Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.
Human adipose-derived mesenchymal stem cells (hASCs) are an ideal cell source for regenerative medicine due to their capabilities of multipotency and the readily accessibility of adipose tissue. They have been found residing in a relatively low oxygen tension microenvironment in the body, but the physiological condition has been overlooked in most studies. In light of the escalating need for culturing hASCs under their physiological condition, this review summarizes the most recent advances in the hypoxia effect on hASCs. We first highlight the advantages of using hASCs in regenerative medicine and discuss the influence of hypoxia on the phenotype and functionality of hASCs in terms of viability, stemness, proliferation, differentiation, soluble factor secretion, and biosafety. We provide a glimpse of the possible cellular mechanism that involved under hypoxia and discuss the potential clinical applications. We then highlight the existing challenges and discuss the future perspective on the use of hypoxic-treated hASCs.
Cardiac myofibroblast differentiation, as one of the most important cellular responses to heart injury, plays a critical role in cardiac remodeling and failure. While biochemical cues for this have been extensively investigated, the role of mechanical cues, e.g., extracellular matrix stiffness and mechanical strain, has also been found to mediate cardiac myofibroblast differentiation. Cardiac fibroblasts in vivo are typically subjected to a specific spatiotemporally changed mechanical microenvironment. When exposed to abnormal mechanical conditions (e.g., increased extracellular matrix stiffness or strain), cardiac fibroblasts can undergo myofibroblast differentiation. To date, the impact of mechanical cues on cardiac myofibroblast differentiation has been studied both in vitro and in vivo. Most of the related in vitro research into this has been mainly undertaken in two-dimensional cell culture systems, although a few three-dimensional studies that exist revealed an important role of dimensionality. However, despite remarkable advances, the comprehensive mechanisms for mechanoregulation of cardiac myofibroblast differentiation remain elusive. In this review, we introduce important parameters for evaluating cardiac myofibroblast differentiation and then discuss the development of both in vitro (two and three dimensional) and in vivo studies on mechanoregulation of cardiac myofibroblast differentiation. An understanding of the development of cardiac myofibroblast differentiation in response to changing mechanical microenvironment will underlie potential targets for future therapy of cardiac fibrosis and failure.
Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2) or in situ normoxia (2% O2). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.
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