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Project description:BackgroundHuman multipotent progenitor cells (hiMPCs) created from induced pluripotent stem cells (iPSCs) represent a new cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) are needed to enhance transforming growth factor-β (TGFβ)-induced hiMPC chondrogenesis. In contrast, TGFβ alone is sufficient to result in robust chondrogenesis of human primary mesenchymal stromal cells (hMSCs). Currently, the mechanism underlying this difference between hiMPCs and hMSCs has not been fully understood.MethodsIn this study, we first tested different growth factors alone or in combination in stimulating hiMPC chondrogenesis, with a special focus on chondrocytic hypertrophy. The reparative capacity of hiMPCs-derived cartilage was assessed in an osteochondral defect model created in rats. hMSCs isolated from bone marrow were included in all studies as the control. Lastly, a mechanistic study was conducted to understand why hiMPCs and hMSCs behave differently in responding to TGFβ.ResultsChondrogenic medium supplemented with TGFβ3 and BMP6 led to robust in vitro cartilage formation from hiMPCs with minimal hypertrophy. Cartilage tissue generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and resulted in a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFβ3 induced phosphorylation of both Smad2/3 and Smad1/5 in hMSCs, but only activated Smad2/3 in hiMPCs. Supplementing BMP6 activated Smad1/5 and significantly enhanced TGFβ's compacity in inducing hiMPC chondrogenesis. The chondro-promoting function of BMP6 was abolished by the treatment of a BMP pathway inhibitor.ConclusionsThis study describes a robust method to generate chondrocytes from hiMPCs with low hypertrophy for hyaline cartilage repair, as well as elucidates the difference between hMSCs and hiMPCs in response to TGFβ. Our results also indicated the importance of activating both Smad2/3 and Smad1/5 in the initiation of chondrogenesis.
| S-EPMC9763539 | biostudies-literature
Project description:Differences in the intrinsic chondrogenic potential of human mesenchymal stromal cells and iPSC-derived multipotent cells
| PRJNA809311 | ENA
Project description:Umbilical cord blood mesenchymal stromal/stem cells (UCB-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) have chondrogenic potential and are alternative sources to standard surgically derived bone marrow or adipose tissue collection for cartilage engineering. However, the majority of comparative studies explore neonatal MSCs potential only on ISCT benchmark assays accounting for some bias in the reproducibility between in vitro and in clinical studies. Therefore, we characterized equine UCB-MSCs and UCM-MSCs and investigated with particular attention their chondrogenesis potential in 3D culture with BMP-2 + TGF-ß1 in normoxia or hypoxia. We carried out an exhaustive characterization of the extracellular matrix generated by both these two types of MSCs after the induction of chondrogenesis through evaluation of hyaline cartilage, hypertrophic and osteogenic markers (mRNA, protein and histology levels). Some differences in hypoxia sensitivity and chondrogenesis were observed. UCB-MSCs differentiated into chondrocytes express an abundant, dense and a hyaline-like cartilage matrix. By contrast, despite their expression of cartilage markers, UCM-MSCs failed to express a relevant cartilage matrix after chondrogenic induction. Both MSCs types also displayed intrinsic differences at their undifferentiated basal status, UCB-MSCs expressing higher levels of chondrogenic markers whereas UCM-MSCs synthesizing higher amounts of osteogenic markers. Our results suggest that UCB-MSCs should be preferred for ex-vivo horse cartilage engineering. How those results should be translated to in vivo direct cartilage regeneration remains to be determined through dedicated study.
| S-EPMC6138671 | biostudies-literature
Project description:The precise predictions of the differentiation direction and potential of mesenchymal stromal cells (MSCs) are an important key to the success of regenerative medicine. The expression levels of fate-determining genes may provide tools for predicting differentiation potential. The expression levels of 95 candidate marker genes and glycosaminoglycan (GAG) contents after chondrogenic induction in 10 undifferentiated ilium and 5 jaw MSC cultures were determined, and their correlations were analyzed. The expression levels of eight genes before the induction of chondrogenic MSC differentiation were significantly correlated with the GAG levels after induction. Based on correlation patterns, the eight genes were classified into two groups: group 1 genes (AURKB, E2F1, CDKN2D, LIF, and ACLY), related to cell cycle regulation, and group 2 genes (CD74, EFEMP1, and TGM2), involved in chondrogenesis. The expression levels of the group 2 genes were significantly correlated with the ages of the cell donors. The expression levels of CDKN2D, CD74, and TGM2 were >10-fold higher in highly potent MSCs (ilium MSCs) than in MSCs with limited potential (jaw MSCs). Three-dimensional (3D) scatter plot analyses of the expression levels of these genes showed reduced variability between donors and confirmed predictive potential. These data suggest that group 2 genes are involved in age-dependent decreases in the chondrogenic differentiation potential of MSCs, and combined 3D analyses of the expression profiles of three genes, including two group 2 genes, were predictive of MSC differentiation potential.
| S-EPMC6199884 | biostudies-literature
Project description:Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.
| S-EPMC8672196 | biostudies-literature
Project description:Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives, particularly extracellular vesicles (EVs), for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs, alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction, indicating a potential mechanism for their immunomodulatory effects. Furthermore, iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally, iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably, priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics, given their potent immunomodulatory and regenerative properties.
| S-EPMC11480492 | biostudies-literature
Project description:BackgroundThe use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion.MethodsIn this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays.ResultsOverall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs.ConclusionsOur results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.
| S-EPMC7059393 | biostudies-literature
Project description:Multipotent mesenchymal stromal cells (MSCs) can be considered an accessible therapeutic tool for regenerative medicine. Here, we compared the growth kinetics, immunophenotypic and immunomodulatory properties, gene expression and secretome profile of MSCs derived from human adult bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and Wharton's jelly (WJ-MSCs) cultured in clinically-relevant conditions, with the focus on the neuroregenerative potential. All the cell types were positive for CD10/CD29/CD44/CD73/CD90/CD105/HLA-ABC and negative for CD14/CD45/CD235a/CD271/HLA-DR/VEGFR2 markers, but they differed in the expression of CD34/CD133/CD146/SSEA-4/MSCA-1/CD271/HLA-DR markers. BM-MSCs displayed the highest immunomodulatory activity compared to AT- and WJ-MSCs. On the other hand, BM-MSCs secreted the lower content and had the lower gene expression of neurotrophic growth factors compared to other cell lines, which may be caused by the higher sensitivity of BM-MSCs to nutrient limitations. Despite the differences in growth factor secretion, the MSC secretome derived from all cell sources had a pronounced neurotrophic potential to stimulate the neurite outgrowth of DRG-neurons and reduce the cell death of neural stem/progenitor cells after H2O2 treatment. Overall, our study provides important information for the transfer of basic MSC research towards clinical-grade manufacturing and therapeutic applications.
| S-EPMC7062771 | biostudies-literature
Project description:Current regenerative strategies used for cartilage repair rely on biomaterial functionality as a scaffold for cells that may have potential in chondrogenic differentiation. The purpose of the research was to investigate the biocompatibility of enzymatically treated alginate/chitosan hydrosol sponges and their suitability to support chondrogenic differentiation of human adipose derived multipotent stromal cells (hASCs). The alginate/chitosan and enzyme/alginate/chitosan sponges were formed from hydrosols with various proportions and were used as a biomaterial in this study. Sponges were tested for porosity and wettability. The porosity of each sponge was higher than 80%. An equal dose of alginate and chitosan in the composition of sponges improved their swelling ability. It was found that equal concentrations of alginate and chitosan in hydrosols sponges assure high biocompatibility properties that may be further improved by enzymatic treatment. Importantly, the high biocompatibility of these biomaterials turned out to be crucial in the context of hydrosols' pro-chondrogenic function. After exposure to the chondrogenic conditions, the hASCs in N/A/C and L/A/C sponges formed well developed nodules and revealed increased expression of collagen type II, aggrecan and decreased expression of collagen type I. Moreover, in these cultures, the reactive oxygen species level was lowered while superoxide dismutase activity increased. Based on the obtained results, we conclude that N/A/C and L/A/C sponges may have prospective application as hASCs carriers for cartilage repair.
| S-EPMC6431914 | biostudies-literature
Project description:ObjectivesMesenchymal stem/stromal cells (MSCs) are a well-established cell source for cartilage engineering, but challenges remain as differentiation often results in chondrocyte hypertrophy. Chondrogenic potential also varies with MSC source and donor age. We assessed the chondrogenic potential of first-trimester and term placental MSCs and compared their response to commonly used bone marrow MSCs (BM-MSCs).DesignMSCs were isolated from first-trimester and term placentae. BM-MSCs were commercially obtained. Chondrogenesis was induced by micromass culture in commercial chondrogenic media for 7, 14, or 21 days. Pellets were assessed for glycosaminoglycan (GAG) content, and types I, II, and X collagen. Gene expression was profiled using Qiagen RT2 human MSC arrays.ResultsAt day 0, first-trimester and term MSCs expression levels of many chondrogenic genes to BM-MSC after 21 days of culture. Only first trimester MSCs showed significant changes in chondrogenic gene expression during induction compared to day 0 undifferentiated MSCs (greater BMP4, KAT2B, and reduced GDF6 expression). Additionally, first-trimester MSCs showed significantly greater expression of ABCB1 (at days 14 and 21) and BMP4 (at days 7, 14, 21) compared with term MSCs. Both first-trimester and term pellets showed increased GAG content over time and term MSCs had significantly GAG greater compared with BM-MSCs at days 7 and 14. Type II collagen was present in all pellets but unlike BM-MSCs, type I collagen was not observed in first-trimester or term MSC pellets.ConclusionsThese data highlight differences in BM-MSC and placental MSC chondrogenesis and demonstrate that placental MSCs may be an alternative cell source.
| S-EPMC8804733 | biostudies-literature