Hiroshi Mizuno

Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based Therapies

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.

Concise Review: Adipose‐Derived Stem Cells as a Novel Tool for Future Regenerative Medicine

The potential use of stem cell‐based therapies for the repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions for a number of diseases. The use of either embryonic stem cells (ESCs) or induced pluripotent stem cells in clinical situations is limited due to cell regulations and to technical and ethical considerations involved in the genetic manipulation of human ESCs, even though these cells are, theoretically, highly beneficial. Mesenchymal stem cells seem to be an ideal population of stem cells for practical regenerative medicine, because they are not subjected to the same restrictions. In particular, large number of adipose‐derived stem cells (ASCs) can be easily harvested from adipose tissue. Furthermore, recent basic research and preclinical studies have revealed that the use of ASCs in regenerative medicine is not limited to mesodermal tissue but extends to both ectodermal and endodermal tissues and organs, although ASCs originate from mesodermal lineages. Based on this background knowledge, the primary purpose of this concise review is to summarize and describe the underlying biology of ASCs and their proliferation and differentiation capacities, together with current preclinical and clinical data from a variety of medical fields regarding the use of ASCs in regenerative medicine. In addition, future directions for ASCs in terms of cell‐based therapies and regenerative medicine are discussed. STEM CELLS 2012;30:804–810

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy.

BACKGROUND AIMS Transplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model. METHODS ASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10(5) cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed. RESULTS ASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group. CONCLUSIONS Compared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.

Improved viability of random pattern skin flaps through the use of adipose-derived stem cells.

Background: Flap necrosis caused by inadequate blood supply is a common postoperative complication in reconstructive surgery. Because a putative stem cell population within the adipose tissue has been found to possess angiogenic potential, the authors sought to determine whether these cells might selectively induce neovascularization and increase the viability of random pattern skin flaps. Methods: Adipose-derived stem cells were isolated from the inguinal fat pads of ICR mice and expanded ex vivo for three passages. After the elevation of cranially based random pattern skin flaps (3 cm long and 1 cm wide), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)–labeled adipose-derived stem cells were then injected into the pedicle base (group A) or 1.5 cm distal to the pedicle (group B). Medium containing no adipose-derived stem cells, mature adipocytes, or basic fibroblast growth factor were injected in three other control groups separately. (n = 10 for each group). Millimetric measurements were taken at postoperative day 7 for evaluation of flap viability. Specimens were harvested for histologic analyses. Results: Adipose-derived stem cells led to a statistically significant increase in flap viability in both group A and group B compared with the control and the adipocyte groups. Histologic examination also demonstrated a statistically significant increase in capillary density in both group A and group B. Moreover, some of the endothelial cells were stained positively for DiI. Conclusions: These findings suggest that adipose-derived stem cells have a potential for enhancing the blood supply of random pattern skin flaps. This mechanism might be both the direct differentiation of adipose-derived stem cells into endothelial cells and the indirect effect of angiogenic growth factor released from adipose-derived stem cells.

Accelerated wound healing in healing-impaired db/db mice by autologous adipose tissue-derived stromal cells combined with atelocollagen matrix.

Adipose tissue-derived stromal cells (ATSCs) have recently gained widespread attention as a potential alternate source to bone marrow–derived mesenchymal stem cells with a proliferative capacity and a similar ability to undergo multilineage differentiation. In this study, we evaluated the effectiveness of freshly isolated autologous ATSCs-containing atelocollagen matrix with silicon membrane (ACMS) on wound healing of diabetic (db/db) mice. Cultured ATSCs from (db/db) mice secreted significant amounts of growth factors and cytokines, which are suitable for wound repair. Two full thickness round skin defects were made on the backs of healing-impaired db/db mice. Freshly isolated autologous ATSCs-containing ACMS or ACMS alone were applied to the wounds. Twelve mice were treated and then killed at 1 or 2 weeks (n = 6 each). Histologic sections of the wounds were prepared at each time period after treatment. Histologic examination demonstrated significantly advanced granulation tissue formation, capillary formation, and epithelialization in diabetic healing-impaired wounds treated with autologous ATSCs-containing ACMS, compared with mice treated with ACMS alone. These results suggested that transplantation of autologous ATSCs-containing ACMS significantly accelerated wound healing in diabetic healing-impaired db/db mice.

Therapeutic angiogenesis by autologous bone marrow cell implantation for refractory chronic peripheral arterial disease using assessment of neovascularization by 99mTc-tetrofosmin (TF) perfusion scintigraphy.

We investigated efficacy and safety of implantation of autologous bone marrow mononuclear cells plus platelets, including endothelial progenitor cells (EPCs), for recovering refractory chronic peripheral arterial disease (PAD) using visual and quantitative analyses by 99mTc-tetrofosmin (TF) perfusion scintigraphy, and also investigated various quantitative assessments objectively. We performed 12 consecutive cases and 19 limbs and hands with severe chronic PAD that were almost Fontaine class IV (11/12 cases, about 92%) in this trial. This treatment was very effective in relieving severe pain of PAD, especially for Buergers disease. We used a visual analog scale (VAS) for measurement of pain level. The maximum pain level before implantation was 66.5 ± 5.0 mm, and it decreased to 12.1 ± 2.2 mm after implantation (p < 0.001). Rest pain in legs and fingers was resolved in 11 cases (11/12 cases, 92%). All patients could measure pain-free walking time on a treadmill, which improved remarkably (140 ± 53 s before implantation vs. 451 ± 74 s after implantation, p = 0.034). Resting ankle brachial pressure index (ABI) in legs implanted with bone marrow mononuclear cells was also improved (0.65 ± 0.08 before implantation vs. 0.73 ± 0.07 after implantation, p = 0.055). According to 99mTc-TF perfusion scintigraphy, the proximal area (region from knee to ankle) was 1.32 ± 0.10 before implantation versus 1.56 ± 0.11 after implantation (p = 0.007). 99mTc-TF perfusion scintigraphy in the distal area (region from ankle to end of toes, or from wrist to end of fingers) was 0.79 ± 0.06 before implantation versus 0.83 ± 0.06 after implantation (p = 0.29). Ischemic legs and hands that were injected showed increased perfusion blood flow. 99mTc-TF perfusion scintigraphy was effective to estimate visual and quantitative analysis of collateral vessels in neovascularization. We were successful with this new treatment for the most severe, chronic PAD that was not curable by any of the current treatments. Thus, this therapeutic angiogenesis could be a new strategy for saving severe ischemic limbs and hands.

Enhanced healing of mitomycin C-treated wounds in rats using inbred adipose tissue-derived stromal cells within an atelocollagen matrix.

The aim of this study was to evaluate the potential accelerating effects of an adipose tissue‐derived stromal cells (ATSC)‐containing atelocollagen matrix with silicone membrane (ACMS) for repairing mitomycin C‐treated healing‐impaired wounds. Mitomycin C was applied to full‐thickness skin incisions in this study to create a healing‐impaired wound model in rat. After thoroughly washing out the mitomycin C from the wound, ACMS alone or ATSC‐containing ACMS was applied to the wounds. Histological sections of the wounds were then prepared at indicated time periods after the treatments. These results indicated significantly advanced granulation tissue and capillary formations in the healing‐impaired wounds treated with ATSC‐containing ACMS compared with those treated with ACMS alone. Thus, this study suggested that transplantation of inbred ATSC‐containing ACMS is effective for repairing healing‐impaired wounds.

Differentiated and undifferentiated adipose-derived stem cells improve function in rats with peripheral nerve gaps

The effect of differentiated and undifferentiated adipose-derived stem cells on the repair of peripheral nerve gaps was studied. Adipose-derived stem cells were maintained in differentiation medium for 2 weeks. The expression of Schwann cell proteins S-100, nerve growth factor receptor (NGFR) p75 and integrin β4 was examined by immunofluorescence staining and real time-polymerase chain reaction (real time-PCR) at the end of the differentiation period. A 10-mm gap on the left sciatic nerves of 20 Fischer rats was created and bridged with silicone tube (group I), silicone tube filled with collagen gel (group II), nerve graft (group III), silicone tube filled with adipose-derived stem cells (group IV) and silicone tube filled with differentiated adipose-derived stem cells (group V). In vitro, the positivity of differentiated adipose-derived stem cells for S-100, NGFR p75 and integrin β4 by immunofluorescence staining was 31%, 27% and 12%, respectively. Fold changes by real time-PCR in comparison with undifferentiated cells were 48.4, 168.7 and 284.85, respectively. In vivo, a walking track analysis did not yield any statistically significant differences after 3 months postoperatively; however, after 6 months, group IV (sciatic function index (SFI) = -49.1 ± 13.1) and V (SFI = -52.6 ± 5.7) showed significant improvement compared to other groups (I: -73.3 ± 5.07, II: -79.6 ± 12.01, III: -74.8 ± 12.89) (p < 0.05). Nerve conduction velocity after 6 months was higher in groups IV (4.44 ± 0.3 mm ms(-1)), V (4.25 ± 0.3 mm ms(-1)) and III (4 ± 0.3 mm ms(-1)) than in groups I (2.5 ± 2.25 mm ms(-1)) and II (2.35 ± 1.58 mm ms(-1)) (p > 0.05). Myelin fibre density and myelinated fibre/unmyelinated fibre ratio were significantly higher in the midnerve and the distal nerve in groups IV and V (p < 0.05). These results reveal the therapeutic potential of adipose-derived stem cells in nerve reconstruction.

Comparison of readily available scaffolds for adipose tissue engineering using adipose-derived stem cells

The purpose of this study was to investigate which of the three readily available scaffold materials would be suitable for adipose tissue engineering when implanted with adipose-derived stem cells (ASCs) in vivo. ASCs isolated from green fluorescence protein (GFP) transgenic mice were incubated in an adipogenic medium and then seeded onto type I collagen sponge, non-woven polyglycolic acid or hyaluronic acid gel. The constructs were harvested and evaluated histologically and immunohistochemically 4 and 8 weeks after subcutaneous implantation into athymic mice. The gene expression of peroxisome-proliferator-activated receptor gamma2 (PPAR-gamma2), the adipocyte-specific transcriptional factor, was also investigated by using reverse transcription-polymerase chain reaction. Histological examination showed that more adipose-tissue-like construct was regenerated when using type I collagen sponge than when the other scaffolds were used. Moreover, immunohistostaining revealed that some of the adipocytes on the type I collagen construct expressed GFP. PPAR-gamma2 gene expression in the induced ASCs in the type I collagen sponge was observed. These findings suggest that type I collagen sponge may be the most suitable among the three readily available scaffolds for adipogenesis.

The Effect of Adipose-Derived Stem Cells on Ischemia-Reperfusion Injury: Immunohistochemical and Ultrastructural Evaluation

Background: Advances in the treatment of reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. The authors examined the direct and indirect effects of adipose-derived stem cells on ischemia-reperfusion injury on a skin flap model to determine the in vivo differentiation of adipose-derived stem cells to endothelial cells; the levels of vascular endothelial growth factor (VEGF), transforming growth factor-&bgr;, and fibroblast growth factor; and the ultrastructural changes apparent with scanning electron microscopy to clarify the initial events and the following cascades. Methods: Two identical cranial based random flaps with a dimension of 1 × 5 cm were elevated on the dorsums of 20 ICR mice. The left flap was designated as the control and the right flap was injected with adipose-derived stem cells. The flaps were then subjected to 6 hours of ischemia by clamping the pedicle, and then reperfusion. Results: The mean viable flap length in the control and experimental groups was 15.2 ± 3.4 mm and 24.4 ± 2.9 mm, respectively. The mean viable flap area in the control and experimental groups was 12.9 ± 4.1 mm2 and 21.8 ± 3.7 mm2, respectively. The in vivo differentiation of adipose-derived stem cells to endothelial cells was observed. The immunohistochemical stainings, VEGF, transforming growth factor-&bgr;, and fibroblast growth factor revealed increased levels in the experimental groups. Scanning electron microscopy indicated mild injury in the experimental group. Conclusions: The adipose-derived stem cells could prevent ischemia-reperfusion injury, mainly by regulating the growth factors. Although VEGF was the foremost inhibitor of injury, the overall cascade was enhanced by adipose-derived stem cells, with the help of the other growth factors.