Zeni Alfonso

PLASTICITY OF HUMAN ADIPOSE STEM CELLS TOWARD ENDOTHELIAL CELLS AND CARDIOMYOCYTES

Recent preclinical and clinical studies have suggested that adult stem cells have the ability to promote the retention or restoration of cardiac function in acute and chronic ischemia. Published clinical studies have used autologous donor cells, including skeletal muscle myoblasts, cultured peripheral blood cells, or bone marrow cells. However, our research and that of others indicates that human adipose tissue is an alternative source of cells with potential for cardiac cell therapy. These findings include the presence of cells within adipose tissue that can differentiate into cells expressing a cardiomyocytic or endothelial phenotype, as well as angiogenic and antiapoptotic growth factors. This potential is supported by preclinical studies in large animals.

Expression of cardiomyocytic markers on adipose tissue-derived cells in a murine model of acute myocardial injury

Animal and early clinical studies have provided evidence suggesting that intracoronary administration of autologous bone marrow-derived cells results in improved outcome following myocardial infarction. Animal studies with cultured marrow stromal cells (MSC) have provided similar data. Cells with properties that are similar to MSC have been identified in adipose tissue. Other groups have demonstrated in vivo differentiation of adipose tissue-derived cells (ADC) into cells exhibiting biochemical and functional markers of cardiac myocytes, including spontaneous beating. Based on these observations, the objective of the present study was to determine whether ADC might undergo similar differentiation in vivo in the context of myocardial injury.ADC were isolated from subcutaneous adipose tissue of Rosa26 mice (which express the beta-galactosidase transgene in almost every tissue) and injected into the intraventricular chamber of B6129S recipient mice immediately following induction of myocardial cryoinjury. Groups of recipients were euthanized at 24 hours, 7 and 14 days post surgery and examined for the presence of donor-derived cells within the heart.Beta-gal positive cells were identified in the infarcts of ADC-treated animals. No staining was observed in uninjured myocardium or in infarcts of control animals. Immunohistochemical analysis revealed co-expression of beta-gal with Myosin Heavy Chain, Nkx2.5 and with Troponin I. Co-expression of beta-galactosidase with Connexin 43, CD31, von Willebrand factor, MyoD or CD45 was not detected.Thus, these data indicate that adipose tissue contains a population of cells that has the ability to engraft injured myocardium and that this engraftment is associated with expression of cardiomyocytic markers by donor-derived cells.

ADIPOSE-DERIVED STEM CELLS

Human adipose tissue has been shown to contain a population of cells that possesses extensive proliferative capacity and the ability to differentiate into multiple cell lineages. These cells are referred to as adipose tissue-derived stem cells (ADSCs) and are generally similar, though not identical, to mesenchymal stem cells (also referred to as marrow stromal cells). ADSCs for research are most conveniently extracted from tissue removed during an elective cosmetic liposuction procedure but may also be obtained from resected adipose tissue. This chapter describes surgical procedures associated with improved ADSC recovery and the processes by which aspirated adipose tissue is washed and digested with collagenase to yield a heterogeneous population from which ADSCs can be expanded. The large volume of tissue obtained from a liposuction procedure (average approximately 2 L), combined with the relatively high frequency of ADSC within the digestate, yields substantially more stem cells than can be realized from marrow without extensive expansion in culture.

Differences in stem and progenitor cell yield in different subcutaneous adipose tissue depots

BACKGROUND Human adipose tissue has been shown to contain multipotent cells with properties similar to mesenchymal stromal cells. While there have been many studies of the biology of these cells, no study has yet evaluated issues associated with tissue harvest. METHODS Adipose tissue was obtained from the subcutaneous space of the abdomen and hips of 10 donors using both syringe and pump-assisted liposuction. Tissue was digested with collagenase and then assayed for the presence of different stem and progenitor cell types using clonogenic culture assays, including fibroblast colony-forming unit (CFU-F) and alkaline phosphatase-positive colony-forming unit (CFU-AP). Paired analysis of samples obtained from the same individual was used to compare harvest method and site. RESULTS Syringe suction provided significantly greater recovery of adipocytes and a non-significant trend towards improved recovery of cells in the adipocyte-depleted fraction. There was considerable donor-to-donor variation in stem cell recovery. However, paired analysis of tissue obtained from different subcutaneous sites in the same donor showed that tissue harvested from the hip yielded 2.3-fold more CFU-F/unit volume and a 7-fold higher frequency of CFU-AP than that obtained from the abdomen. These differences were statistically significant. DISCUSSION Harvest site influences the stem and progenitor cell content of subcutaneous adipose tissue.

Fresh and cryopreserved, uncultured adipose tissue-derived stem and regenerative cells ameliorate ischemia–reperfusion-induced acute kidney injury

Background. Acute kidney injury (AKI) represents a major clinical problem with high mortality and limited causal treatments. The use of cell therapy has been suggested as a potential modality to improve the course and outcome of AKI. Methods. We investigated the possible renoprotection of freshly isolated, uncultured adipose tissue-derived stem and regenerative cells (ADRCs) before and after cryopreservation in a rat ischemia–reperfusion (I–R) model of AKI. Results. We demonstrated that ADRC therapy drastically reduced mortality (survival 100% vs. 57%, ADRC vs. controls, respectively) and significantly reduced serum creatinine (sCr on Day 3: 3.03 ± 1.58 vs. 7.37 ± 2.32 mg/dL, ADRC vs. controls, respectively). Histological analysis further validated a significantly reduced intratubular cast formation, ameliorated acute tubular epithelial cell necrosis and mitigated macrophage infiltration. Furthermore, a reduced RNA expression of CXCL2 and IL-6 was found in the ADRC group which could explain the reduced macrophage recruitment. Use of cryopreserved ADRCs resulted in an equally high survival (90% vs. 33% in the control group) and similarly improved renal function (sCr on Day 3: 4.64 ± 2.43 vs. 7.24 ± 1.40 mg/dL in controls). Conclusions. Collectively, these results suggest a potential clinical role for ADRC therapy in patients with AKI. Importantly, cryopreservation of ADRCs could offer an autologous treatment strategy for patients who are at high risk for AKI during planned interventions.

Uncultured adipose-derived regenerative cells (ADRCs) seeded in collagen scaffold improves dermal regeneration, enhancing early vascularization and structural organization following thermal burns

OBJECTIVE Advances in tissue engineering have yielded a range of both natural and synthetic skin substitutes for burn wound healing application. Long-term viability of tissue-engineered skin substitutes requires the formation and maturation of neo-vessels to optimize survival and biointegration after implantation. A number of studies have demonstrated the capacity of Adipose Derived Regenerative Cells (ADRCs) to promote angiogenesis and modulate inflammation. On this basis, it was hypothesized that adding ADRCs to a collagen-based matrix (CBM) (i.e. Integra) would enhance formation and maturation of well-organized wound tissue in the setting of acute thermal burns. The purpose of this study was to evaluate whether seeding uncultured ADRCs onto CBM would improve matrix properties and enhance healing of the grafted wound. METHODS Full thickness thermal burns were created on the backs of 8 Gottingen mini-swine. Two days post-injury wounds underwent fascial excision and animals were randomized to receive either Integra seeded with either uncultured ADRCs or control vehicle. Wound healing assessment was performed by digital wound imaging, histopathological and immunohistochemical analyses. RESULTS In vitro analysis demonstrated that freshly isolated ADRCs adhered and propagated on the CBM. Histological scoring revealed accelerated maturation of wound bed tissue in wounds receiving ADRCs-loaded CBM compared to vehicle-loaded CBM. This was associated with a significant increase in depth of the wound bed tissue and collagen deposition (p<0.05). Blood vessel density in the wound bed was 50% to 69.6% greater in wounds receiving ADRCs-loaded CBM compared to vehicle-loaded CBM (p=0.05) at day 14 and 21. In addition, ADRCs delivered with CBM showed increased blood vessel lumen area and blood vessel maturation at day 21(p=0.05). Interestingly, vascularity and overall cellularity within the CBM were 50% and 45% greater in animals receiving ADRC loaded scaffolds compared to CBM alone (p<0.05). CONCLUSIONS These data demonstrate that seeding uncultured ADRCs onto CBM dermal substitute enhances wound angiogenesis, blood vessel maturation and matrix remodeling.

Development of a combined radiation and full thickness burn injury minipig model to study the effects of uncultured adipose-derived regenerative cell therapy in wound healing

Abstract Purpose: To develop an approach that models the cutaneous healing that occurs in a patient with full thickness thermal burn injury complicated by total body radiation exposure sufficient to induce sub-lethal prodromal symptoms. An assessment of the effects of an autologous cell therapy on wound healing on thermal burn injury with concomitant radiation exposure was used to validate the utility of the model. Methods: Göttingen minipigs were subjected to a 1.2 Gy total body irradiation by exposure to a 6 MV X-ray linear accelerator followed by ∼10 cm2 full thickness burns (pre-heated brass block with calibrated spring). Three days after injury, wounds were excised to the underlying fascia and each animal was randomized to receive treatment with autologous adipose-derived regenerative cells (ADRC) delivered by local or intravenous injection, or vehicle control. Blood counts were used to assess radiation-induced marrow suppression. All animals were followed using digital imaging to assess wound healing. Full-thickness biopsies were obtained at 7, 14, 21 and 30 days’ post-treatment. Results: Compared to animals receiving burn injury alone, significant transient neutropenia and thrombocytopenia were observed in irradiated subjects with average neutrophil nadir of 0.79 × 103/μl (day 15) and platelet nadir of 60 × 103/μl (day 12). Wound closure through a combination of contraction and epithelialization from the wound edges occurred over a period of approximately 28 days’ post excision and treatment. Re-epithelialization was accelerated in wounds treated with ADRC (mean 3.5-fold increase at 2 weeks post-treatment relative to control). This acceleration was accompanied by an average 67% increase in blood vessel density and 30% increase in matrix (collagen) deposition. Similar results were observed when ADRC were injected either directly into the wound or by intravenous administration. Conclusions: Although preliminary, this study provides a reproducible minipig model of thermal burn injury complicated by myelosuppressive total body irradiation that utilizes standardized procedures to evaluate novel countermeasures for potential use following attack by an improvised nuclear device.