Gene expression data of human dermal pilla cells, human induced pluripotent stem cell-derived CD271+CD90+ mesenchymal stem cells before and after the induction toward dermal papilla cells
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ABSTRACT: To assess if human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) could be induce to acquire dermal papilla (DP) properties (especially hair inductive capacity), hiPSCs were initially programmed in serum-free MSC medium with FGF, TGFbeta and PDGF. Subsequently, hiMSCs were exposed to retinoic acid and activators of WNT, BMP and FGF signaling pathways. Global gene expression profiles were compared among primarily cultured human DP cells, hiPSC-MSCs before and after induction. Human dermal papillae were microdissected and cultured from different donors. Induction from hiPSCs to MSCs and subsequent sorting of CD271+CD90+ subpopulation and thier induction to DP was performed using WD39 hiPSC lines in two independent experimentations to generate hiPSC-MSC-1,hiPSC-MSC-2 ipDPSC-1 and ipDPSC-2. Funding: The Human Stem Cells Informatization Project of the Ministry of Health, Labour and Welfare, Japan
Project description:To assess if human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) could be induce to acquire dermal papilla (DP) properties (especially hair inductive capacity), hiPSCs were initially programmed in serum-free MSC medium with FGF, TGFbeta and PDGF. Subsequently, hiMSCs were exposed to retinoic acid and activators of WNT, BMP and FGF signaling pathways. Global gene expression profiles were compared among primarily cultured human DP cells, hiPSC-MSCs before and after induction.
Project description:This experiment investigates differences in miRNA expression between different populations of human mesenchymal stem cells (MSCs), which are a potential treatment for multiple sclerosis (MS). Previously it has been reported that MSCs derived from human olfactory mucosa (OM-MSCs) may be a better candidate for the treatment of MS than MSCs derived from bone marrow (BM-MSCs), due to a better ability to promote CNS myelination in vitro. In this study, miRNA profiling of OM-MSCs and BM-MSCs was undertaken to investigate the differences between these two cell types in relation to their prospective therapeutic use.
Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. To analyse whether the BM-MSC and Fat-MSC (ASC) differentiation stage may define the sarcoma phenotype, RbloxP/loxPp53loxP/loxP BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage and both Rb and p53 were excised with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre) at different stages (day 0 and 10) along osteogenic differentiation. NSG mice were inoculated subcutaneously with 5M-CM-^W10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using: WT BM-MSCs and ASCs, Rb-/-p53-/- BM-MSCs and ASCs previously differentiated to the osteogenic lineage for 10 days and a tumor cell line derived from p53-/-Rb-/- BM-MSC differentiated to the osteogenic lineage for 10 days.
Project description:Although different sarcomas have been modeled in mice upon expression of fusion oncogenes in MSCs, sarcomagenesis has not been successfully modeled in human MSCs (hMSCs). We report that FUS-CHOP, a hallmark fusion gene in mixoid liposarcoma (MLS), has an instructive role in lineage commitment, and its expression in hMSC sequentially immortalized/transformed with up to 5 oncogenic hits (p53 and Rb deficiency, hTERT over-expression, c-myc stabilization and H-RASv12 mutation) drives the formation of serially transplantable MLS. This is the first model of sarcoma based on the expression of a sarcoma-associated fusion protein in hMSC, and allowed us to unravel the differentiation processes and signaling pathways altered in the MLS-initiating cells. This study will contribute to test novel therapeutic approaches, and constitutes a proof-of-concept to employ hMSCs as target cell for modeling other fusion gene-associated human sarcomas. Wild type (MSC-0H) or transformed (MSC-5H) BM-hMSCs were transduced with concentrated viral particles expressing either pRRL-EF1?-PGK-GFP (empty vector; GFP) or pRRL-EF1?-FUS-CHOP-PGK-GFP (FUSCHOP expressing vector; FC) in order to generate MSC-0H-GFP, MSC-0H-FC, MSC-5H-GFP and MSC-5H-FC cell lines. MSC-0H-GFP and MSC-0H-FC cells did not develop tumors, meanwhile MSC-5H-GFP cells gave rise to undifferentiated sarcomas and MSC-5H-FC originated mixoid liposarcoma tumors when inoculated into immunedeficient mice. Several cell lines were derived from tumors developed from MSC-5H-GFP (T-5H-GFP-1 to -3 cell lines) and MSC-5H-FC (T-5H-FC-1 to -3 cell lines) cells. Gene expression analysis was performed using MSC-0H, MSC-5H and T-5H cell lines and lists of differentially expressed genes were created by comparing the gene expression profiles of MSC-0H-FC, MSC-5H-FC and T-5H-FC cell types to the control MSC-0H-GFP cells.
Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. BM-MSC and ASC cultures were established from 3 mouse strains: (a) WT, (b) p53loxP/loxP and (c) p53loxP/loxP RbloxP/loxP. The corresponding mutant cells were generated by excision of the LoxP-flanked sequences by infection of all MSC cultures with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre). NSG mice were inoculated subcutaneously with 5×10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using BM-MSCs and ASCs from all genotypes, as well as tumor cell lines derived from p53-/- and p53-/-Rb-/- BM-MSC and ASC cultures.
Project description:Human induced pluripotent stem cells provide an unlimited, scalable source of youthful tissue progenitors and secretome for regenerative therapies. The aim of our study was to assess the potential of conditioned medium (CM) derived from hiPSC-mesenchymal progenitors (hiPSC-MPs) to stimulate osteogenic differentiation of adult and aged human bone marrow-mesenchymal stromal cells (MSCs). In addition, we evaluated whether extended cultivation or osteogenic pre-differentiation of hiPSC-MPs could enhance the CM stimulatory activity.
Project description:Mesenchymal stem cells (MSCs) are an attractive therapeutic tool for tissue engineering and re-generative medicine due to their regenerative and trophic properties. The best-known and most widely used are bone marrow MSCs, but they are now being harvested and developed from a wide range of adult and perinatal tissues. MSCs from different sources are believed to have dif-ferent secretion potentials and production, which may influence their therapeutic effects. To prove it, we performed a quantitative proteomic analysis based on the TMT technique of MSCs from 3 different sources: wharton’s jelly (WJ), dental pulp (DP) and bone marrow (BM). Our analysis has focused on MSC biological properties of interest for tissue engineering. We identified a total of 611 human proteins differentially expressed. WJ-MSCs shown the greatest variation compared to the other sources. WJ produced more extra-cellular matrix (ECM) proteins and ECM-affiliated proteins and appeared to more able to modulate the inflammatory and immune response. BM-MSCs display enhanced differentiation and paracrine communication capabilities. DP-MSC appeared to promote exosome production. The results obtained confirm the existence of differences between WJ, DP and BM-MSC and the need to select the MSC origin according to the therapeutic objective sought.
Project description:Bone-marrow mesenchymal stem cells (MSCs) are plastic adherent cells that can differentiate into various tissue lineages, including osteoblasts, adipocytes and chondrocytes. However, this progenitor property is not shared by all cells within the MSC population. In addition, MSCs vary in their proliferation capacities and expression of markers. Because of heterogeneity of CD146 expression in the MSC population, we compared CD146-/Low and CD146High cells under clonal and non-clonal (sorted MSCs) conditions to determine whether this expression is associated with specific functions. CD146-/Low and CD146High MSCs did not differ in colony-forming unit-fibroblast number, osteogenic and adipogenic differentiation or in vitro hematopoietic supportive activity. However, CD146-/Low clones proliferated slightly but significantly faster than did CD146High clones. In addition, a strong expression of CD146 molecule was associated with a commitment towards a vascular smooth muscle cell lineage with upregulation of calponin-1 expression. Thus, within a bone-marrow MSC population, certain subpopulations characterized by high expression of CD146, are committed toward a vascular smooth muscle cell lineage. Clonal MSC were selected on the basis of CD146 level at their surface (CD146Low and CD146 High) and non-clonal MSC were compared from 4 different donors.
Project description:Ex-vivo expanded mesenchymal stromal cells (MSCs) are increasingly used for paracrine support of hematopoietic stem cell (HSC) regeneration, but inconsistent outcomes have been the huddle for on-going clinical trials. Here, we hypothesized that the heterogeneity in the niche activity of manufactured MSCs can be a parameter for variable outcomes in MSC-based cell therapy. We first screened MSC culture medium and found that serum batches caused larger variations in colony forming unit-fibroblast (CFU-F) content of MSCs than individual donor variations. The culture conditions supporting high (stimulatory) and low (non-stimulatory) CFU-F caused distinct niche activity of MSCs; MSCs under stimulatory condition exhibited higher level expression of cross-talk molecules (Jagged-1 and CXCL-12) and higher support for HSCS during long-term culture than MSCs under non-stimulatory culture. Moreover, the effects of MSCs enhancing hematopoietic engraftment were only visible when HSCs were co-transplanted with MSCs expanded under stimulatory, but not non-stimulatory conditions. However, these differences of MSCs were readily reversed by switching the culture mediums, indicating their distinct functional state, rather than clonal heterogeneity. Accordingly, transcriptomic analysis showed distinct gene set enrichment between the different MSCs and revealed distinct upstream signaling pathways such as inhibition of P53 and activation of ATF4 for MSCs under stimulatory conditions. Taken together, our study shows that the heterogeneity in the niche activity of MSCs can be created during ex-vivo expansion to cause a difference in the hematopoietic engraftment and raise the possibility that MSCs can be pre-screened for more predictable outcomes in clinical trials of MSCs. Total RNA obtained from isolated human mesenchymal stromal cells. To compare stimulatory (SS) serum and non-stimulatory (NSS) serums, MSCs had been maintained in each serum media were sub-cultured for at least two passages before analysis.
Project description:Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels.1-5 While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals,6,7 the identity of these signals is unknown. Here, by utilizing two functionally distinct human MSC clones, we found that so-called “pericytic” MSCs secrete the pro-angiogenic neurovascular guidance molecule SLIT3,8 which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, “non-pericytic” MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs, and knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway regulates MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications. 4 samples with 2 replicates each