Decreased Insulin Sensitivity in Telomerase-Immortalized Mesenchymal Stem Cells Affects Efficacy and Outcome of Adipogenic Differentiation in vitro.
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ABSTRACT:
SUBMITTER: Kulebyakin K
PROVIDER: S-EPMC8371914 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human mesenchymal stem cells (MSCs) are a heterogeneous subset of nonhematopoietic multipotent stromal stem cells and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes, as well as ectodermal and endodermal lineages. Human umbilical cord (UC) is one of the most promising sources of MSCs. However, the molecular and cellular characteristics of UC-derived MSCs (UC-MSCs) require extensive investigations, which are hampered by the limited lifespan and the diminished potency over passages. Here, we used the piggyBac transposon-based simian virus 40 T antigen (SV40T) immortalization system and effectively immortalized UC-MSCs, yielding the iUC-MSCs. A vast majority of the immortalized lines are positive for MSC markers but not for hematopoietic markers. The immortalization phenotype of the iUC-MSCs can be effectively reversed by flippase recombinase-induced the removal of SV40T antigen. While possessing long-term proliferation capability, the iUC-MSCs are not tumorigenic in vivo. Upon bone morphogenetic protein 9 (BMP9) stimulation, the iUC-MSC cells effectively differentiate into osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo, which is indistinguishable from that of primary UC-MSCs, indicating that the immortalized UC-MSCs possess the characteristics similar to that of their primary counterparts and retain trilineage differentiation potential upon BMP9 stimulation. Therefore, the engineered iUC-MSCs should be a valuable alternative cell source for studying UC-MSC biology and their potential utilities in immunotherapies and regenerative medicine.
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Project description:The effects of 5,10-Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) viability, morphology, physiology and differentiation capacity were investigated in this study. For this purpose, primary hBM-MSCs with wild type (WT, C/C), heterozygote (HTZ, C/T) and homozygote (HMZ, T/T) for the MTHFR gene were obtained with ethical committee permission and donor informed. Mutations were detected using RFLP and Sanger sequencing methods from genomic DNA isolated from cells, colonization properties were investigated by CFU-F test and proliferative differences were investigated by MTT test. Adipogenic, osteogenic, and chondrogenic differentiation were induced to study changes in their differentiation potentials, and the results were statistically analyzed using one-way ANOVA with Graphpad Prism. A total of 13 donors were screened and there were no differences in the hBM-MSC markers and in vitro morphologies of the cells. While there were significant differences between WT and HTZ as a result of the CFU-F test, there were no significant differences in the MTT test after 24 and 48 h. As a result of differentiation tests, it was found that adipogenic differentiation was significantly more in HMZ cells than WT cells. Osteogenic and chondrogenic differentiation results did not give statistically significant results. As a result of these experiments, adipogenic differentiation was found to be affected by the MTHFR genotype in hBM-MSCs.
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Project description:The umbilical cord (UC) matrix is a source of multipotent mesenchymal stem cells (MSCs) that have adipogenic potential and thus can be a model to study adipogenesis. However, existing variability in adipocytic differentiation outcomes may be due to discrepancies in methods utilized for adipogenic differentiation. Additionally, functional characterization of UCMSCs as adipocytes has not been described. We tested the potential of three well-established adipogenic cocktails containing IBMX, dexamethasone, and insulin (MDI) plus indomethacin (MDI-I) or rosiglitazone (MDI-R) to stimulate adipocyte differentiation in UCMSCs. MDI, MDI-I, and MDI-R treatment significantly increased peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer binding protein alpha (C/EBPα) mRNA and induced lipid droplet formation. However, MDI-I had the greatest impact on mRNA expression of PPARγ, C/EBPα, FABP4, GPD1, PLIN1, PLIN2, and ADIPOQ and lipid accumulation, whereas MDI showed the least. Interestingly, there were no treatment group differences in the amount of PPARγ protein. However, MDI-I treated cells had significantly more C/EBPα protein compared to MDI or MDI-R, suggesting that indomethacin-dependent increased C/EBPα may contribute to the adipogenesis-inducing potency of MDI-I. Additionally, bone morphogenetic protein 4 (BMP4) treatment of UCMSCs did not enhance responsiveness to MDI-induced differentiation. Finally to characterize adipocyte function, differentiated UCMSCs were stimulated with insulin and downstream signaling was assessed. Differentiated UCMSCs were responsive to insulin at two weeks but showed decreased sensitivity by five weeks following differentiation, suggesting that long-term differentiation may induce insulin resistance. Together, these data indicate that UCMSCs undergo adipogenesis when differentiated in MDI, MDI-I, and MDI-R, however the presence of indomethacin greatly enhances their adipogenic potential beyond that of rosiglitazone. Furthermore, our results suggest that insulin signaling pathways of differentiated UCMSCs are functionally similar to adipocytes.
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Project description:Adipogenesis is a tightly regulated cellular process that involves the action of multiple signaling pathways. Characterization of regulators that are associated with adipose development is crucial to understanding the mechanisms underlying obesity and other metabolic disorders. Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein that was first described as a neurotrophic factor. The role of PEDF in lipid metabolism was established when adipose triglyceride lipase (ATGL), a major triglyceride hydrolase, was characterized as its binding partner. In this study, we investigated the downstream effects of PEDF on adipogenic differentiation using rat adipose-derived stem cells (AdSCs) and the mouse pre-adipocyte cell line 3T3-L1. Knocking down PEDF in differentiating cells resulted in elevated levels of ATGL and CD36, as well as other adipogenic markers, with a concomitant increase in adipocyte number. CD36, a scavenger receptor for a variety of ligands, regulated proliferation and lipogenic gene expression during adipogenesis. The CD36 increase due to PEDF down-regulation might be a result of elevated PPAR?. We further demonstrated that PEDF expression was regulated by dexamethasone, a synthetic glucocorticoid that is widely used for adipogenesis at the transcriptional level. Taken together, our findings highlight that PEDF negatively regulates adipogenesis through the regulation of various signaling intermediates, and it may play a crucial role in lipid metabolic disorders.
| S-EPMC6321369 | biostudies-literature
Project description:BackgroundAn understanding of the mechanism underlying adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) will provide new therapeutic approaches for many diseases, including osteoporosis. This study aimed to investigate the role of miR-431 in adipogenic differentiation of hMSCs.MethodshMSCs were induced for adipogenic differentiation and miR-431 was detected by polymerase chain reaction (PCR). hMSCs were transfected by miR-431 or small interfering RNA (siRNA) for insulin receptor substance 2 (IRS2). The expression of IRS2 was detected by PCR and Western blot analysis. The targeting of the 3'-untranslated region (UTR) of IRS2 by miR-431 was examined by luciferase assay.ResultsmiR-431 expression was decreased during adipogenesis of hMSCs. Overexpression of miR-431 inhibited adipogenic differentiation, accompanied by the downregulation of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), two key regulators of adipogenesis. Moreover, miR-431 decreased both protein and mRNA levels of IRS2. The expression of IRS2 was increased during adipogenic differentiation of hMSCs in conjunction with decreased levels of miR-431, and knockdown of IRS2 in hMSCs inhibited adipogenic differentiation. Luciferase assay confirmed that miR-431 targeted the 3'-UTR of IRS2 in hMSCs.ConclusionsThis is the first study to show that miR-431 inhibits adipogenic differentiation of hMSCs via targeting IRS2.
| S-EPMC6117893 | biostudies-literature
Project description:Human mesenchymal stem cells (MSCs) are adult multipotent stem cells which can be isolated from bone marrow, adipose tissue as well as other tissues and have the capacity to differentiate into a variety of mesenchymal cell types such as adipocytes, osteoblasts and chondrocytes. Differentiation of stem cells into mature cell types is guided by growth factors and hormones, but recent studies suggest that metabolic shifts occur during differentiation and can modulate the differentiation process. We therefore investigated mitochondrial biogenesis, mitochondrial respiration and the mitochondrial membrane potential during adipogenic differentiation of human MSCs. In addition, we inhibited mitochondrial function to assess its effects on adipogenic differentiation. Our data show that mitochondrial biogenesis and oxygen consumption increase markedly during adipogenic differentiation, and that reducing mitochondrial respiration by hypoxia or by inhibition of the mitochondrial electron transport chain significantly suppresses adipogenic differentiation. Furthermore, we used a novel approach to suppress mitochondrial activity using a specific siRNA-based knockdown of the mitochondrial transcription factor A (TFAM), which also resulted in an inhibition of adipogenic differentiation. Taken together, our data demonstrates that increased mitochondrial activity is a prerequisite for MSC differentiation into adipocytes. These findings suggest that metabolic modulation of adult stem cells can maintain stem cell pluripotency or direct adult stem cell differentiation.
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Project description:Despite many advances in tissue engineering, there are still significant challenges associated with restructuring, repairing, or replacing damaged tissue in the body. Currently, a major obstacle has been trying to develop a scaffold for cartilage tissue engineering that provides the correct mechanical properties to endure the loads associated with articular joints as well as promote cell-scaffold interactions to aid in extracellular matrix deposition. In addition, adipogenic tissue engineering is widely growing due to an increased need for more innovative reconstructive therapies following adipose tissue traumas and cosmetic surgeries. Recently, lipoaspirate tissue has been identified as a viable alternative source for mesenchymal stem cells because it contains a supportive stroma that can easily be isolated. Adipose derived stem cells (ADSCs) can differentiate into a variety of mesodermal lineages including the adipogenic and chondrogenic phenotypes. Biodegradable polymeric scaffolds have been shown to be a promising alternative and stem cells have been widely used to evaluate the compatibility, viability, and bioactivity of these materials. Polycaprolactone is a bioresorbable polymer, which has been widely used for biomedical and tissue engineering applications. The fundamental concept behind successful synthetic tissue-engineered scaffolds is to promote progenitor cell migration, adhesion, proliferation, and induce differentiation, extracellular matrix synthesis, and finally integration with host tissue. In this study, we investigated the adhesion, proliferation, and chondrogenic and adipogenic differentiation of ADSCs on nanowire surfaces. A solvent-free gravimetric template technique was used to fabricate polycaprolactone nanowires surfaces. The results indicated that during the growth period i.e., initial 7 days of culture, the nanowire surfaces (NW) supported adhesion and proliferation of the cells that had elongated morphologies. However, cell on surfaces without nanowires had non-elongated morphologies. Further, immunofluorescence imaging of marker proteins showed that the nanowires surfaces did not appear to support chondrogenic differentiation whereas supported adipogenic differentiation of ADSCs.
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Project description:Bone marrow-derived mesenchymal stem cells (BMSCs) exhibit multi-lineage differentiation potential and robust proliferative capacity. The late stage of differentiation signifies the functional maturation and characterization of specific cell lineages, which is crucial for studying lineage-specific differentiation mechanisms. However, the molecular processes governing late-stage BMSC differentiation remain poorly understood. This study aimed to elucidate the key biological processes involved in late-stage BMSC differentiation. Publicly available transcriptomic data from human BMSCs were analyzed after approximately 14 days of osteogenic, adipogenic, and chondrogenic differentiation. Thirty-one differentially expressed genes (DEGs) associated with differentiation were identified. Pathway enrichment analysis indicated that the DEGs were involved in extracellular matrix (ECM)-receptor interactions, focal adhesion, and glycolipid biosynthesis, a ganglion series process. Subsequently, the target genes were validated using publicly available single-cell RNA-seq data from mouse BMSCs. Lamc1 exhibited predominant distribution in adipocytes and osteoblasts, primarily during the G2/M phase. Tln2 and Hexb were expressed in chondroblasts, osteoblasts, and adipocytes, while St3gal5 was abundantly distributed in stem cells. Cell communication analysis identified two receptors that interact with LAMCI. q-PCR results confirmed the upregulation of Lamc1, Tln2, Hexb, and St3gal5 during osteogenic differentiation and their downregulation during adipogenic differentiation. Knockdown of Lamc1 inhibited adipogenic and osteogenic differentiation. In conclusion, this study identified four genes, Lamc1, Tln2, Hexb, and St3gal5, that may play important roles in the late-stage differentiation of BMSCs. It elucidated their interactions and the pathways they influence, providing a foundation for further research on BMSC differentiation.
| S-EPMC11344058 | biostudies-literature
Project description:The expression of hTERT, the catalytic subunit of telomerase, immortalizes normal human urothelial cells (NHUC). Expression of a modified hTERT, without the ability to act in telomere maintenance, did not immortalize NHUC, confirming that effects at telomeres are required for urothelial immortalization. Previous studies indicate that inhibition of telomerase has an immediate effect on urothelial carcinoma (UC) cell line viability, before sufficient divisions to account for telomere attrition, implicating non-telomere effects of telomerase in UC. We analyzed the effects of telomerase on gene expression in isogenic mortal and hTERT-transduced NHUC. hTERT expression led to consistent alterations in the expression of genes predicted to be of phenotypic significance in tumorigenesis. A subset of expression changes were detected soon after transduction with hTERT and persisted with continued culture. These genes (NME5, PSCA, TSPYL5, LY75, IGFBP2, IGF2, CEACAM6, XG, NOX5, KAL1, and HPGD) include eight previously identified as polycomb group targets. TERT-NHUC showed overexpression of the polycomb repressor complex (PRC1 and PRC4) components, BMI1 and SIRT1, and down-regulation of multiple PRC targets and genes associated with differentiation. TERT-NHUC at 100 population doublings, but not soon after transduction, showed increased saturation density and an attenuated differentiation response, indicating that these are not acute effects of telomerase expression. Some of the changes in gene expression identified may contribute to tumorigenesis. Expression of NME5 and NDN was down-regulated in UC cell lines and tumors. Our data supports the concept of both telomere-based and non-telomere effects of telomerase and provides further rationale for the use of telomerase inhibitors in UC.
| S-EPMC3437422 | biostudies-literature
Project description:ObjectivesAdipose tissue engineering is one of the hottest topics in the field of regenerative medicine. Fat tissue has been considered as an abundant and accessible source of adult stem cells by tissue engineers, since it gives rise to adipose stem cells. However, recent reports have pointed out that adipose tissue, as a secretory and endocrine organ, might secrete cytokines that regulate body functions such as metabolism, infammation and more. In this study, we aim to investigate the adipogenic-inducing factors secreted by fat tissue.Materials and methodsConditioned medium were collected by culturing fat tissue fragments in plastic flasks. Mesenchymal stem cells (MSCs) cultured in conditioned medium (CM) to test the adipogenic-inducing factors. Oil red O staining, reverse transcription/polymerase chain reaction and immunocytofluorescent staining were performed to examine the differentiation of MSCs in CM.ResultsMSCs cultured in CM of adipose tissue spontaneously differentiated into adipocytes. Furthermore, supplementation of insulin or dexamethasone to CM accelerated the process of lipid accumulation of differentiated MSCs.DiscussionResults from this study demonstrated that fat tissues secrete small molecules, which induce adipogenic differentiation of MSCs.ConclusionsOur study provides clues for improving adipose tissue engineering by using fragmented adipose tissue as sources of fat-inducing factors.
| S-EPMC6496604 | biostudies-literature