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Human mesenchymal stem cells (hMSCs) have been demonstrated to exhibit characteristic cytosolic Ca2+ ([Ca2+]i) oscillations in response to physicochemical factors, including soluble factors, matrix proteins and microenvironment. It has been postulated that the calcium oscillation at the early stages of hMSC differentiation may serve as a biophysical predictor. The goal of the present study is therefore aimed at elucidating the matrix proteinmediated [Ca2+]i oscillation that occurs in the early stages of hMSC osteogenic differentiation. On the collagen substrates of different stiffness and concentrations, intracellular [Ca2+]i oscillations of hMSCs exhibited rapid spikes on the hard substrates and the opposite on the soft substrate. Cells seeded on a hard substrate also expressed signi ficantly high levels of osteogenic gene markers, suggesting a key role of intracellular calcium in the transduction of ECM signaling to the hMSC differentiation. Our findings revealed that the characteristics of [Ca2+]i oscillation are regulated by the biochemical and mechanical properties of the collagen substrate. Furthermore, β1-integrin and associated signaling proteins found in focal adhesions (FAs) were involved in the signaling pathways. Because the [Ca2+]i signaling and stemcell differentiation appear closely correlated, the regulation of [Ca2+]i signaling through the modification of engineeredECMmay provide a controllable exogenous technique to direct the fate of hMSCs.
This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.
Mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after Parkinson’s disease. MSCs have significant advantages over other stem cell types, and greater potential for immediate clinical application. The aim of this study was to investigate whether MSCs from the human placenta could be induced to differentiate into dopaminergic cells. MSCs from the human placenta were isolated by digestion and density gradient fractionation, and their cell surface glycoproteins were analyzed by flow cytometry. These MSCs were cultured under conditions promoting differetiation into adipocytes and osteoblasts. Using a cocktail that includes basic fibroblast growth factor (bFGF), all trans retinoic acid (RA), ascorbic acid (AA) and 3-isobutyl-1-methylxanthine (IBMX), the MSCs were induced in vitro to become dopamine (DA) neurons. Then, the expression of the mRNA for the Nestin and tyrosine hydroxylase (TH) genes was assayed via RT-PCR. The expression of the Nestin, dopamine transporter (DAT), neuronal nuclear protein (NeuN) and TH proteins was determined via immunofluorescence. The synthesized and secreted DA was determined via ELISA. We found that MSCs from the human placenta exhibited a fibroblastoid morphology. Flow cytometric analyses showed that the MSCs were positive for CD44 and CD29, and negative for CD34, CD45, CD106 and HLA-DR. Moreover, they could be induced into adipocytes and osteocytes. When the MSCs were induced with bFGF, RA, AA and IBMX, they showed a change in morphology to that of neuronal-like cells. The induced cells expressed Nestin and TH mRNA, and the Nestin, DAT, NeuN and TH proteins, and synthesized and secreted DA. Our results suggest that MSCs from the human placenta have the ability to differentiate into dopaminergic cells.
The expression of predominant housekeeping genes used in RT-qPCR can vary during development and differentiation. The frequently used housekeeping genes (ACTB, GAPDH, 18S rRNA, EF1α and RPL 13a) were evaluated during an early stage of the osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (mMSCs) (under normal conditions or treated with CCG-4986) to identify housekeeping genes whose expression remained constant during osteogenic differentiation. When we used RGS4 mRNA, which was determined as copy number per μg of total RNA, to normalize gene expression, we observed that the relative EF1α expression profile was consistent with RGS4 expression after treatment with CCG-4986. All the relative expression profiles of the EF1α, 18S rRNA, and RPL13a housekeeping genes were consistent with RGS4 profiles determined by measuring mRNA copies under normal osteogenic differentiation conditions. The expression profiles calibrated by ACTB and GAPDH were not consistent with those determined using mRNA copy number in untreated cells or cells treated with CCG-4986 under osteogenic differentiation conditions. Under normal osteogenic differentiation conditions, EF1α, 18S rRNA, and RPL 13a are suitable housekeeping genes for RT-qPCR analysis. However, EF1α is the only suitable gene upon CCG-4986 treatment.
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