Forum Shah

Adipose-derived stromal/stem cells: a primer.

Until recently, the complexity of adipose tissue and its physiological role was not well appreciated. This changed with the discovery of adipokines such as leptin. The cellular composition of adipose tissue is heterogeneous and changes as a function of diabetes and disease states such as diabetes. Tissue engineers view adipose tissue as a rich source of adult stromal/stem cells isolated by collagenase digestion. In vitro and in vivo studies have documented that adipose stromal/stem cells are multipotent, with the ability to differentiate along the adipocyte, chondrocyte, osteoblast and other lineage pathways. The adipose stromal/stem cells secrete a wide range of cytokines and growth factors with potential paracrine actions. Furthermore, adipose stromal/stem cells exert immunomodulatory functions when added to mixed lymphocyte reactions, suggesting that they can be transplanted allogeneically. This review article focuses on these mechanisms of adipose stromal/stem cell action and their potential utility as cellular therapeutics.

A non-enzymatic method for isolating human adipose tissue-derived stromal stem cells

BACKGROUND AIMS The isolation of human adipose stromal/stem cells (ASCs) currently relies on the use of the enzyme collagenase, which digests the triple helix region of peptide bonds in the collagen of adipose tissue. Collagenase is an expensive reagent derived from a bacterial source, and its use in isolating ASCs is a time-consuming procedure. This experiment evaluated the extraction of ASCs without an enzymatic digest. METHODS We used a simple method of washing adipose tissue to isolate and characterize the cells and compared this method with the enzymatic procedure in terms of processing time, stem cell yield, differentiation potential and immunophenotype. RESULTS Based on fluorescence activated cell sorting analysis, the stromal vascular fractions isolated with the washing method displayed a distinct and potentially favorable immunophenotype relative to the collagenase digestion. This difference may reflect the absence of chemical alteration of the cells by collagenase digestion. Independent of the isolation procedure, the resulting passaged ASCs were comparable based on immunophenotype and adipogenic and osteogenic differentiation potential. CONCLUSIONS Although using collagenase substantially increases cell yield, the two methods yield a similar cell product.

Comparison of human adult stem cells from adipose tissue and bone marrow in the treatment of experimental autoimmune encephalomyelitis

IntroductionWhile administration of ex vivo culture-expanded stem cells has been used to study immunosuppressive mechanisms in multiple models of autoimmune diseases, less is known about the uncultured, nonexpanded stromal vascular fraction (SVF)-based therapy. The SVF is composed of a heterogeneous population of cells and has been used clinically to treat acute and chronic diseases, alleviating symptoms in a range of tissues and organs.MethodsIn this study, the ability of human SVF cells was compared with culture-expanded adipose stem cells (ASCs) and bone-derived marrow stromal cells (BMSCs) as a treatment of myelin oligodendrocyte glycoprotein (35–55)-induced experimental autoimmune encephalitis in C57Bl/6J mice, a well-studied multiple sclerosis model (MS). A total of 1 × 106 BMSCs, ASCs, or SVF cells were administered intraperitoneally concomitantly with the induction of disease. Mice were monitored daily for clinical signs of disease by three independent, blinded investigators and rated on a scale of 0 to 5. Spinal cords were obtained after euthanasia at day 30 and processed for histological staining using luxol fast blue, toluidine blue, and hematoxylin and eosin to measure myelin and infiltrating immune cells. Blood was collected from mice at day 30 and analyzed by enzyme-linked immunosorbent assay to measure serum levels of inflammatory cytokines.ResultsThe data indicate that intraperitoneal administration of all cell types significantly ameliorates the severity of disease. Furthermore, the data also demonstrate, for the first time, that the SVF was as effective as the more commonly cultured BMSCs and ASCs in an MS model. All cell therapies also demonstrated a similar reduction in tissue damage, inflammatory infiltrates, and sera levels of IFNγ and IL-12. While IFNγ levels were reduced to comparable levels between treatment groups, levels of IL-12 were significantly lower in SVF-treated than BMSC-treated or ASC-treated mice.ConclusionsBased on these data, it is evident that SVF cells have relevant therapeutic potential in an animal model of chronic MS and might represent a valuable tool for stem cell-based therapy in chronic inflammatory disease of the central nervous system. SVF offers advantages of direct and rapid isolation procedure in a xenobiotic-free environment.

Adipocytes and the Regulation of Bone Remodeling: A Balancing Act

Abstract Throughout life, a balance exists within the marrow cavity between adipose tissue and bone. Each tissue derives from a common progenitor cell known both as a “bone marrow-derived multipotent stromal cell” and as a “mesenchymal stem cell” (BMSC). The majority of in vitro and in vivo data suggest that BMSCs differentiate into adipocytes or osteoblasts in a reciprocal manner. For example, while ligand induction of the transcription factors peroxisome proliferator-activated receptor γ initiates BMSC adipogenesis, it suppresses osteogenesis. Nevertheless, this hypothesis may oversimplify a complex regulatory paradigm. The picture may be further complicated by the systemic impact of extramedullary adipose depots on bone via the secretion of protein adipokines and lipid metabolites. This review focuses on past and current literature examining the mechanisms governing the adipose–bone interface.

Tissue-engineered autologous grafts for facial bone reconstruction.

Anatomically shaped living bone formed in a portable bioreactor using autologous cells and bone matrix repaired the facial ramus-condyle unit in pigs. Saving face Restoring your reputation after a social gaffe may be challenging, but perhaps welcomed in comparison to saving face through restoration of actual bone structure. A delicate and precise process, facial bone reconstruction currently uses bone grafts from the same patient. Cell- and biomaterial-based approaches could benefit this field by providing personalized grafts for deformities of all shapes and sizes. Bhumiratana and colleagues therefore designed a maxillofacial reconstructive strategy centered on a combination of stem cells, decellularized bone, and a custom-designed perfusion bioreactor. The bone was first shaped to the defect in the ramus-condyle unit of minipigs, which have similar jaw anatomies and weight-bearing properties as humans. Then, stem cells were cultured on the bone for several weeks. To mimic the manufacturing and transport chain that could be involved in reconstructing human facial bones, then authors shipped the bioreactor with the living bone inside the site of surgery. Paired histological and image analysis showed that the implanted scaffold material integrated with host tissue, formed new bone, and was vascularized extensively, but only if cells were present. Growing such anatomically correct, large-scale bone constructs could vastly improve regenerative medicine options for patients with craniofacial bone deformities. Facial deformities require precise reconstruction of the appearance and function of the original tissue. The current standard of care—the use of bone harvested from another region in the body—has major limitations, including pain and comorbidities associated with surgery. We have engineered one of the most geometrically complex facial bones by using autologous stromal/stem cells, native bovine bone matrix, and a perfusion bioreactor for the growth and transport of living grafts, without bone morphogenetic proteins. The ramus-condyle unit, the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatán minipigs (human-scale preclinical model). We used clinically approved decellularized bovine trabecular bone as a scaffolding material and crafted it into an anatomically correct shape using image-guided micromilling to fit the defect. Autologous adipose-derived stromal/stem cells were seeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immature bone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure, integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration than either nonseeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility of facial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents a platform for personalized bone tissue engineering.

Comparison of infrapatellar and subcutaneous adipose tissue stromal vascular fraction and stromal/stem cells in osteoarthritic subjects

Since inflammatory mechanisms have been postulated to link obesity to osteoarthritis, the current study evaluated the ratio of immune cells to multipotent stromal cells within the infrapatellar fat pad (IPFP) and subcutaneous adipose tissue (SQ) of the knee; each depot has potential as a source of regenerative cells. The immunophenotypes of stromal vascular fraction (SVF) and adipose‐derived stem cells (ASCs) of the IPFP and SQ were determined in tissues from osteoarthritic subjects (n = 7) undergoing total knee replacement. Based on a subset of surface antigens, the immunophenotype of ASCs from SQ of OA subjects was not significantly different from that of relatively healthy and leaner subjects undergoing elective liposuction surgery. Flow‐cytometry comparison of SVF cell populations in the IPFP of OA subjects resembled those within the subjects own matched SQ, with the exception of the endothelial marker CD31+, which was significantly greater in cells from SQ. In the OA subjects, lower numbers of capillary‐like structures and higher numbers of stromal and alkaline phosphatase colony‐forming units in the IPFP vs SQ were consistent with this finding; however, ASCs from both depots in OA subjects exhibited comparable adipogenic and osteogenic differentiation potential. Thus, the IPFP contains an ASC and immune cell population similar to that of donor‐matched SQ, making it an alternative ASC source for tissue regeneration. Further studies will be needed to determine whether IPFP immune cell infiltrates play an aetiological role in osteoarthritis equivalent to that shown in diabetes associated with obesity. Copyright

Human Adipose-Derived Stromal/Stem Cell Isolation, Culture, and Osteogenic Differentiation

Annually, more than 200,000 elective liposuction procedures are performed in the United States and over a million worldwide. The ease of harvest and abundance make human adipose-derived stromal/stem cells (hASCs) isolated from lipoaspirates an attractive, readily available source of adult stem cells that have become increasingly popular for use in many studies. Here, we describe common methods for hASC culture, preservation, and osteogenic differentiation. We introduce methods of ceramic, polymer, and composite scaffold synthesis with a description of morphological, chemical, and mechanical characterization techniques. Techniques for scaffold loading are compared, and methods for determining cell loading efficiency and proliferation are described. Finally, we provide both qualitative and quantitative techniques for in vitro assessment of hASC osteogenic differentiation.

Comparison of Stromal/Stem Cells Isolated from Human Omental and Subcutaneous Adipose Depots: Differentiation and Immunophenotypic Characterization

The emerging field of regenerative medicine has identified adipose tissue as an abundant source of stromal/stem cells for tissue engineering applications. Therefore, we have compared the differentiation and immunophenotypic features of adipose-derived stromal/stem cells (ASC) isolated from either omental or subcutaneous adipose depots. Human tissue samples were obtained from bariatric and plastic surgical practices at a university-affiliated teaching hospital and a private practice, respectively, with informed patient consent. Primary cultures of human ASC were isolated from adipose specimens within 24 h of surgery and culture expanded in vitro. The passaged ASC were induced to undergo adipogenic or osteogenic differentiation as assessed by histochemical methods or evaluated for surface antigen expression profiles by flow cytometry. ASC yields per unit weight of tissue were comparable between omental and subcutaneous depots. At passage 0, the immunophenotype of omental and subcutaneous ASC were not significantly different with the exception of CD105 and endoglin, a component of the transforming growth factor β receptor. The adipogenic differentiation of omental ASC was less robust than that of subcutaneous ASC based on in vitro histochemical and PCR assays. Although the yield and immunophenotype of ASC from omental adipose depots resembled that of subcutaneous ASC, omental ASC displayed significantly reduced adipogenic differentiation capacity following chemical induction. Further studies are necessary to evaluate and optimize the differentiation function of omental ASC in vitro and in vivo. Pending such analyses, omental ASC should not be used interchangeably with subcutaneous ASC for regenerative medical applications.

Human adipose-derived mesenchymal stromal cell pigment epithelium-derived factor cytotherapy modifies genetic and epigenetic profiles of prostate cancer cells.

BACKGROUND AIMS Adipose-derived mesenchymal stromal cells (ASCs) are promising tools for delivery of cytotherapy against cancer. However, ASCs can exert profound effects on biological behavior of tumor cells. Our study aimed to examine the influence of ASCs on gene expression and epigenetic methylation profiles of prostate cancer cells as well as the impact of expressing a therapeutic gene on modifying the interaction between ASCs and prostate cancer cells. METHODS ASCs were modified by lentiviral transduction to express either green fluorescent protein as a control or pigment epithelium-derived factor (PEDF) as a therapeutic molecule. PC3 prostate cancer cells were cultured in the presence of ASC culture-conditioned media (CCM), and effects on PC3 or DU145. Ras cells were examined by means of real-time quantitative polymerase chain reaction, EpiTect methyl prostate cancer-focused real-time quantitative polymerase chain reaction arrays, and luciferase reporter assays. RESULTS ASCs transduced with lentiviral vectors were able to mediate expression of several tumor-inhibitory genes, some of which correlated with epigenetic methylation changes on cocultured PC3 prostate cancer cells. When PC3 cells were cultured with ASC-PEDF CCM, we observed a shift in the balance of gene expression toward tumor inhibition, which suggests that PEDF reduces the potential tumor-promoting activity of unmodified ASCs. CONCLUSIONS These results suggest that ASC-PEDF CCM can promote reprogramming of tumor cells in a paracrine manner. An improved understanding of genetic and epigenetic events in prostate cancer growth in response to PEDF paracrine therapy would enable a more effective use of ASC-PEDF, with the goal of achieving safer yet more potent anti-tumor effects.

The Relative Functionality of Freshly Isolated and Cryopreserved Human Adipose-Derived Stromal/Stem Cells

The capability of multipotent mesenchymal stem cells to maintain cell viability, phenotype and differentiation ability upon thawing is critical if they are to be banked and used for future therapeutic purposes. In the present study, we examined the effect of 9-10 months of cryostorage on the morphology, immunophenotype, colony-forming unit (CFU) and differentiation capacity of fresh and cryopreserved human adipose-derived stromal/stem cells (ASCs) from the same donors. Cryopreservation did not reduce the CFU frequency and the expression levels of CD29, CD73, CD90 and CD105 remained unchanged with the exception of CD34 and CD45; however, the differentiation capacity of cryopreserved ASCs relative to fresh cells was significantly reduced. While our findings suggest that future studies are warranted to improve cryopreservation methods and agents, cryopreserved ASCs retain sufficient features to ensure their practical utility for both research and clinical applications.