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Abstracts: Stem Cells from Adipose Tissue

de la Garza-Rodea AS. Myogenic properties of human mesenchymal stem cells derived from three different sources. Cell Transplant, 2011 Jun 7, PubMed.

Mesenchymal stem cells (MSCs) of mammals have been isolated from many tissues and are characterized by their aptitude to differentiate into bone, cartilage and fat. Differentiation into cells of other lineages like skeletal muscle, tendon/ligament, nervous tissue and epithelium has been attained with MSCs derived from some tissues. Whether such abilities are shared by MSCs of all tissues is unknown. We therefore compared for three human donors the myogenic properties of MSCs from adipose tissue (AT), bone marrow (BM) and synovial membrane (SM). Our data show that human MSCs derived from the three tissues differ in phenotype, proliferation capacity and differentiation potential. The division rate of AT derived MSCs (AT-MSCs) was distinctly higher than that of MSCs from the other two tissue sources. In addition, clear donor-specific differences in the long-term maintenance of MSC proliferation ability were observed. Although similar in their in vitro fusogenic capacity with murine myoblasts, MSCs of the three sources contributed to a different extent to skeletal muscle regeneration in vivo. Transplanting human AT-, BM- or SM-MSCs previously transduced with a lentiviral vector encoding β-galactosidase into cardiotoxin-damaged tibialis anterior muscles (TAMs) of immunodeficient mice revealed that at 30 days after treatment the frequency of hybrid myofibers was highest in the TAMs treated with AT-MSCs. Our finding of human-specific β-spectrin and dystrophin in hybrid myofibers containing human nuclei argues for myogenic programming of MSCs in regenerating murine skeletal muscle. For the further development of MSC-bases treatments of myopathies, AT-MSCs appear to be the best choice in view of their efficient contribution to myoregeneration, their high ex vivo expansion potential and because their harvesting is less demanding than that of BM- or SM-MSCs.

Zuk PA, Zhu M, Ashjian P, et al. Human Adipose Tissue Is a Source of Multipotent Stem Cells. Molecular Biology of the Cell 2002; 13: 4279.

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering 2001; 7 (2): 211.

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

Huang JI, Beanes SR, Zhu M, Lorenz HP, Hedrick MH, Benhaim P. Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells. Plast Reconstr Surg 2002; 109 (3): 1033.

Human liposuction aspirates contain pluripotent adipose-derived mesodermal stem cells that have previously been shown to differentiate into various mesodermal cell types, including osteoblasts and chondrocytes. To develop an autologous research model of bone and cartilage tissue engineering, the authors sought to determine whether rat inguinal fat pads contain a similar population of osteochondrogenic precursor cells. It was hypothesized that the rat inguinal fat pad contains adipose-derived multipotential cells that resemble human adipose-derived mesodermal stem cells in their osteochondrogenic capacity. To test this, the authors assessed the ability of cells isolated from the rat inguinal fat pad to differentiate into osteoblasts and chondrocytes by a variety of lineage-specific histologic stains. Rat inguinal fat pads were isolated and processed from Sprague-Dawley rats into fibroblast-like cell population. Cell cultures were placed in pro-osteogenic media containing dexamethasone, ascorbic acid, and beta-glycerol phosphate. Osteogenic differentiation was assessed at 2, 4, and 6 weeks. Alkaline phosphatase activity and von Kossa staining were performed to assess osteoblastic differentiation and the production of a calcified extracellular matrix. Cell cultures were also placed in prochondrogenic conditions and media supplemented with transforming growth factor-beta1, insulin, transferrin, and ascorbic acid. Chondrogenic differentiation was assessed at 2, 7, and 14 days by the presence of positive Alcian blue staining and type II collagen immunohistochemistry. Cells placed in osteogenic conditions changed in structure to a more cuboidal shape, formed bone nodules, stained positively for alkaline phosphatase activity, and secreted calcified extracellular matrix by 2 weeks. Cells placed in chondrogenic conditions formed cartilaginous nodules within 48 hours that stained positively for Alcian blue and type II collagen. The authors identified the rat inguinal fat pad as a source of osteochondrogenic precursors and developed a straightforward technique to isolate osteochondrogenic precursors from a small animal source. This relatively easily obtained source of osteochondrogenic cells from the rat may be useful for study of tissue engineering strategies and the basic science of stem cell biology.

Mitchell S. Fat stem cells can become nerve cells. United Press International. Durham, 2002.

Stem cells obtained from human fat tissues are capable of being transformed into nerve cells that could be used to treat brain and spinal cord injuries, scientists reported Friday. The research furthers the possibility this virtually limitless supply of stem cells could carry the same benefits as the controversial embryonic stem cells, which potentially can become any type of cell and treat diseases ranging from diabetes to Parkinson's. "These are a very attractive source of cells for stem cell therapies," Henry Rice, a pediatric surgeon at Duke University and senior author of the paper, told United Press International. The cells are "readily accessible by liposuction, inexhaustible and can be taken from the same person who needs it," which minimizes the chance that the body will reject the cells. "This is a big leap," Rice said, because the finding shows for the first time that fat or adipose stem cells are capable of becoming cells of a different lineage. Although previous research has shown fat stem cells could be transformed into fat, cartilage and bone cells, these tissues are all closely related. Nerve cells, however, are of a completely different lineage. Brain and spinal cord cells generally do not regenerate, "so to get cells that are potentially capable of repairing injuries to these regions are of potential clinical benefit to lots of people," Rice told UPI. Pat Zuk, research director of the Regenerative Bioengineering and Repair Lab at the University of California at Los Angeles, which last year completed the original research showing the existence of adipose stem cells, told UPI, "This (finding) adds a layer of plasticity to adipose stem cells." Zuk noted her lab also has completed a soon-to-be-published study demonstrating adipose stem cells can be turned into nerve cells. "In another five to 10 years adult stem cells will have the same potential as embryonic stem cells," she said. The scientists used the now-common technique of liposuction to obtain stem cells from human fat or adipose tissue. Then they grew the cells in the presence of various chemicals and growth factors chosen specifically to spur the development of nerve cells. The stem cells began to "take on a shape and morphology" similar to nerve cells, and they also started to express proteins characteristic of cells in the central nervous system, Rice said.

Stashower M, Smith K, Williams J, Skelton H. Stromal Progenitor cells present within liposuction and reduction abdominoplasty fat for autologous transfer to aged skin. Dermatol Surg 1999; 25 (12): 945.

Autologous fat is used for direct transfer to locally replace fat, as well as for use in intradermally the treatment of rhytids in aged skin. Liposuction material from four patients and fat from abdominoplasty from five patients was processed by homogenization and centrifugation to separate mature lipocytes from other stromal cell populations in its associated extracellular matrix, and then to separate this from the blood and cellular debris. the cellular layer was evaluated histologically and with the immunohistochemical antibodies for CD34, SM-actin, S-100 protein, MIB-1, Bcl-2, factor XIIIa. The cellular layer showed spindle cells, some small vascular structures, a small amount of mature fat, and extracellular matrix. CD34 showed diffuse staining of most spindle and endothelial cells in all sections, factor XIIIa showed only focal staining of spindle and dendritic cells, and Bcl-2 showed light to moderate staining in scattered cells within the cellular component. S-100 protein, SM-actin, and MIB-1 were negative.

Hicok KC, Zhou Y, Pucilowski Y. Adipose derived adult stem (ADAS) cells differentiate into bone synthesizing osteoblasts in vivo. Presentation #M233

The presence in bone marrow of multi-potential cells capable of osteoblastic differentiation is now well established. For reasons of accessibility and low abundance, these cells are also sought in other more “accessible” tissues. Adult subcutaneous fat tissue is an excellent and abundant source of multi-potent cells. In vitro, adipose-derived adult stem (ADAS) cells express bone marker proteins including alkaline phosphatase, type I collagen, osteoprontin, and osteocalcin and produce a mineralized matrix as shown by alizarin red staining. In this study, ADAS cell ability to form bone in vivo was determined. ADAS cells isolated from liposuction waste of two different donors and a control osteoblastic cell line, hFOB 1.19, were expanded for 1 week in either DMEM:F12 plus 10% (v/v) FBS or in this media plus an osteoinductive cocktail (10 nM 1,25 dihydroxy-vitamin D3, 10 nM dexamethasone and 50 microg/ml ascorbic acid). 3x 10(6) cells were loaded onto either hydroxy-apatite/tricalcium phosphate (HA/TCP) cubes or the collagen/HA/TCP composite matrix, Collagraft, and were implanted subcutaneously into SCID mice. After 6 weeks, implants were removed, fixed, demineralized and sectioned for hematoxylin and eosin staining. Bone formation was observed in 75% of HA/TCP implants loaded with ADAS cells from both donors. Only 17% of Collagraft implants were positive for the presence of bone matrix. While 50% of HA/TCP implants loaded with hFOB 1.19 cells formed bone, when Collagraft was loaded with hFOB 1.19 cells, intermatrix spaces displayed a high degree of adipose tissue formation. Immunostaining of serial sections for human nuclear antigen )HNA) demonstrated that cells embedded within and lining regions of bone were of human origin. Little or no bone formation was observed in the control HA/TCP or Collagraft matrices without cells. In summary, the data demonstrates the ability of ADAS cells to form bone matrix in vivo. Because of their abundance and accessibility, ADAS cells may provide an important and renewable source of osteoblasts for tissue engineering based therapeutics of bone injury and disease.