Aim
To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate.
Background
The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations. Mesenchymal stromal cells (MSCs) derived from the Wharton's Jelly (WJ) tissue can be used as a source for obtaining vascular smooth muscle cells (VSMCs), while the human umbilical arteries (hUAs) can serve as a scaffold for blood vessel engineering.
Conclusion
VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, hUAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.
Methods
WJ-MSCs were isolated and expanded until passage (P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid, followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9, NANOG homeobox, OCT4 and GAPDH, was performed. In addition, immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated hUAs.
Results
WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into "osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression (> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, hUAs were isolated and decellularized. Based on histological analysis, decellularized hUAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized hUAs with VSMCs was performed for 3 wk. Decellularized hUAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and sGAG contents in repopulated hUAs with VSMCs. Specifically, total hydroxyproline and sGAG content after the 1st, 2nd and 3rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 μg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1 μg sGAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups (P < 0.05).