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Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering.

ABSTRACT: 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. AIM:To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate. 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 ?g hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1 ?g sGAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups (P < 0.05). 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.

SUBMITTER: Mallis P

PROVIDER: S-EPMC7118289 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering.

Mallis Panagiotis P Papapanagiotou Aggeliki A Katsimpoulas Michalis M Kostakis Alkiviadis A Siasos Gerasimos G Kassi Eva E Stavropoulos-Giokas Catherine C Michalopoulos Efstathios E

World journal of stem cells 20200301 3

<h4>Background</h4>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.<h4>Aim</h4>To develop VSMCs from WJ-MSCs utilizing umbilical cord blood pl ...[more]

PMID: 32266052

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Project description:BackgroundHuman platelet lysate (HPL) is emerging as the preferred xeno-free supplement for the expansion of mesenchymal stromal cells (MSCs) for bone tissue engineering (BTE) applications. Due to a growing demand, the need for standardization and scaling-up of HPL has been highlighted. However, the optimal storage time of the source material, i.e., outdated platelet concentrates (PCs), remains to be determined. The present study aimed to determine the optimal storage time of PCs in terms of the cytokine content and biological efficacy of HPL.MethodsDonor-matched bone marrow (BMSCs) and adipose-derived MSCs (ASCs) expanded in HPL or fetal bovine serum (FBS) were characterized based on in vitro proliferation, immunophenotype, and multi-lineage differentiation. Osteogenic differentiation was assessed at early (gene expression), intermediate [alkaline phosphatase (ALP) activity], and terminal stages (mineralization). Using a multiplex immunoassay, the cytokine contents of HPLs produced from PCs stored for 1-9 months were screened and a preliminary threshold of 4 months was identified. Next, HPLs were produced from PCs stored for controlled durations of 0, 1, 2, 3, and 4 months, and their efficacy was compared in terms of cytokine content and BMSCs' proliferation and osteogenic differentiation.ResultsBMSCs and ASCs in both HPL and FBS demonstrated a characteristic immunophenotype and multi-lineage differentiation; osteogenic differentiation of BMSCs and ASCs was significantly enhanced in HPL vs. FBS. Multiplex network analysis of HPL revealed several interacting growth factors, chemokines, and inflammatory cytokines. Notably, stem cell growth factor (SCGF) was detected in high concentrations. A majority of cytokines were elevated in HPLs produced from PCs stored for ≤ 4 months vs. > 4 months. However, no further differences in PC storage times between 0 and 4 months were identified in terms of HPLs' cytokine content or their effects on the proliferation, ALP activity, and mineralization of BMSCs from multiple donors.ConclusionsMSCs expanded in HPL demonstrate enhanced osteogenic differentiation, albeit with considerable donor variation. HPLs produced from outdated PCs stored for up to 4 months efficiently supported the proliferation and osteogenic differentiation of MSCs. These findings may facilitate the standardization and scaling-up of HPL from outdated PCs for BTE applications.

Project description:Purpose: Obtaining sufficient numbers of cells in a short time is a major goal of cell culturing in cell therapy and tissue engineering. However, current bidimensional (2D) culture methods are associated to several limitations, including low efficiency and the loss of key cell differentiation markers on cultured cells. Methods: In the present work, we have designed a novel biofabrication method based on a three-dimensional (3D) culture system (FIBRIAGAR-3D). Human Wharton's jelly mesenchymal stromal cells (HWJSC) were cultured in 3D using 100%, 75%, 50%, and 25% concentrations of fibrin-agarose biomaterials (FA100, FA75, FA50 and FA25 group) and compared with control cells cultured using classical 2D systems (CTR-2D). Results: Our results showed a significant increase in the number of cells generated after 7 days of culture, with cells displaying numerous expansions towards the biomaterial, and a significant overexpression of the cell proliferation marker KI67 was found for the FA75 and FA100 groups. TUNEL and qRT-PCR analyses demonstrated that the use of FIBRIAGAR-3D was not associated with an induction of apoptosis by cultured cells. Instead, the 3D system retained the expression of typical phenotypic markers of HWJSC, including CD73, CD90, CD105, NANOG and OCT4, and biosynthesis markers such as types-I and IV collagens, with significant increase of some of these markers, especially in the FA100 group. Finally, our analysis of 8 cell signaling molecules revealed a significant decrease of GM-CSF, IFN-g, IL2, IL4, IL6, IL8, and TNFα, suggesting that the 3D culture system did not induce the expression of pro-inflammatory molecules. Conclusion: These results confirm the usefulness of FIBRIAGAR-3D culture systems to increase cell proliferation without altering cell phenotype of immunogenicity and opens the door to the possibility of using this novel biofabrication method in cell therapy and tissue engineering of the human cornea, oral mucosa, skin, urethra, among other structures.

Dataset Information

Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering.

Publications

Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering.

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