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Adipose: Current Research on Isolation or Production of Therapeutic Cells

Adipose-derived stem cells, or ASCs, are a unique population of stem cells isolated from adipose tissue. These multipotent stem cells present an alternative to the widely used embryonic and hematopoietic stem cell lineages for laboratory and clinical applications in regenerative medicine. Adipose tissue is abundant in the body and exists in several forms, including bone marrow, mammary tissue, and mechanical, brown (multilocular), and white (unilocular) adipose tissues.

Traditional methods of gathering stem cells include the controversial isolation of embryonic stem cells and the painful process of procuring stem cells from bone marrow. In contrast to these methods, an efficient manner of isolating and producing reliable stem cell populations from abundant, easily accessible adipose tissue presents an appealing alternative for clinical therapeutic applications. Thus far, adipose stem cells have been shown capable of multipotent mesodermal differentiation, as well as potential endodermal and ectodermal lineages in vitro.

Evidence for Adipocyte Precursor Cells

Progressive osseous heteroplasia (POH), a pathologic condition that leads to heterotopic bone formation within subcutaneous adipose and, eventually, muscle tissue, demonstrates the presence of adipocytes, osteoblasts, and chondrocytes upon histologic examination of the resultant lesions. Pathologic evidence from this rare, autosomal-dominant, inherited genetic defect suggests that ADCs are at least capable of differentiation into the aforementioned mesodermal lineages. Along with POH, lupus and Paget disease are also known to present with calcification of subcutaneous adipose, providing further evidence for the presence of multipotent ASCs within adipose tissue. Additionally, research using ligand-induced adipogenesis for the chemotherapeutic treatment of liposarcomas suggests that these cancers may derive from a stem cell progenitor; stimulation by both long-chain fatty acids and synthetic steroid compounds induces adipocyte formation from liposarcoma-derived cells. Furthermore, radioactive tracing to measure adipocyte turnover rates in obese patients indicates a lifespan of six to 15 months for these fully differentiated adult adipocytes, a value that seems to indicate the presence of a controlled replacement mechanism of mature cells by resident stem cell–adipocyte precursors. Evaluation of individuals who have undergone rapid weight loss through either metabolic or procedural means also supports this concept: Not only do existing adipocytes increase in volume but new adipocytes emerge in a homeostatic process to maintain a relatively constant level of adipose tissue within the organism.

Origin of ASCs

Adipose tissue is abundant in the body and exists in several forms, including bone marrow, mammary tissue, and mechanical, brown, and white adipose tissues. It is speculated that ASCs arrive in adipose tissue via distribution of circulating fibroblasts derived from the bone marrow that then colonize the respective adipose tissues throughout the body. Transplanted bone marrow-derived fibroblasts similar to the ones already discussed have been shown capable of differentiating into adipocytes upon proper chemical stimulation and in the presence of a lipid-rich diet; however, the exact origin and distribution of ASC populations remains unknown. Regarding the relative abundance of stem cell populations within the body’s various adipose depots, current evidence suggests that richer concentrations of progenitor cells exist in the arm and abdomen relative to samples taken from the thigh or breast. Further studies, along with effective isolation techniques, are still needed to optimize processes for procuring ASCs in the most efficient manner from donors.

Cell Isolation From Adipose Tissue

Older methods of isolating cells from adipose tissue involved thoroughly rinsing minced animal fat pads, digestion with collagenases, and centrifugation to separate the desired stromal vascular fraction (SVF) that contained the processed lipoaspirate cells within a heterogeneous mixture. Finally, the plastic-adherent cells within the isolated SVF were selectively purified on the surface of tissue-culture flasks to enrich the concentration of adipocyte precursors.

Researchers are developing more efficient isolation methods that use advances in liposuction and reconstructive plastic surgery; in this process, plastic surgeons use a cannula to infuse subcutaneous adipose tissue with an anesthetic-containing saline solution. The procedure produces aspirations of adipose-tissue fragments containing viable adipocyte precursors within the SVF. Following collection of tissue samples, centrifugation at 1,200 G optimizes the ASC fraction recovered from the liposuction aspirate. However, surgical procedures involving ultrasound-assisted liposuction have shown adverse effects on the quantity of viable cells abstracted via the procedure. Once isolated, ASCs double in vitro within two to four days. Since massive quantities of tissue must be handled to isolate significant quantities of desired cells, methods such as rotating, temperature-controlled collagenase incubators, and bag-within-a-bag sieves are being tested to assist in procuring the desired cellular fraction from liposuction aspirate samples. Thanks to the development of these novel techniques for efficiently isolating ASCs, larger-scale commercial isolation methods are in development and becoming a realistic possibility for clinical application.

Purification and Identification

To identify a stem cell, researchers evaluate the presence and absence of various surface marker proteins, or antigens, which are important for immune recognition by leukocytes. Studies analyzing the immunophenotype of cells abstracted from liposuction aspirate find much consistency in the surface markers of these adipose-derived cells, indicating that there is, in fact, a unique population of adipocyte precursors present within the aspirate. Among the antigens used to identify stem cell populations, ASCs have been identified as positive for CD29, CD34, CD54, CD90, CD105, CD166, and human leukocyte antigen (HLA)-ABC markers; they are negative for CD31, CD45, CD106, CD146, and HLA-DR markers. Additionally, ASCs may be purified from the heterogeneous subcutaneous adipose and vascular fraction SC+VF by exploiting their plastic-adherent characteristic and their multipotent differentiation potential.

Multipotency of ASCs

It is now well known that adipose-derived stem cells can differentiate into adipocytes, chondrocytes, and osteoblasts. Not only are they capable of this multipotency, but their clonogenicity has been established as well. That is, a single ASC has been shown capable of cloning itself and then further differentiating into multiple lineages, eliminating the possibility of multiple precursors producing the respective observed lineages. Beyond the mesodermal tripotency seen in conditions of pathologic calcification, researchers have more recently successfully induced in vivo ASC differentiation into neurogenic ectodermal cells consistent with neurons, oligodendrocytes, and Schwann cells. Other confirmatory studies have elicited ASC commitment to hepatogenic, pancreaticogenic, myogenic, hematopoietic supporting, and endodermal lineages by targeting various chemical inductive factors to the cells.

Both endogenous and synthetic chemicals have successfully induced differentiation into determinate cell lineages. Cardiomyocytes have been induced using the iron transporter, transferrin, interleukins 3 and 6, as well as vascular endothelial growth factor. Endothelial cells, on the other hand, result from ASC exposure to basic fibroblast growth factor, and epidermal growth factor. Differentiation into many cell lineages indicates that ASCs contain vast potential, perhaps far wider than that initially suggested by pathological evidence; it is even theorized that these cells are pluripotent, much like embryonic stem cells.

Clinical Trials With Therapeutic ASCs

Compared to hematopoietic stem cells, ASCs exhibit greater long-term genetic stability and are more immunoprivileged (i.e., evidence suggests they are effective at preventing severe graft-versus-host disease). Therefore, presently, these cells seem potentially safer and more effective than their adult stem cell counterparts. Currently, dozens of clinical trials are underway to evaluate their efficacy in various regenerative treatments. Immunosuppressive studies have shown that ASCs suppress T-cell–mediated immunity and inflammation while activating regulatory T cells, which downregulate inflammatory mediators and reduce the tissue response of inflammatory cells. Other clinical trials are investigating applications in cardiovascular and hepatic disease, type 1 and 2 diabetes, amyotrophic lateral sclerosis, multiple sclerosis, immunosuppression, limb ischemia, and bone reconstruction.

Regenerative capacities for lumpectomy patients and perianal fistulas are also being investigated. Current theories on the mechanism of action for ASCs include the paracrine secretion of signaling molecules like cytokines or growth factors that would guide the differentiation of local cells to the necessary type to speed recovery; in the treatment of ischemia, ASCs may help remove toxins by producing antioxidants and free-radical scavengers to aid in tissue recovery.

While many clinical trials are underway to address the safety and efficacy of ASCs, much remains to be seen regarding the potential these versatile cells have in therapeutic and regenerative settings. The vast array of possible treatments being explored will undoubtedly continue to revolutionize the management of disease.

Krishna S. Vyas

Brett Austin

University of Kentucky College of Medicine

See Also: Adipose: Development and Regeneration Potential; Adipose: Stem and Progenitor Cells in Adults; Pluripotent Stem Cells, Embryonic; Stem Cell Potency.