BackgroundMesenchymal stem cells (MSCs) are multipotent stem cells able to differentiate into different cell lineages. However, MSCs represent a subpopulation of a more complex cell composition of stroma cells contained in mesenchymal tissue. Due to a lack of specific markers, it is difficult to distinguish MSCs from other more mature stromal cells such as fibroblasts, which, conversely, are abundant in mesenchymal tissue. In order to find more distinguishing features between MSCs and fibroblasts, we studied the phenotypic and functional features of human adipose-derived MSCs (AD-MSCs) side by side with normal human dermal fibroblasts (HNDFs) in vitroMethodsAD-MSCs and HNDFs were cultured, expanded and phenotypically characterized by flow cytometry (FC). Immunofluorescence was used to investigate cell differentiation. ELISA assay was used to quantify angiogenic factors and chemokines release. Cultures of endothelial cells (ECs) and a monocyte cell line, U937, were used to test angiogenic and anti-inflammatory properties.ResultsCultured AD-MSCs and HNDFs display similar morphological appearance, growth rate, and phenotypic profile. They both expressed typical mesenchymal markers-CD90, CD29, CD44, CD105 and to a minor extent, the adhesion molecules CD54, CD56, CD106 and CD166. They were negative for the stem cell markers CD34, CD146, CD133, CD117. Only aldehyde dehydrogenase (ALDH) was expressed. Neither AD-MSCs nor HNDFs differed in their multi-lineage differentiation capacity; they both differentiated into osteoblast, adipocyte, and also into cardiomyocyte-like cells. In contrast, AD-MSCs, but not HNDFs, displayed strong angiogenic and anti-inflammatory activity. AD-MSCs released significant amounts of VEGF, HGF and Angiopoietins and their conditioned medium (CM) stimulated ECs proliferation and tube formations. In addition, CM-derived AD-MSCs (AD-MSCs-CM) inhibited adhesion molecules expression on U937 and release of RANTES and MCP-1. Finally, after priming with TNFα, AD-MSCs enhanced their anti-inflammatory potential; while HNDFs acquired pro-inflammatory activity.ConclusionsAD-MSCs cannot be distinguished from HNDFs in vitro by evaluating their phenotypic profile or differentiation potential, but only through the analysis of their anti-inflammatory and angiogenic properties. These results underline the importance of evaluating the angiogenic and anti-inflammatory features of MSCs preparation. Their priming with inflammatory cytokines prior to transplantation may improve their efficacy in cell-based therapies for tissue regeneration.
IntroductionHuman adipose-derived stromal cells (hASCs), due to their relative feasibility of isolation and ability to secrete large amounts of angiogenic factors, are being evaluated for regenerative medicine. However, their limited culture life span may represent an obstacle for both preclinical investigation and therapeutic use. To overcome this problem, hASCs immortalization was performed in order to obtain cells with in vitro prolonged life span but still maintain their mesenchymal marker expression and ability to secrete angiogenic factors.MethodshASCs were transduced with the human telomerase reverse transcriptase (hTERT) gene alone or in combination with either SV-40 or HPV E6/E7 genes. Mesenchymal marker expression on immortalized hASCs lines was confirmed by flow cytometry (FC), differentiation potential was evaluated by immunocytochemistry and ELISA kits were used for evaluation of angiogenic factors. Green fluorescent protein (GFP) gene transduction was used to obtain fluorescent cells.ResultsWe found that hTERT alone failed to immortalize hASCs (hASCs-T), while hTERT/SV40 (hASCs-TS) or hTERT/HPV E6/E7 (hASCs-TE) co-transductions successfully immortalized cells. Both hASCs-TS and hASCs-TE were cultured for up to one year with a population doubling level (PDL) up to 100. Comparative studies between parental not transduced (hASCs-M) and immortalized cell lines showed that both hASCs-TS and hASCs-TE maintained a mesenchymal phenotypic profile, whereas differentiation properties were reduced particularly in hASCs-TS. Interestingly, hASCs-TS and hASCs-TE showed a capability to secrete significant amount of HGF and VEGF. Furthermore, hASCs-TS and hASCs-TE did not show tumorigenic properties in vitro although some chromosomal aberrations were detected. Finally, hASCs-TS and hASCs-TE lines were stably fluorescent upon transduction with the GFP gene.ConclusionsHere we demonstrated, for the first time, that hASCs, upon immortalization, maintain a strong capacity to secrete potent angiogenic molecules. By combining hASCs immortalization and their paracrine characteristics, we have developed a “hybridoma-like model” of hASCs that could have potential applications for discovering and producing molecules to use in regenerative medicine (process scale-up).In addition, due to the versatility of these fluorescent-immortalized cells, they could be employed in in vivo cell-tracking experiments, expanding their potential use in laboratory practice.
Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would use less resource and time and it could possibly contribute to simplification of GMP procedures.
Human adipose tissue has proven to be an abundant, accessible, and rich source of adult mesenchymal stromal cells, suitable for tissue engineering and regenerative medicine. However, a major complication in fully investigating these cells may derive from their limited life span.Although methods to isolate, expand, and immortalize these cells have been widely reported in the literature, exhaustive explanations on the problems that can be encountered during these processes and how these can be solved have never been described. It is of fundamental importance to follow a common protocol to achieve reliable and reproducible results. Here, we describe a protocol to isolate and expand human adipose stromal cells from specimens obtained from tissue biopsies and liposuction surgical interventions. Finally, we broadly describe the cell immortalization technique, and particular attention is paid to some of the apparently "secondary" aspects.
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