Vascular morphogenesis of adipose-derived stem cells is mediated by heterotypic cell-cell interactions.
ABSTRACT Adipose-derived stromal/stem cells (ASCs) are a promising cell source for vascular-based approaches to clinical therapeutics, as they have been shown to give rise to both endothelial and perivascular cells. While it is well known that ASCs can present a heterogeneous phenotypic profile, spontaneous interactions among these subpopulations that result in the formation of complex tissue structures have not been rigorously demonstrated. Our study reports the novel finding that ASCs grown in monolayers in the presence of angiogenic cues are capable of self-assembling into complex, three-dimensional vascular structures. This phenomenon is only apparent when the ASCs are seeded at a high density (20,000 cells/cm(2)) and occur through orchestrated interactions among three distinct subpopulations: CD31-positive cells (CD31+), α-smooth muscle actin-positive cells (αSMA+), and cells that are unstained for both these markers (CD31-/αSMA-). Investigations into the kinetics of the process revealed that endothelial vessel-like structures initially arose from individual CD31+ cells through proliferation and their interactions with CD31-/αSMA- cells. During this period, αSMA+ cells proliferated and appeared to migrate toward the vessel structures, eventually engaging in cell-cell contact with them after 1 week. By 2 weeks, the lumen-containing CD31+ vessels grew greater than a millimeter in length, were lined with vascular basement membrane proteins, and were encased within a dense, three-dimensional cluster of αSMA+ and CD31-/αSMA- cells. The recruitment of αSMA+ cells was largely due to platelet-derived growth factor (PDGF) signaling, as the inhibition of PDGF receptors substantially reduced αSMA+ cell growth and vessel coverage. Additionally, we found that while hypoxia increased endothelial gene expression and vessel width, it also inhibited the growth of the αSMA+ population. Together, these findings underscore the potential use of ASCs in forming mature vessels in vitro as well as the need for a further understanding of the heterotypic interactions among ASC subpopulations.
- SourceAvailable from: Gail E Kilroy[show abstract] [hide abstract]
ABSTRACT: With the emergence of regenerative medicine, many researchers have turned to fat tissue as a source of adipose-derived stem cells (ASCs). Because freshly collected adipose tissue is not always readily available, there will be a need for improved cryopreservation methods to reproducibly maintain ASC viablility and multipotentiality in long-term storage. This study examines the efficiency of conventional dimethyl sulphoxide cryopreservation methods by measuring the maintenance of differentiation potential after one freeze cycle. Additionally, we analysed the viability of ASCs as a function of varying cell concentrations in cryopreservation media. We evaluated four distinct colony-forming unit assays (fibroblast, alkaline phosphatase, adipocyte and osteoblast) to monitor quantitatively the differentiation potential in ASCs after one freeze cycle. We found that the post-thaw viability was a function of storage concentration and that an optimal viability was observed for a concentration of 0.5 x 10(6) cells/ml cryopreservation medium.Journal of Tissue Engineering and Regenerative Medicine 01/2007; 1(4):322-4. · 2.83 Impact Factor
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ABSTRACT: In vivo endothelial commitment of adipose stem cells (ASCs) has scarcely been reported, and controversy remains on the contribution of ASCs to vascularization. We address the epigenetic commitment of ASCs to the endothelial lineage. We report a bisulfite sequencing analysis of CpG methylation in the promoters of two endothelial-cell-specific genes, CD31 and CD144, in freshly isolated and in cultures of ASCs before and after induction of endothelial differentiation. In contrast to adipose tissue-derived endothelial (CD31(+)) cells, freshly isolated ASCs display a heavily methylated CD31 promoter and a mosaically methylated CD144 promoter despite basal transcription of both genes. Methylation state of both promoters remains globally stable upon culture. Endothelial stimulation of ASCs in methylcellulose elicits phenotypic changes, marginal upregulation of CD31, and CD144 expression and restrictive induction of a CD31(+)CD144(+) immunophenotype. These events are accompanied by discrete changes in CpG methylation in CD31 and CD144 promoters; however, no global demethylation that marks CD31(+) cells and human umbilical vein endothelial cells occurs. Immunoselection of CD31(+) cells after endothelial stimulation reveals consistent demethylation of one CpG immediately 3' of the transcription start site of the CD31 promoter. Adipogenic or osteogenic differentiation maintains CD31 and CD144 methylation patterns of undifferentiated cells. Methylation profiles of CD31 and CD144 promoters suggest a limited commitment of ASCs to the endothelial lineage. This contrasts with the reported hypomethylation of adipogenic promoters, which reflects a propensity of ASCs toward adipogenic differentiation. Analysis of CpG methylation at lineage-specific promoters provides a robust assessment of epigenetic commitment of stem cells to a specific lineage.Stem Cells 05/2007; 25(4):852-61. · 7.70 Impact Factor
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ABSTRACT: Adipose-derived stromal (stem) cells (ASC) have been shown to be of great therapeutic use in pre-clinical studies in diverse fields, but a standard expansion method has not been established. We investigated the effects of an endothelial growth medium (EGM-2) on ASC, focusing on proliferation and differentiation potentials. ASC were cultured in EGM-2 and DMEM. Doubling time and total cell number were compared between the two media. The proliferative effect of each growth factor supplemented in EGM-2 was also examined. Cultured cells in each medium were examined for surface marker expression using flow cytometry. Differentiation into the adipogenic, chondrogenic and osteogenic lineages was analyzed after culture in each medium. ASC cultured with EGM-2 proliferated much more rapidly (10(5) times in 2 weeks) and reached the stationary phase earlier than those cultured with DMEM. Among the supplements contained in EGM-2, only fibroblast growth factor-2 (FGF-2) significantly promoted proliferation of ASC, although the proliferative effect of FGF-2 was much less than that of EGM-2, suggesting a synergism among other supplement factors. Flow cytometry and differentiation assays suggested that ASC cultured in EGM-2 preserved immunophenotype and differentiation capacity for at least three mesenchymal lineages (adipogenic, chondrogenic and osteogenic), similar to those cultured with DMEM. The present expansion method markedly accelerates proliferation of ASC, preserving their multipotent differentiation capacities, and lays the groundwork for establishing a practical route to mega-expansion of ASC for clinical applications.Cytotherapy 02/2007; 9(8):738-45. · 3.06 Impact Factor