Myocardial Patch Formation by Three-Dimensional Culture of Bone Marrow Mesenchymal Stem Cells with 3-Hydroxybutyrate-Co-4-Hydroxybutyrate Under Hypoxia

Abstract

Herein we researched the effects of a hypoxic microenvironment on bone marrow mesenchymal stem cells (BM-MSCs) on poly 3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)] and present a theoretical basis for development of cell transplantation. Mouse bone marrow mesenchymal stem cells were isolated by whole bone marrow culture and surface antigens were analyzed by flow cytometry of passage 5 cells. P(3HB-co-4HB) and bone marrow mesenchymal stem cells were prepared as stem cell patches randomly divided into normoxia (control, 20% oxygen) and hypoxia (3% oxygen) groups. After 24 h, the patch was used for experiments. Cell proliferation was determined by CCK-8 assays. Adhesion, survival, and growth of cells on patches were observed by scanning electron microscopy. Expression of hypoxia-inducible factor-1α (HIF-1α) was tested by real-time quantitative PCR and western blotting. At 2 weeks after addition of cardiomyocyte differentiation inducer 5-azacytidine, cardiac troponin T (cTnT) expression was detected by immunofluorescence. After 24 h, the proliferation of the hypoxic group was considerably greater compared with the normoxic group (n = 12,P < 0 05). SEM demonstrated that the number of viable cells in the hypoxic group was higher than that in the normoxic group. Adhesion between cells and the patch was firm and cell morphology was normal in the hypoxic group. Significant upregulation of HIF-1α mRNA was observed by real-time quantitative PCR after 12 h (P < 0 05). HIF-1αprotein expression in the hypoxia group was considerably higher than that in the normoxia group. cTnT expression in the hypoxic group was more pronounced than that in the normoxic group. Our results show that a hypoxic microenvironment promotes the adhesion, survival, proliferation, and myocardial differentiation of bone marrow mesenchymal stem cells on a P(3HB-co-4HB) patch, which may be mediated by the HIF-1α; pathway.