Adipose-derived stem cells: Isolation, expansion and differentiation☆
Introduction
By definition, a stem cell is characterized by its ability to undergo self-renewal and its ability to undergo multilineage differentiation and form terminally differentiated cells. Ideally, a stem cell for regenerative medicinal applications should meet the following set of criteria: (i) should be found in abundant quantities (millions to billions of cells); (ii) can be collected and harvested by a minimally invasive procedure; (iii) can be differentiated along multiple cell lineage pathways in a reproducible manner; (iv) can be safely and effectively transplanted to either an autologous or allogeneic host [1].
Tissue specific stem cells comprise the second group and are derived from specific organs, such as brain, gut, lung, liver, adipose tissue and bone marrow [2]. It has become evident that these stem cells persist in adult tissues, although they represent a rare population localized in small niches [3]. Postnatal (adult) stem cells are not totipotent; however, they are pluripotent. These cells retain a broad differentiation potential, but their developmental potential is more restricted than embryonic stem cells. Adult stem cells were initially thought to have the differentiation capacity limited to their tissue of origin, however recent studies have demonstrated that stem cells have the capacity to differentiate into cells of mesodermal, endodermal and ectodermal origins [4], [5], [6], [7], [8], [9], [10]. The plasticity of MSCs most often refers to the inherent ability retained within stem cells to cross lineage barriers and to adopt the phenotypic, biochemical and functional properties of cells unique to other tissues. Adult MSCs can be isolated from bone marrow and adipose tissue. With the increased incidence of obesity in the U.S. and abroad, subcutaneous adipose tissue is abundant and readily accessible [11], [12]. Approximately 400,000 liposuction surgeries are performed in the U.S. each year and these procedures yield anywhere from 100 ml to >3 L of lipoaspirate tissue [12]. This material is routinely discarded. As discussed below, adipose-derived stem cells are multipotent and hold promise for a range of therapeutic applications.
Adipocytes develop from mesenchymal cells via a complex cascade of transcriptional and non-transcriptional events that occurs throughout human life. Stromal cells that have preadipocyte characteristics can be isolated from adipose tissue of adult subjects, propagated in vitro and induced to differentiate into adipocytes [13], [14], [15], [16]. Adipocyte differentiation is a complex process accompanied by coordinated changes in cell morphology, hormone sensitivity and gene expression that have been studied primarily in murine preadipocyte cell lines rather than in human preadipocytes. This protocol describes primary in vitro culture of stromal cell isolated from either large or small quantities of human adipose tissue. While historically in the literature adipose-derived stromal cells have been termed “pre-adipocytes” [14], [15], there is a growing appreciation that they are multipotent, with chondrogenic, neurogenic and osteogenic capability [14], [15], [17], [18], [19].
A variety of names have been used to describe the plastic adherent cell population isolated from collagenase digests of adipose tissue. The following terms have been used to identify the same adipose tissue cell population: adipose-derived stem/stromal cells (ASCs), adipose derived adult stem (ADAS) cells, adipose derived adult stromal cells, adipose derived stromal cells (ADSC), adipose stromal cells (ASC), adipose mesenchymal stem cells (AdMSC), lipoblast, pericyte, pre-adipocyte, processed lipoaspirate (PLA) cells. The use of this diverse nomenclature has lead to significant confusion in the literature. To address this issue, the International Fat Applied Technology Society reached a consensus to adopt the term “adipose-derived stem cells” (ASCs) to identify the isolated, plastic-adherent, multipotent cell population. The ASCs have been distinguished from the plastic adherent adult stem/progenitor cells from bone marrow originally referred to as fibroblastoid colony forming units, then in the hematological literature as marrow stromal, subsequently as mesenchymal stem cells, and most recently as multipotent mesenchymal stromal cells (MSCs).
Section snippets
Isolation of mesenchymal stem cells from adipose tissue
The initial methods to isolate cells from adipose tissue were pioneered by Rodbell and colleagues in the 1960s [20], [21], [22]. They minced rat fat pads, washed extensively to remove contaminating hematopoietic cells, incubated the tissue fragments with collagenase and centrifuged the digest, thereby separating the floating population of mature adipocytes from the pelleted stromal vascular fraction (SVF) (Fig. 1). The SVF consisted of a heterogeneous cell population, including circulating
Culture and expansion of ASCs
Seventy-two hours after plating, aspirate the entire medium from the wells. Again, it is essential to note that the percent of preadipocytes obtained from the SVF after digestion is patient-dependent. If the SVF does not expand well, increase the FBS contained in the stromal medium to 25%; however this may promote premature adipogenesis. Signs of deterioration such as granularity around the nucleus, cytoplasmic vacuolations and/or detachment of the cells from the plastic surface may indicate
Freezing and long-term storage of ASCs
ASCs should be harvested at 80% confluence for freezing. To collect cells, remove the culture medium and replace with a small volume of sterile, warm PBS. Remove the PBS and replace with trypsin-EDTA solution. Incubate the culture dish at 37 °C for at least 5 min, or until approximately 90% of the cells have detached from the bottom of the dish. Progress can be monitored under a microscope; the treated cells will be rounded and floating. Add an equal volume of stromal medium to inactivate the
Mesenchymal differentiation assays for ASCs
ASCs display multipotency, meaning they retain the ability to differentiate into cell types of multiple different lineages (Fig. 2, Fig. 3). The multipotency of these cells has generated interest in their potential therapeutic value for regenerative medicine. Differentiation of these cells can be directed by the addition of specific cocktails of chemical inducers or cytokines. The differentiation efficiency is patient dependent. The age of the donor can be a factor, since some studies suggest
Neural differentiation of ASCs
Neurospheres are generated as described in Kang et al. [34]. Undifferentiated adipose stem cells cultured at high densities will spontaneously form spherical clumps of cells that can be isolated in 0.25% trypsin/2.21 mM EDTA. Free-floating neurospheres released from the cell culture surface into the culture medium can also be collected. The spheres of cells are transferred to an ultra low cluster culture dish and cultured in neurobasal medium supplemented with B27 (1:50), 20 ng/ml bFGF and 20
Conclusion
In summary, ASCs provide unique opportunities for investigating novel treatments for a vast array of inherited and acquired diseases. In addition, ASCs may also provide an opportunity to identify new molecular targets for drug discovery. In this review article, a series of detailed protocols for the isolation, characterization, expansion and differentiation of ASCs has been provided. These protocols can readily be adapted to adjust for differences in the size of the adipose tissue sample. Many
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The research was supported by the National Center for Research Resources, National Institutes of Health, Grant No. RR00164, and a grant from the State of Louisiana Millennium Health Excellence Fund and the Louisiana Gene Therapy Research Consortium.