro expansion.
Project description:We have employed whole genome microarray to identify changes in gene expression in human bone marrow MSCs (hMSC-TERT) during adipocytic differentiation in culture for 7 days