| Background:Human bone marrow mesenchymal stem cells (BM-MSCs) are a group of clonogenic cells capable of self-renewal and multi-lineage differentiation into mesoderm-type cells. As the key precursor cells of marrow microenvironment, BM-MSCs are of great importance in maintaining hematopoietic and immune homeostasis. Notch signaling is a developmental pathway that regulates several fundamental cellular processes including cell fate and cell differentiation through PI3K/Akt pathway. Previous studies have demonstrated that phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathways play a vital role in the process of autophagy. However, there is a little known about the role of Notch signaling and autophagy via PI3K/Akt/mTOR during the adipogenic differentiation of human bone marrow mesenchymal stem cells.Objective:This study was designed to investigate the role of Notch signaling and autophagy in adipogenic differentiation of human bone marrow derived MSCs (BM-MSCs), and to reveal the possible underlying mechanism during the process.Methods:(1) BM-MSCs were isolated and purified from bone marrow by adherent culture; BM-MSCs were induced to differentiate into adipocytes and osteoblasts, which were identified with corresponding staining and specific genes; immunophenotyping of BM-MSCs was analyzed by flow cytometry. (2) Real-time PCR and Western blot detected relative gene expression analysis of Notch signaling and autophagy during adipogenic differentiation of BM-MSCs. (3) To explore the effect of Notch signaling pathway in BM-MSCs by spotting 0 μM,2.5 μM,5 μM and 10 μM DAPT, screening the best inhibitory concentrations of DAPT. (4) Red Oil O staining and PPARy, C/EBPa mRNA were assessed the BM-MSCs adipocyte differentiation ability by adding Notch inhibitor DAPT (5 μM). (5) Autophagy inhibitor 3-MA (5 mM) or CQ (20 μM) either alone or in combination with DAPT to test DAPT promoted the adipogenic differentiation of BM-MSCs through the activation of autophagy. (6) Adipogenic differentiation of BM-MSCs was associated with time-dependent modulation of PI3K/Akt/mTOR signaling was detected by Western blot. (7) MDC, TEM and Western blot were used to clarify DAPT induced autophagy in BM-MSCs via inhibition of PI3K/Akt/mTOR pathway. (8) Autophagy inhibitor 3-MA (5 mM) or CQ (20 μM repressed DAPT-induced autophagy were detected by Western blot and confocal microscopy.Results:(1) Elongated spindle-shaped or rhomboid BM-MSCs grew colonially and displayed a rather homogeneous confluent population. The cells possessed multipotent differentiation potential, as they could be induced into adipocytes and osteoblasts under standard in vitro differential condition. Cells showed positive expression of CD44, CD73, CD90, CD105 and CD166, but negative for CD14, CD19, CD34, CD45 and HLA-DR. All fulfilled the minimal phenotypic and function criteria proposed by International Society for Cellular Therapy. (2) There were a reduction of NOTCH1, NOTCH2, NOTCH3, DLL1, DLL3, DLL4, DLK1, DLK2 and HEY1 mRNA (P<0.05), whereas no changes were observed in NOTCH4, Jagged1, Jagged2, HES1 and HEY2 gene expression during BM-MSCs adipogenic differentiation. (3) Adipogenic differentiation of BM-MSCs induced early autophagy. (4) The expression of Notch signaling in BM-MSCs could be effectively inhibited by 5 μM DAPT. (5) Added Notch inhibitor DAPT (5 μM) during adipogenic differentiation of BM-MSCs, Red Oil O staining and PPARy, C/EBPa mRNA were assessed the pro-adipogenic differentiation of DAPT. (6) BM-MSCs were incubated with autophagy inhibitor 3-MA (5 mM) or CQ (20 μM) either alone or in combination with DAPT during adipogenic differentiation, through Oil Red O and PPARγ and C/EBPa mRNA to investigate the DAPT-induced adipogenesis is dependent on autophagy activation. (7) DAPT induced autophagy in BM-MSCs via inhibition of PI3K/Akt/mTOR pathwayConclusion:We first confirmed the down-regulation of Notch gene expression during BM-MSCs adipocyte differentiation and adipogenic differentiation of BM-MSCs induced early autophagy. Inhibition of Notch signaling by DAPT could promote BM-MSCs adipogenesis mediated by autophagy involving PTEN-PI3K/Akt/mTOR pathway.Background:Mesenchymal stem cells (MSCs) represent a promising young-state stem cell source for tissue engineering and regeneration due to the inherent potential of these cells to differentiate into various cell types. MSCs can be isolated from a variety of tissues such as bone marrow, fat, umbilical cord and placental. Human umbilical cord mesenchymal stem cells (hUC-MSCs) were isolated from umbilical cord with easily accessible and fewer ethical constrains, have emerged as a leading candidate source for regeneration. Under inducing media, MSCs could differentiate into adipocytes and osteoblasts. Galectins are part of the lectin super-family and have emerged an important regulator of diverse function in cell biology including immune homeostasis, pathogen recognition, neurogenesis and vascular permeability. Recent studies demonstrated that Gal-8 promoted cell differentiation and supported adipose tissue formation. However, the role of galectins in hUC-MSCs differentiation is not clear.Objective:This study aims to investigate the expression of Galectins during the adipogenic differentiation and osteogenic differentiation of human umbilical cord mesenchymal stem cells, which will offer a new strategy for the differentiation of hUC-MSCs in future application.Methods:(1) hUC-MSCs were isolated from umbilical cord obtained from donors. hUC-MSCs were induced to differentiate into adipocytes and osteoblasts, which were identified with corresponding staining; immunophenotyping of BM-MSCs were tested by flow cytometry. (2) The expression of Galectins at mRNA level in hUC-MSCs was measured by RT-PCR. (3) The levels of Gal-1, Gal-3, Gal-8 and Gal-9 mRNA were detected during hUC-MSCs adipogenesis and osteogenesis (0 d,1 d,3 d,7 d,14 d and 21 d) by Real-time PCR. (4) The levels of secretory Gal-1 and Gal-9 in hUC-MSCs adipogenesis and osteogenesis culture supernatants were detected by ELISA.Results:(1) The isolated hUC-MSCs expressed D29, CD73, CD90, CD105, CD44, CD166 and HLA-ABC, meanwhile, negative for CD14, CD31, CD45, CD133 and HLA-DR. Culured with inducing media, they could differentiate into adipocytes and osteoblasts. (2) mRNA of Gal-1, Gal-3, Gal-7, Gal-8, Gal-9, Gal-10, Gal-12 and Gal-13 were detected in hUC-MSCs, besides the abundant expression of gal-1, gal-3, Gal-8 and gal-9 were highly expressed. (3) The expression of Gal-1 and Gal-9 mRNA was significantly decreased during hUC-MSCs adipogenesis and osteogenesis. Meanwhile, the expression of Gal-3 and Gal-8 mRNA was significantly increased during hUC-MSCs adipogenesis and osteogenesis. (4) hUC-MSCs could express secretory Gal-1 and Gal-9, which may be associated with differentiation of hUC-MSCs.Conclusion:The expression of Gal-1, Gal-3, Gal-8 and Gal-9 was significantly changed during hUC-MSCs adipogenesis and osteogenesis. All these data laid the foundation for further detecting the differentiation mechanism of hUC-MSCs. |
PDF Full Text Request