Biological Characteristics of Human Skeletal Muscle-Derived Pericytes/Perivascular Cells and Their Supporting Effects on Hematopoietic Stem Cell in Vitro

Mesenchymal stem/stromal cells (MSCs) as precursor cells of bone marrow stromal cells, in addition to the formation of the bone marrow microenvironment, secrete a variety of blood-related factors, promote hematopoietic recovery and reconstruction. However, mesenchymal stem/stromal cells are extremely rare within human bone marrow, which limit their experimental and clinical applications. Our previous study demonstrated that MSCs were derived from pericytes/perivascular cells (PCs), and PCs were the precursor of MSCs. PCs are widely distributed, especially in skeletal muscle tissue, which are rich in content, easy to be harvested with minimal damage, and rapid cell proliferation. However, very few researches have been done on whether human skeletal muscle-derived pericytes/perivascular cells (hMD-PCs) are involved in HSPCs expansion/proliferation, differentiation and possible hematopoietic regulation mechanisms. Based on the above research background and research ideas, in current study, we intend to isolate, purify and characterize the CD146⁺ hMD-PCs and further investigate their supporting effect on human umbilical cord blood CD34⁺ cells in vitro.

Methods 1. hMD-PCs with phenotype CD146⁺CD56^-CD34^-CD144^-CD45^- were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting (MP-FACS), and their biological characteristic were conducted by detecting the surface marker of MSCs and inducing multilineage differentiation. 2. Human UCB CD34⁺cells were sorted by immunomagnetic beads, and then established the in vitro expansion culture system of UCB CD34⁺ cells co-culture with CD146⁺ hMD-PCs (experimental group), human bone marrow MSCs for feeder layer (positive control) and UCB CD34⁺ cells alone culture system (blank control). After 1 week, 2 weeks and 4 weeks of co-culture, the number of cells, the colony formation ability and immunophenotype were analysed. Result 1.CD146⁺ hMD-PCs were sorted by MP-FACS and the purity was (91.5±1.85)% (n=5); CD146⁺ hMD-PCs expressed mesenchymal surface markers CD73, CD90, CD105, CD44, and did not express hematopoietic cell and endothelial cell marker CD45, CD34, CD31. After induced culture, CD146⁺ hMD-PCs can differentiate into osteoblasts, chondrogenesis, adipocytes and myoblasts; 2.UCB CD34⁺ cells were sorted by magnetic beads which number was (9.18±3.50)×10⁵, and then co-cultured with CD146⁺ hMD-PCs and human BM-MSCs at a density of 5×10⁴/well, respectively. After 1, 2 and 4 weeks of co-culture, there were no statistically significant differences in cell number, colony formation ability and immunophenotype (CD45⁺cells, CD34⁺CD33^-cells, CD14⁺cells, CD10⁺/CD19⁺cells) between CD146⁺ hMD-PCs for feeder layer culture system and BM-MSCs for feeder layer culture system (P>0.05, n=6). However, after 1 week, 2 weeks of co-culture, the both cells number of CD146⁺ hMD-PCs and human BM-MSCs culture system were significantly different from the blank control group, (P<0.01) and (P<0.001) respectively. The colony culture could not be performed due to low cell number of the blank control group. Conclusion CD146+ hMD-PCs, like human BM-MSCs, have hematopoietic support capacity in vitro; therefore CD146+ hMD-PCs can be used as another source of stromal cells for the expansion of hematopoietic stem cells.