Published online Mar 26, 2020. doi: 10.4252/wjsc.v12.i3.203
Peer-review started: November 1, 2019
First decision: December 12, 2019
Revised: January 17, 2020
Accepted: January 31, 2020
Article in press: January 31, 2020
Published online: March 26, 2020
Small diameter vascular grafts can be applied in a wide variety of diseases, but mostly in cardiovascular disease (CAD). Globally, CAD affects more than 18 million people, and it is estimated that more than 500000 bypass surgeries are performed. Until now, autologous saphenous vein transplants, or conduits made of Dacron or ePTFE, represent the gold standard strategy. However, severe side effects, including impaired patency, immune reaction and intima hyperplasia, may be accompanied by their use. For this purpose, the decellularization of human umbilical arteries and the repopulation with vascular smooth muscle cells (VSMCs) in order to obtain fully functional vascular grafts, could represent an alternative approach. VSMCs are a cellular population responsible for vasoconstriction and vasodilation. Recently, the development of VSMCs has been proposed using induced pluripotent stem cell (iPSCs) technology. However, iPSCs have not been approved for broad human use. In this way, an alternative approach using platelet lysate from umbilical cord blood (UCB-PL) may be applied in the differentiation process of VSMCs from mesenchymal stromal cells (MSCs). It is known that UCB-PL contains significant amounts of growth factors such as TGF-β1, PDGFA, FGF2, IFN-γ and TNF-α, which have previously been used in several differentiation protocols. The aim of this study was to establish the differentiation process of VSMCs from MSCs derived from the Wharton’s Jelly tissue (WJ-MSCs) using the UCB-PL. Then, the differentiated VSMCs were used for repopulation experiments of decellularized human umbilical arteries (hUAs) to produce fully functional small diameter vascular grafts.
Until now, the development of VSMCs is accomplished using exogenous supplementation of several growth factors or through iPSC technology. However, both approaches may cause allergic reactions or could even be tumorigenic. Indeed, a great number of growth factors are derived from animals. Additionally, iPSC technology has not received full approval from the Food and Drug Administration for human use, due to the use of c-Myc, which may lead to tumor development. In order to overcome these issues, the differentiation of VSMCs from MSCs using UCB-PL and ascorbic acid has been proposed. It has been shown in the past that specific growth factors, especially TGF-β1, could promote the differentiation of VSMCs. UCB-PL contains several growth factors, including TGF-β1, PDGF-A, FGF2, IFN-γ and TNF-α, and in combination with ascorbic acid may lead to the successful development of VSMCs.
The main objective of this study was the successful differentiation of VSMCs obtained from WJ-MSCs using UCB-PL. Secondary objectives were the production of small diameter vascular grafts in hUAs using the decellularization method. In addition, the repopulation of decellularized vessels with the produced VSMCs, which may result in functional vessels, was also evaluated in this study.
Initially, WJ-MSCs were isolated from hUCs and expanded until they reached P4. Characterization of WJ-MSCs was performed according to the criteria of the International Society for Cell and Gene Therapy, including morphological evaluation, trilineage differentiation and flow cytometry analysis. Then, the differentiation of VSMCs was performed. To do this, WJ-MSCs were cultured in a medium containing UCB-PL and ascorbic acid for 3 wk. Gene expression profiles of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9, NANOG, OCT4, and GAPDH by RT-PCR, PCR and gel electrophoresis were evaluated. Further analysis included the indirect immunofluorescence of VSMCs using antibodies against ACTA2 and MYH11. The production of vascular grafts was performed using the decellularization of hUAs. Then histological (H & E, SR and TB stains) and biochemical analyses (hydroxyproline, sGAG, DNA content) in decellurized hUAs were applied. Finally, the repopulation of decellularized hUAs with VSMCs through static seeding was performed. Repopulated vessels were analyzed histologically (H & E, MYH11/DAPI) and biochemically (hydroxyproline, DNA content and ADP/ATP ratio). In addition, the proliferation of VSMCs in repopulated vessels was immunohistochemically evaluated using Ki67 and proliferating cell nuclear antigen.
WJ-MSCs were successfully isolated and expanded from hUCs. Their spindle-shaped morphology was retained until they reached P4. Total cell number, CDT and PD of WJ-MSCs at P4 was > 12 × 106 cells, 36 ± 3 h and 6 ± 1, respectively. WJ-MSCs fulfilled the criteria of the International Society for Cell and Gene Therapy, indicating successful differentiation towards “osteogenic”, “adipogenic” and “chondrogenic” lineages, positive expression (> 95%) for CD73, CD90 and CD105, and negative expression (< 3%) for CD34, CD45 and HLA-DR. WJ-MSCs were successfully differentiated into VSMCs using UCB-PL and ascorbic acid. Differentiated VSMCs expressed ACTA2, MYOCD, MYH11 and TGLN. In addition, early and late VSMCs markers such as ACTA2 and MYH11 were evaluated according to indirect immunofluorescence analyses. HUAs were effectively decellularized and characterized by the preservation of ECM proteins, while no cell and nuclei materials were evident. Statistically significant differences were observed between non-decellularized and decellularized hUAs regarding the hydroxyproline (P < 0.001), sGAG (P < 0.001) and DNA (P < 0.001) content. Decellularized hUAs were successfully repopulated by the produced VSMCs, as it was indicated by histological analysis (H and E, MYH11/DAPI). Repopulated vessels were characterized by elevated levels of hydroxyproline (86 ± 8 μg hydroxyproline/mg of dry tissue weight), sGAG (3 ± 1 μg sGAG / mg of dry tissue weight), and DNA (554 ± 49 ng DNA/mg of dry tissue weight) content after 3 wk of cultivation. In addition, the key proliferation markers Ki67 and proliferating cell nuclear antigen were positively expressed by VSMCs in repopulated vessels, according to immunohistochemistry results.
VSMCs can be successfully produced from WJ-MSCs using UCB-PL in combination with ascorbic acid. Unlike current approaches, including the exogenous supplementation of growth factors or the use of iPSC technology, no such approaches were applied to this study. UCB-PL contains significant amounts of key growth factors required for VSMC differentiation. In addition, ascorbic acid supplementation to the differentiation medium appears to enhance the underlying mechanism. Besides, the successful production of VSMCs and the development of functional small diameter vascular grafts were assessed. HUAs were efficiently decellularized, and could serve as potential scaffolds for blood vessel engineering. To obtain functional small diameter vascular grafts, the decellularized hUAs were repopulated with the produced VSMCs. Finally, the repopulated vessels were characterized for their similar properties to the hUAs before the decellularization process. Taking into consideration the above data, hUCs could be a rich source both for cellular populations and vessel conduits. Additionally, this study brings into light a safer differentiation process that can possibly be used for the production of patient-specific VSMCs. It is known that the circulatory system of CAD patients is primarily affected. The isolation of VSMCs from patient vessel biopsies, which can be used for vascular graft engineering, is not efficient. On the contrary, MSCs (in adults) that are presented both in bone marrow and adipose tissue, can be isolated and differentiated into VSMCs with the current protocol, and can thus potentially be used in blood vessel engineering. In this way, and unlike the complicated and expensive approaches of the past, the production of fully functional blood vessels is one step closer to its clinical application.
The next step of this study will be focused on the use of the repopulated (with VSMCs) vascular grafts in an animal model, in order to better evaluate their functionality. Small blood vessel engineering is one of the milestones of personalized regenerative medicine. To this direction, the production of patient-specific small diameter vascular grafts under good manufacturing practice conditions, that can be readily accessible, will be of great importance.