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MicroRNA Profile of Human Bone Marrow Mesenchymal Stem Cells during Hepatic Differentiation and Therapy.

ABSTRACT: Background and Aims: MicroRNAs (miRNAs) play important roles in hepatocyte differentiation from human bone marrow mesenchymal stem cells (hBMSCs) and the therapeutic application in vivo. However, the mechanisms of miRNA regulation are still unknown. This study aimed to profile the miRNA basis for improving the function of hBMSC-differentiated hepatocyte-like cells (hBMSC-Heps). Methods: Characteristic miRNAs of hBMSC-Heps were identified by transcriptome sequencing and validated by quantitative real-time polymerase chain reaction (qRT-PCR). An in vivo hBMSC transplantation model was used to assess the regulatory effects of miRNAs on liver regeneration during hBMSC therapy in pigs with fulminant hepatic failure (FHF). The biological functions of significant miRNA molecules were confirmed by transfection of miRNA activators or inhibitors into hBMSCs during hepatogenic differentiation. Results: The transcriptome of hBMSC-Heps showed characteristics distinct from those of undifferentiated hBMSCs. A total of 77 miRNAs were significantly differentially expressed in hBMSC-Heps at day 10 and day 20 after hBMSC differentiation that were directly related to the functions of hepatocytes. Among the top 10 significantly differentially expressed and the top 10 most abundant miRNAs, nine miRNAs that exhibited a pattern of gradual change were chosen for further analysis. The expression of nine miRNAs was confirmed by qRT-PCR in vitro and showed the same changing trends in vivo in an hBMSC transplantation model in pigs. Functional experiments with these miRNAs showed that activators of hsa-miR-26b-5p and hsa-miR-148a-3p and an inhibitor of hsa-miR-423-3p were sufficient to improve the differentiation of hBMSCs into hepatocyte-like cells. Conclusions: Transcriptome profiles of miRNA revealed the basis of the differentiation and development of hBMSC-Heps. Manipulation of three miRNAs (hsa-miR-26b-5p, hsa-miR-148a-3p and hsa-miR-423-3p) significantly improved hepatocyte generation and liver regeneration, indicating the potential of these miRNAs for future clinical applications.

SUBMITTER: Jiang J

PROVIDER: S-EPMC8692113 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHuman bone marrow-derived mesenchymal stem cells (hBMSCs) have chondrocyte differentiation potential and are considered to be a cell source for cell-transplantation-mediated repair of cartilage defects, including those associated with osteoarthritis (OA). However, chondrocyte hypertrophic differentiation is a major obstacle for the application of hBMSCs in articular cartilage defect treatment. We have previously shown that microRNA-27b (miR-27b) inhibits hypertrophy of chondrocytes from rat knee cartilage. In this study, we investigated the role of miR-27b in chondrocyte hypertrophic differentiation of hBMSCs.MethodsChondrogenic marker and microRNA expression in hBMSC chondrogenic pellets were evaluated using RT-qPCR and immunohistochemistry. The hBMSCs were transfected with miR-27b before inducing differentiation. Gene and protein expression levels were analyzed using RT-qPCR and western blot. Coimmunoprecipitation was used to confirm interaction between CBFB and RUNX2. Luciferase reporter assays were used to demonstrate that CBFB is a miR-27b target. Chondrogenic differentiation was evaluated in hBMSCs treated with shRNA targeting CBFB. Chondrogenic hBMSC pellets overexpressing miR-27b were implanted into cartilage lesions in model rats; therapeutic effects were assessed based on histology and immunohistochemistry.ResultsThe hBMSCs showed typical MSC differentiation potentials. During chondrogenic differentiation, collagen 2 and 10 (COL2 and COL10), SOX9, and RUNX2 expression was upregulated. Expression of miR-140, miR-143, and miR-181a increased over time, whereas miR-27b and miR-221 were downregulated. Cartilage derived from hBMSC and overexpressing miR-27b exhibited higher expression of COL2 and SOX9, but lower expression of COL10, RUNX2, and CBFB than did the control cartilage. CBFB and RUNX2 formed a complex, and CBFB was identified as a novel miR-27b target. CBFB knockdown by shRNA during hBMSC chondrogenic differentiation led to significantly increased COL2 and SOX9 expression and decreased COL10 expression. Finally, miR-27b-overexpressing hBMSC chondrogenic pellets had better hyaline cartilage morphology and reduced expression of hypertrophic markers and tend to increase repair efficacy in vivo.ConclusionMiR-27b plays an important role in preventing hypertrophic chondrogenesis of hBMSCs by targeting CBFB and is essential for maintaining a hyaline cartilage state. This study provides new insights into the mechanism of hBMSC chondrocyte differentiation and will aid in the development of strategies for treating cartilage injury based on hBMSC transplantation.

Project description:Bone marrow mesenchymal stem cells (BMSCs) nowadays are regarded as promising candidates in cell-based therapy for the regeneration of damaged bone tissues that are either incurable or intractable due to the insufficiency of current therapies. Recent studies suggest that BMSCs differentiate into osteoblasts, and that this differentiation is regulated by some specific patterns of epigenetic modifications, such as DNA methylation. However, the potential role of DNA methylation modification in BMSC osteogenic differentiation is unclear. In this study, we performed a genome-wide study of DNA methylation between the noninduced and induced osteogenic differentiation of BMSCs at day 7. We found that the majority of cytosines in a CpG context were methylated in induced BMSCs. Our results also revealed that, along with the induced osteogenic differentiation in BMSCs, the average genomic methylation levels and CpG methylation in transcriptional factor regions (TFs) were increased, the CpG methylation level of various genomic elements was mainly in the medium-high methylation section, and CpG methylation levels in the repeat element had highly methylated levels. The GO analysis of differentially methylated region- (DMR-) associated genes (DMGs) showed that GO terms, including cytoskeletal protein binding (included in Molecular Function GO terms), skeletal development (included in Biological Process GO terms), mesenchymal cell differentiation (included in Biological Process GO terms), and stem cell differentiation (included in Biological Process), were enriched in the hypermethylated DMGs. Then, the KEGG analysis results showed that the WNT pathway, inositol phosphate metabolism pathway, and cocaine addiction pathway were more correlative with the DMRs during the induced osteogenic differentiation in BMSCs. In conclusion, this study revealed the difference of methylated levels during the noninduced and induced osteogenic differentiation of BMSCs and provided useful information for future works to characterize the important function of epigenetic mechanisms on BMSCs' differentiation.

Project description:BackgroundLong non-coding RNA (lncRNA) plays crucial role in osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), involving in regulation of competing endogenous RNA (ceRNA) mechanisms and conduction of signaling pathways. However, its mechanisms are poorly understood. This study aimed to investigate lncRNAs, miRNAs and mRNAs expression profiles in rat BMMSCs (rBMMSCs) osteogenic differentiation, screen the potential key lncRNA-miRNA-mRNA networks, explore the putative functions and identify the key molecules, as the basis of studying potential mechanism of rBMMSCs osteogenic differentiation driven by lncRNA, providing molecular targets for the management of bone defect.MethodsHigh-throughput RNA sequencing (RNA-seq) was used to determine lncRNAs, miRNAs, and mRNAs expression profiles at 14-day rBMMSCs osteogenesis. The pivotal lncRNA-miRNA and miRNA-mRNA networks were predicted from sequencing data and bioinformatic analysis, and the results were exported by Cytoscape 3.9.0 software. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used for functional exploration. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to validate lncRNAs, miRNAs and mRNAs.ResultsrBMMSCs were identified, and the osteogenic and adipogenic differentiation ability were detected. A total of 8634 lncRNAs were detected by RNA-seq, and 1524 differential expressed lncRNAs, of which 812 up-regulated and 712 down-regulated in osteo-inductive groups compared with control groups. 30 up-regulated and 61 down-regulated miRNAs, 91 miRNAs were differentially expressed in total. 2453 differentially expressed mRNAs including 1272 up-expressed and 1181 down-expressed were detected. 10 up-regulated lncRNAs were chosen to predict 21 down-regulated miRNAs and 650 up-regulated mRNAs. 49 lncRNA-miRNA and 1515 miRNA-mRNA interactive networks were constructed. GO analysis showed the most important enrichment in cell component and molecular function were "cytoplasm" and "protein binding", respectively. Biological process related to osteogenic differentiation such as "cell proliferation", "wound healing", "cell migration", "osteoblast differentiation", "extracellular matrix organization" and "response to hypoxia" were enriched. KEGG analysis showed differentially expressed genes were mainly enriched in "PI3K-Akt signaling pathway", "Signaling pathway regulating pluripotency of stem cells", "cGMP-PKG signaling pathway", "Axon guidance" and "Calcium signaling pathway". qRT-PCR verified that lncRNA Tug1, lncRNA AABR07011996.1, rno-miR-93-5p, rno-miR-322-5p, Sgk1 and Fzd4 were consistent with the sequencing results, and 4 lncRNA-miRNA-mRNA networks based on validations were constructed, and enrichment pathways were closely related to "PI3K-Akt signaling pathway", "Signaling pathway regulating pluripotency of stem cells" and "Wnt signaling pathway".ConclusionslncRNAs, miRNAs and mRNAs expression profiles provide clues for future studies on their roles for BMMSCs osteogenic differentiation. Furthermore, lncRNA-miRNA-mRNA networks give more information on potential new mechanisms and targets for management on bone defect.

Project description:The production of a clinically useful engineered cartilage is an outstanding and unmet clinical need. High-throughput RNA sequencing provides a means of characterizing the molecular phenotype of populations of cells and can be leveraged to better understand differences among source cells, derivative engineered tissues, and target phenotypes. In this study, small RNA sequencing is utilized to comprehensively characterize the microRNA transcriptomes (miRNomes) of native human neonatal articular cartilage and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) differentiating into cartilage organoids, contrasting the microRNA regulation of engineered cartilage with that of a promising target phenotype. Five dominant microRNAs are upregulated during cartilage organoid differentiation and disproportionately regulate transcription factors: miR-148a-3p, miR-140-3p, miR-27b-3p, miR-140-5p, and miR-181a-5p. Two microRNAs that dominate the miRNomes of hBM-MSCs, miR-21-5p and miR-143-3p, persist throughout the differentiation process and may limit the ability of these cells to differentiate into an engineered cartilage resembling target native articular cartilage. By using predictive bioinformatics tools and antagomir inhibition, these persistent microRNAs are shown to destabilize the mRNA of genes with known or potential roles in cartilage biology including FGF18, TGFBR2, TET1, STOX2, ARAP2, N4BP2L1, LHX9, NFIA, and RPS6KA5. These results shed light on the extent to which only a few microRNAs contribute to the complex regulatory environment of hBM-MSCs for engineered tissues. Impact statement MicroRNAs are emerging as important controlling elements in the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). By using a robust bioinformatic approach and further validation in vitro, here we provide a comprehensive characterization of the microRNA transcriptomes (miRNomes) of a commonly studied and clinically promising source of multipotent cells (hBM-MSCs), a gold standard model of in vitro chondrogenesis (hBM-MSC-derived cartilage organoids), and an attractive in vivo target phenotype for clinically useful engineered cartilage (neonatal articular cartilage). These analyses highlighted a specific set of microRNAs involved in the chondrogenic program that could be manipulated to acquire a more robust articular cartilage-like phenotype. This characterization provides researchers in the cartilage tissue engineering field a useful atlas with which to contextualize microRNA involvement in complex differentiation pathways.

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MicroRNA Profile of Human Bone Marrow Mesenchymal Stem Cells during Hepatic Differentiation and Therapy.

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