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In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells.

ABSTRACT: Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow-derived cells were CD11b?CD34?CD44?CD45?CD90? and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrations of VEGF (EGM?+?VEGF; 50 ng/ml). Endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE-cadherin, PECAM-1, VEGF-R1 and VEGF-R2 after 10 days of stimulation. Cells cultured in EGM?+?VEGF medium demonstrated higher amounts of DiI-AcLDL-positive cells and enhanced the presence of vWF (90%), VE-Cadherin- (60%) and PECAM-1 (48%)-positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5-2-fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM?+?VEGF medium when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in vitro inducing conditions. The differentiated cells would provide new options for re-endothelialization following interventional procedures and tissue engineering.

SUBMITTER: Pankajakshan D

PROVIDER: S-EPMC4089894 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells.

Pankajakshan Divya D Kansal Vikash V Agrawal Devendra K DK

Journal of tissue engineering and regenerative medicine 20120518 11

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow-derived cells were CD11b⁻CD34⁻CD44⁺CD45⁻CD90⁺ and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrat ...[more]

PMID: 22605545

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Project description:Bone-marrow mesenchymal stem cells (BMSC) are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC). The abundance and ease of harvest make the adipose-derived stem cells (ASC) an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ?0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition, functional analysis data might indicate differences in therapeutic application.

Project description:Silica nonwoven fabrics (SNFs) with enough mechanical strength are candidates as implantable scaffolds. Culture of cells therein is expected to affect the proliferation and differentiation of the cells through cell-cell and cell-SNF interactions. In this study, we examined three-dimensional (3D) SNFs as a scaffold of mesenchymal stem cells (MSCs) for bone tissue engineering applications. The interconnected highly porous microstructure of 3D SNFs is expected to allow omnidirectional cell-cell interactions, and the morphological similarity of a silica nanofiber to that of a fibrous extracellular matrix can contribute to the promotion of cell functions. 3D SNFs were prepared by the sol-gel process, and their mechanical properties were characterized by the compression test and rheological analysis. In the compression test, SNFs showed a compressive elastic modulus of over 1 MPa and a compressive strength of about 200 kPa. These values are higher than those of porous polystyrene disks used for in vitro 3D cell culture. In rheological analysis, the elastic modulus and fracture stress were 3.27 ± 0.54 kPa and 25.9 ± 8.3 Pa, respectively. Then, human bone marrow-derived MSCs were cultured on SNFs, and the effects on proliferation and osteogenic differentiation were evaluated. The MSCs seeded on SNF proliferated, and the thickness of the cell layer became over 80 μm after 14 days of culture. The osteogenic differentiation of MSCs on SNFs was induced by the culture in the commercial osteogenic differentiation medium. The alkaline phosphatase activity of MSCs on SNFs increased rapidly and remained high up to 14 days and was much higher than that on two-dimensional tissue culture-treated polystyrene. The high expression of RUNX2 and intense staining by alizarin red s after differentiation supported that SNFs enhanced the osteogenic differentiation of MSCs. Furthermore, permeation analysis of SNFs using fluorescein isothiocyanate-dextran suggested a sufficient permeability of SNFs for oxygen, minerals, nutrients, and secretions, which is important for maintaining the cell viability and vitality. These results suggested that SNFs are promising scaffolds for the regeneration of bone defects using MSCs, originated from highly porous and elastic SNF characters.

Project description:BackgroundTissue-engineered bone grafts (TEBGs) that undergo vascularization and neurotization evolve into functioning bone tissue. Previously, we verified that implanting sensory nerve tracts into TEBGs promoted osteogenesis. However, the precise mechanisms and interaction between seed cells were not explored. In this study, we hypothesized that neurotization may influence the osteogenesis of TEBGs through vascularization.MethodsWe cultured rat Schwann cells (SCs), aortic endothelial cells (AECs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) and then obtained BM-MSC-derived induced endothelial cells (IECs) and induced osteoblasts (IOBs). IECs and AECs were cultured in an SC-conditioned medium (SC-CM) to assess proliferation, migration, capillary-like tube formation, and angiogenesis, and the vascular endothelial growth factor (VEGF) levels in the supernatants were detected. We established an indirect coculture model to detect the expression of nestin and VEGF receptors in IECs and tissue inhibitor of metalloproteinase (TIMP)-2 in SCs. Then, SCs, IECs, and IOBs were labeled and loaded into a β-tricalcium phosphate scaffold to induce prevascularization, and the scaffold was implanted into a 6-mm-long defect of rat femurs. Three groups were set up according to the loaded cells: I, SCs, and IECs (coculture for 3 days) plus IOBs; II, IECs (culture for 3 days) plus IOBs; III, IOBs. Nestin and TIMP-2 expression and osteogenesis of TEBGs were evaluated at 12 weeks post-implantation through histological and radiological assessments.ResultsWe found that SC-CM promoted IEC proliferation, migration, capillary-like tube formation, and angiogenesis, but no similar effects were observed for AECs. IECs expressed nestin extensively, while AECs barely expressed nestin, and SC-CM promoted the VEGF secretion of IECs. In the coculture model, SCs promoted nestin and VEGF receptor expression in IECs, and IECs inhibited TIMP-2 expression in SCs. The promotion of prevascularized TEBGs by SCs and IECs in group I augmented new bone formation at 6 and 12 weeks. Nestin expression was higher in group I than in the other groups, while TIMP-2 expression was lower at 12 weeks.ConclusionsThis study demonstrated that SCs can promote TEBG osteogenesis via IECs and further revealed the related specific characteristics of IECs, providing preliminary cytological evidence for neurotization of TEBGs.

Project description:BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitor cells localized in the stromal compartment of the bone marrow (BM). The potential of MSC for mesenchymal differentiation has been well documented in different animal models predominantly on rodents. However, information regarding bovine MSC (bMSC) is limited, and the differentiation potential of bMSC derived from fetal BM remains unknown. In the present study we sought to isolate bMSC from abattoir-derived fetal BM and to characterize the multipotent and differentiation potential under osteogenic, chondrogenic and adipogenic conditions by quantitative and qualitative analyses. RESULTS: Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. These cells expressed high levels of MSC surface markers (CD73, CD90, and CD105) and low levels of hematopoietic surface markers (CD34 and CD45). Culture of bMSC under osteogenic conditions during a 27-day period induced up-regulation of the osteocalcin (OC) gene expression and alkaline phosphatase (ALPL) activity, and promoted mineralization of the matrix. Increasing supplementation levels of ascorbic acid to culture media enhanced osteogenic differentiation of bMSC; whereas, reduction of FBS supplementation compromised osteogenesis. bMSC increased expression of cartilage-specific genes aggrecan (ACAN), collagen 2A1 (COL2A1) and SRY (sex-determining region Y) box 9 (SOX9) at Day 21 of chondrogenic differentiation. Treatment of bMSC with adipogenic factors increased levels of fatty acid-binding protein 2 (AP2) mRNA and accumulation of lipid vacuoles after 18 days of culture. NANOG mRNA levels in differentiating bMSC were not affected during adipogenic culture; however, osteogenic and chondrogenic conditions induced higher and lower levels, respectively. CONCLUSIONS: Our analyses revealed the potential multilineage differentiation of bMSC isolated from abattoir-derived fetal BM. NANOG mRNA pattern in differentiating bMSC varied according to differentiation culture conditions. The osteogenic differentiation of bMSC was affected by ascorbic acid and FBS concentrations in culture media. The simplicity of isolation and the differentiation potential suggest that bMSC from abattoir-derived fetal BM are appropriate candidate for investigating MSC biology and for eventual applications for regenerative therapy.

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In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells.

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In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells.

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