Isolation Of Adipose-derived Stem Cells Research Articles

Endothelial Differentiation of Adipose-Derived Stem Cells from Elderly Patients with Cardiovascular Disease

Adipose-derived stem cells (ASCs) possess significant therapeutic potential for tissue engineering and regeneration. This study investigates the endothelial differentiation and functional capacity of ASCs isolated from elderly patients. Isolation of ASCs from 53 patients (50-89 years) revealed that advanced age or comorbidity did not negatively impact stem cell harvest; rather, higher numbers were observed in older donors (>70 years) than in younger. ASCs cultured in endothelial growth medium-2 for up to 3 weeks formed cords upon Matrigel and demonstrated acetylated-low-density lipoprotein and lectin uptake. Further stimulation with vascular endothelial growth factor and shear stress upregulated endothelial cell-specific markers (CD31, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin). Inhibition of the PI(3)K but not mitogen-activated protein kinase pathway blocked the observed endothelial differentiation. Shear stress promoted an anti-thrombogenic phenotype as demonstrated by production of tissue-plasminogen activator and nitric oxide, and inhibition of plasminogen activator inhibitor-1. Shear stress augmented integrin α(5)β(1) expression and subsequently increased attachment of differentiated ASCs to basement membrane components. Finally, ASCs seeded onto a decellularized vein graft resisted detachment despite application of shear force up to 9 dynes. These results suggest that (1) advanced age and comorbidity do not negatively impact isolation of ASCs, and (2) these stem cells retain significant capacity to acquire key endothelial cell traits throughout life. As such, adipose tissue is a practical source of autologous stem cells for vascular tissue engineering.

Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype

The ability to isolate, expand and differentiate adult stem cells into a chondrogenic lineage is an important step in the development of tissue engineering approaches for cartilage repair or regeneration for the treatment of joint injury or osteoarthritis, as well as for their application in plastic or reconstructive surgery. Adipose-derived stem cells (ASCs) provide an abundant and easily accessible source of adult stem cells for use in such regenerative approaches. This protocol first describes the isolation of ASCs from liposuction aspirate. The cell culture conditions provided for ASC expansion provide a large number of multipotent stem cells. Instructions for growth factor-based induction of ASCs into chondrocyte-like cells using either cell pellet or alginate bead systems are detailed. These methods are similar to those published for chondrogenesis of bone marrow-derived mesenchymal stem cells but distinct because of the unique nature of ASCs. Investigators can expect consistent differentiation of ASCs, allowing for slight variation as a result of donor and serum lot effects. Approximately 10-12 weeks are needed for the entire process of ASC isolation, including the characterization of chondrocyte-like cells, which is also described.

Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction

Isolation of adipose-derived stem cells (ASCs) typically involves 8+ hours of intense effort, requiring specialized equipment and reagents. Here, we present an improved technique for isolating viable populations of mesenchymal stem cells from lipoaspirate saline fractions within 30 minutes. Importantly, the cells exhibit remarkable similarities to those obtained using the traditional isolation protocols, in terms of their multipotent differentiation potential and immunophenotype. Reducing the acquisition time of ASCs is critical for advancing regenerative medicine therapeutics, and our approach provides rapid and simple techniques for enhanced isolation and expansion of patient-derived mesenchymal stem cells.

Human adipose-derived mesenchymal stem cells in vitro: evaluation of an optimal expansion medium preserving stemness

The potential of cultured adipose-derived stem cells (ASC) in regenerative medicine and new cell therapeutic concepts has been shown recently by many investigations. However, while the method of isolation of ASC from liposuction aspirates depending on plastic adhesion is well established, a standard expansion medium optimally maintaining the undifferentiated state has not been described. We cultured ASC in five commonly used culture media (two laboratory-made media and three commercially available media) and compared them with a standard medium. We analyzed the effects on cell morphology, proliferation, hepatocyte growth factor (HGF) expression, stem cell marker profile and differentiation potential. Proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a fluorescent assay. Release of HGF was assessed by an immunoassay. Expression of characteristic stem cell-related transcription factors and markers was evaluated by quantitative polymerase chain reaction (qPCR) (Nanog, Sox-2, Rex-1, nestin and Oct-4) and flow cytometry (CD44, CD73, CD90, CD105 and CD166), and differentiation was shown by adipogenic medium. The morphology and expansion of ASC were significantly affected by the media used, whereas none of the media influenced the ASC potential to differentiate into adipocytes. Furthermore, two of the media induced an increase in expression of transcription factors, an increased secretion of HGF and a decrease in CD105 expression. Culture of ASC in one of these two media before using the cells in cell therapeutic approaches may have a benefit on their regenerative potential.

Adipose-derived stem cells: Isolation, expansion and differentiation

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in adipose-derived stem cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.

Leporine-Derived Adipose Precursor Cells Exhibit In Vitro Osteogenic Potential

Adipose-derived stem cells (ASCs) possess osteogenic potential and have been shown to undergo in vitro osteoblastic differentiation and promote bone regeneration in vivo. In this study, we describe the isolation and osteoblastic differentiation of rabbit ASCs and their behavior on a gelatin foam scaffold. These studies will form the basis of future in vivo studies of the osteogenic potential of rabbit ASCs for calvarial defect repair.Adipose-derived stem cells were isolated from New Zealand White rabbits and cultured in osteogenic medium +/- bone morphogenetic protein 2. Osteoblastic differentiation was assessed via histochemical stains for alkaline phosphatase (AP) and extracellular matrix (ECM) calcification. Reverse transcriptase polymerase chain reaction was performed to evaluate the expression of AP and the osteogenic transcription factor Runx2. Adipose-derived stem cells were seeded onto gelatin foam scaffolds at various densities, and cell proliferation was measured fluorometrically. Cells isolated from rabbit adipose tissue exhibited classic ASC morphology. Adipose-derived stem cells cultured in osteogenic medium exhibited more robust staining for AP and ECM calcification compared with ASCs in control medium. Furthermore, this staining was more marked in male ASCs versus female ASCs and also enhanced by bone morphogenetic protein 2. mRNA for AP and Runx2 were also increased in the osteoinduced cells. Theoptimal seeding density was 1 x 10 ASCs on an 8-mm gelatin foam scaffold. We have shown that rabbit ASCs have in vitro osteogenic potential and are compatible with a gelatin foam scaffold. Characteristic features of osteoblasts, such as ECM mineralization and expression of osteogenic genes, were demonstrated in this cell population. In vitro osteoblastic differentiation and scaffold studies are necessary before in vivo trials. The mechanism underlying the sex-based variation in osteoblastic differentiation is unknown but may involve signaling via factors such as estrogen.

125 ISOLATION OF MULTILINEAGE STEM CELLS FROM THE PORCINE MAMMARY FAT PAD

Porcine mammary tissue is a dynamic system that undergoes multiple cycles of growth, differentiation, and regression during the life cycle of a female; however, the mechanisms of mammary morphogenesis are not fully understood. The current hypothesis suggests that epithelial stem cells in the terminal end buds interact with surrounding epithelial and stromal cells during mammary tissue development and regeneration. Because the mammary gland of the virgin animal consists largely of a pad of adipose tissue, we propose that adipose-derived stem cells from the mammary fat pad (MFP) may also play a role in the regeneration process. Therefore, the objective of this experiment was to determine whether multilineage stem cells are present in the MFP of gilts. Adipose tissue from the MFP and back fat (BF) from the loin region were harvested from six nulliparous, cycling Yorkshire gilts, approximately 16 mo of age. Methods for isolation of adipose-derived stem cells were modified from those of Malusky and Wheeler (2004; International Society for Stem Cell Research, Proceedings 2nd Annual Meeting, abstr. 248:124). Presumptive stem cells isolated from each tissue type were maintained in vitro in DMEM supplemented with either 10% newborn calf serum (NCS; Rep 1 and 2) or 10% fetal calf serum (Rep 3). BF and MFP cell types formed colonies of fibroblast-like cells within 3 to 5 d. Growth characteristics are shown in Table 1. When the cells were 80% confluent, they were trypsinized and reseeded into 75-cm2 flasks. When cultured in the presence of NCS, both cell types had a progressive decrease in viability, and these cultures could not be maintained past Passage 4. Differentiation of both cell types (Passage 1) into adipogenic, myogenic, and osteogenic lineages are currently underway. Based on morphological evaluations, both cell types are able to differentiate into the aforementioned lineages; however, differentiation of MFP-derived cells occurred at a slower rate and was less pronounced than that of BF-derived cells. These preliminary findings suggest that the MFP likely contains a population of multipotent stem cells; however, at this time, it is not possible to make meaningful statistical comparisons. Further experiments are needed to fully characterize these cells and determine their role in mammary gland morphogenesis. Table 1. Growth of porcine adipose-derived stem cells obtained from the BF or from the MFP This study was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program and USDA Multistate Project (W-1171).