Purification and Long-Term Expansion of Multipotent Endothelial-Like Cells with Potential Cardiovascular Regeneration

Abstract

Endothelial progenitor cells (EPC) represent a relatively rare cell population, and expansion of sufficient cell numbers remains a challenge. Nevertheless, human adipose-derived stem cells (hASC) can be easily isolated and possess the ability to differentiate into endothelial cells. Here, we propose the isolation and characterization of multipotent endothelial-like cells (ME-LC) with the capacity to maintain their vascular progenitor properties for long periods. hASC were isolated from lipoaspirates and cultured through distinct consecutive culture stages for 2 months to enrich ME-LC: first in Dulbecco's modified Eagle's medium-fetal bovine serum (stage I), followed by a stage of culture in absent of fetal bovine serum (stage II), a culture in SFO3 medium (stage III), and, finally, the culture of ME-LC into collagen IV-coated flasks in endothelial growth medium (EGM-2) (stage IV). ME-LC display increased expression levels of endothelial and hematopoietic lineage markers (CD45, KDR, and CXCR4) and EPC markers (CD34 and CD133), whereas the expression of CD31 was barely detectable. Reverse transcription (RT)-polymerase chain reaction assays showed expression of genes involved in early stages of EPC differentiation and decreased expression of genes associated to differentiated EPC (TIE-2, DLL4, and FLT-1). ME-LC formed capillary-like structures when grown on Matrigel, secreted increased levels of stromal cell-derived factor-1 (SDF-1), and showed the ability to migrate attracted by SDF-1, vascular endothelial growth factor, and hematopoietic growth factor cytokines. Importantly, ME-LC retained the capacity to differentiate into cardiomyocyte-like cells. We present a simplified and efficient method to generate large numbers of autologous ME-LC from lipoaspirates-derived hASC, opening up potential cell-based therapies for cardiovascular regenerative medicine.

Interactions between Adipocytes and Breast Cancer Cells Stimulate Cytokine Production and Drive Src/Sox2/miR-302b–Mediated Malignant Progression

“…S1A for hASC characterization; refs. 24,25). In vitro differentiated adipocytes formed adherent cultures containing abundant lipid droplets (see Fig.…”

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“…30 Mesenchymal stem cells (MSCs) are most commonly used for bone tissue regeneration. Nanoscale modification of the matrix further enhances their growth and differentiation.…”

Fast-Proliferating Adipose Tissue Mesenchymal-Stromal-Like Cells for Therapy

“…Marchal et al [25] have provided credible data in support of an endothelial-like cell type [multipotent endothelial-like cells (ME-LC)] with endothelial and cardiomyocytic differentiation potential that can be selected from hASC populations through an elaborate process including growth in EGM-2. However, while ME-LC and FP-ASC types share endothelial and cardiomyocyte differentiation potential and express common endothelial hematopoietic marker VEGFR (KDR) and EPC marker CD133, contrary to ME-LCs, FPASCs do not express CD34 or CD45.…”

“…However, analysis of contribution of individual growth factors and other components in defined medium has proven difficult due to synergistic effects resulting from their interactions when used in combination [19][20][21][22][23][24]. The use of specific media such as EGM-2 has allowed groups like Marchal et al [25] describe the purification from hASC populations of an endothelial-like cell type with potential application for myocardium repair. This is a key issue in most clinical applications since establishment of a vascular system is essential for tissue repair.…”