Wakako Tsuji

Adipose-derived stem cells: Implications in tissue regeneration

Adipose-derived stem cells (ASCs) are mesenchymal stem cells (MSCs) that are obtained from abundant adipose tissue, adherent on plastic culture flasks, can be expanded in vitro, and have the capacity to differentiate into multiple cell lineages. Unlike bone marrow-derived MSCs, ASCs can be obtained from abundant adipose tissue by a minimally invasive procedure, which results in a high number of cells. Therefore, ASCs are promising for regenerating tissues and organs damaged by injury and diseases. This article reviews the implications of ASCs in tissue regeneration.

Adipogenesis induced by human adipose tissue-derived stem cells.

Adipose tissue-derived stem cells (ASCs), including preadipocytes, may play an important role in de novo adipogenesis and are expected to be a useful external source of cells for adipose tissue engineering. In this study, we examined in vivo adipogenesis up to 24 weeks after implantation, induced by human ASCs that were isolated from adipose tissues and expanded in vitro. ASCs proliferated in vitro in the presence of basic fibroblast growth factor (bFGF), and the number of cells increased by more than 1000-fold at the fourth passage. The ability to differentiate into mature adipocytes was maintained up to the third passage. We incorporated designated numbers of third-passage-expanded cells into a type I collagen scaffold and implanted them into the back of nude mice with or without controlled-release bFGF. After the implantation of 2 x 10(6) ASCs with controlled-release bFGF, the greatest cross-sectional surface area of adipose tissue in the scaffold was 1.19 mm(2) at 12 weeks and 2.14 mm(2) at 24 weeks. About 2 x 10(6) ASCs with controlled-release bFGF was the best condition for total adipogenesis. Immunohistochemical analysis with antihuman vimentin antibody showed that the area of human-origin adipose tissue was maximum in the group with 8 x 10(6) ASCs incorporated in a scaffold at both 12 and 24 weeks. The amount of human-origin adipose tissue increased in all groups with implanted ASCs from 12 to 24 weeks. Only trace of human-origin adipose tissue was observed in other groups implanted ASCs. Our results show that human ASCs not only function as progenitor cells for in vivo adipogenesis, but also induce de novo adipogenesis for long period.

Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells Prolong Graft Survival in Vascularized Composite Allotransplantation

Background Strategies aiming at minimization or elimination of systemic immunosuppression are key immediate goals for clinical expansion of vascularized composite allotransplantation (VCA). We compared the in vitro and in vivo immunomodulatory efficacy of adipose-derived mesenchymal stem cells (AD-MSCs) and bone marrow (BM)–derived MSCs in a rat VCA model. Methods Both cell types were tested in vitro for suppressor function using mixed lymphocyte reactivity assays. AD-MSCs or BM-MSCs were administered intravenously (1 × 106 or 5 × 106 cells/animal) to Lewis rat recipients of mismatched Brown Norway hindlimb transplants. Short course tacrolimus (FK-506) monotherapy was withdrawn at postoperative day 21. In vivo regulatory T-cell induction, peripheral blood chimerism, and microchimerism in lymphatic organs were analyzed. Results AD-MSCs and BM-MSCs exhibited strong dose-dependent suppressor function in vitro, which was significantly more pronounced for AD cells. In vivo, all animals revealed peripheral multi-lineage chimerism at four weeks (P < 0.01) independent of cell type and dosage. Regulatory T-cell levels were increased with both cell types, the most in AD-MSC groups. These immunomodulatory effects were only transient. MSC treatment resulted in long-term (>120 day) allograft survival in 47% of the animals, which correlated with durable microchimerism in BM and spleen. Conclusions AD-MSCs and BM-MSCs exert immunomodulatory effects that prolong survival of immunogenic skin-bearing VCA grafts with short course (21 day) tacrolimus induction therapy. The in vivo findings in terms of allograft survival did not reflect superior immunomodulatory characteristics of AD-MSCs found in vitro.

The Role of Adipose-Derived Stem Cells in Breast Cancer Progression and Metastasis

Conventional breast cancer extirpation involves resection of parts of or the whole gland, resulting in asymmetry and disfiguration. Given the unsatisfactory aesthetic outcomes, patients often desire postmastectomy reconstructive procedures. Autologous fat grafting has been proposed for reconstructive purposes for decades to restore form and anatomy after mastectomy. Fat has the inherent advantage of being autologous tissue and the most natural-appearing filler, but given its inconsistent engraftment and retention rates, it lacks reliability. Implementation of autologous fat grafts with cellular adjuncts, such as multipotent adipose-derived stem cells (ADSCs), has shown promising results. However, it is pertinent and critical to question whether these cells could promote any residual tumor cells to proliferate, differentiate, or metastasize or even induce de novo carcinogenesis. Thus far, preclinical and clinical study findings are discordant. A trend towards potential promotion of both breast cancer growth and invasion by ADSCs found in basic science studies was indeed not confirmed in clinical trials. Whether experimental findings eventually correlate with or will be predictive of clinical outcomes remains unclear. Herein, we aimed to concisely review current experimental findings on the interaction of mesenchymal stem cells and breast cancer, mainly focusing on ADSCs as a promising tool for regenerative medicine, and discuss the implications in clinical translation.

In situ adipogenesis in fat tissue augmented by collagen scaffold with gelatin microspheres containing basic fibroblast growth factor

In situ adipose tissue regeneration in fat tissue by collagen sponges and gelatin microspheres containing basic fibroblast growth factor (bFGF) was investigated. A minced collagen sponge scaffold (1 ml) was incorporated with microspheres containing 10 µg bFGF and administered into a defect of rabbit fat tissues. Adipogenesis at the administered site was evaluated histologically. The adipose tissue regeneration induced by the administration of mixed collagen scaffold and microspheres containing bFGF was significantly stronger than that of either collagen scaffold alone or microspheres containing bFGF alone. The histological area of in situ adipogenesis by the mixed collagen scaffold and microspheres containing bFGF was enhanced over time by repeated administration. It is concluded that the repeated administration of collagen scaffold and microspheres containing bFGF is a promising way to achieve adipose tissue regeneration inside inherent fat tissue. This technique might be applicable for the reconstruction of volume contour deformities by trauma or surgical interventions of adipose tissue in a minimally invasive manner. Copyright

Adipogenesis using human adipose tissue-derived stromal cells combined with a collagen/gelatin sponge sustaining release of basic fibroblast growth factor

We have developed a collagen/gelatin sponge (CGS) that can provide a sustained release of basic fibroblast growth factor (bFGF). In our previous study, it was shown that CGS impregnated with the appropriate dosage of bFGF accelerates dermis‐like tissue formation two or three times earlier than an existing collagen sponge. In this study, adipogenesis was evaluated using CGSs disseminated with adipose tissue‐derived stem cells (ASCs). Human ASCs were primarily isolated from human adipose tissue that was obtained during breast cancer surgery with informed consent at Kyoto University Hospital. ASCs were isolated from collagenase digests of adipose tissue. ASCs were labelled with PKH26. CGSs (8 mm diameter × 3 mm thickness) were impregnated with bFGF (0.1, 1, 7, 14 µg/cm2) or normal saline solution. Then the labelled cells were disseminated (passage 3) on CGSs at a seeding density of 1 × 105 cells/cm2 and implanted into the back subcutis of nude mice. Six weeks after implantation, adipogenesis at the administered site was evaluated. Immunohistological staining with von Willebrand factor (vWf) was performed to evaluate newly formed capillaries. Newly formed adipose tissue was observed macroscopically and histologically in all groups. The weight and area of regenerated adipose tissue were largest in the 1 µg/cm2 bFGF group. Under a fluorescent microscope, newly formed adipose tissue in the bFGF‐administered group was PKH‐positive. These findings show that ASCs differentiated and formed adipose tissue. In this study, we showed that our CGSs impregnated with bFGF could be used as scaffolds with ASCs for adipogenesis. Copyright

The Influence of Timing and Frequency of Adipose-Derived Mesenchymal Stem Cell Therapy on Immunomodulation Outcomes After Vascularized Composite Allotransplantation.

Background Cellular therapies for immunomodulation in vascularized composite allotransplantation (VCA) have gained importance due to their potential for minimization of immunosuppression. Adipose-derived (AD) mesenchymal stem cells (MSCs) especially have shown encouraging potential. We investigated the influence of timing and frequency of AD-MSC treatment on immunologic and graft survival as well as graft vasculopathy outcomes after VCA. Methods Lewis rats received full-mismatched Brown Norway rat hindlimb transplants. Recipient animals were assigned to groups receiving donor-derived AD-MSCs (106 cells/animal) either on postoperative day (POD) 1, POD 4, or repeatedly on POD 4, 8, and 15, and compared to untreated controls. Results Although AD-MSC administration on POD 1 or POD 4, 8, and 15 resulted in 50% long-term graft acceptance, recipients treated on POD 4, and controls rejected before POD 50. All treated animals revealed peripheral blood chimerism (4 weeks), most pronounced after repetitive cell administration (12.92% vs 5.03% [POD 1] vs 6.31% [POD 4]; P < 0.05; all P < 0.01 vs control 1.45%). Chimerism was associated with the generation of regulatory T cells (CD4+CD25highFoxP3+). In vitro mixed lymphocyte reactions revealed modulation of the recipient immune response after AD-MSC treatment. Graft arteries at end point revealed significant differences of arterial intimal thickness between rejecting and AD-MSC–treated animals (P < 0.01). Conclusions Taken together, our results point to the potential for repetitive AD-MSC administration in improving outcomes after VCA. Future studies are warranted into optimization of the dosing and frequency of AD-MSC therapy, either alone or used in, combination with other cell therapies (such as hematopoietic stem cells or bone marrow–derived MSC or dendritic cells) for optimization of appropriate conditioning or maintenance regimens.

Effects of Immunosuppressive Drugs on Viability and Susceptibility of Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells

The immunomodulatory potential of cell therapies using adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) has been studied in vascularized composite allotransplantation (VCA). Most cell therapy-based experimental and clinical protocols integrate some degree of recipient conditioning/induction with antibodies or other immunosuppressive agents. We investigated the susceptibility of ASCs and BM-MSCs to anti-lymphocyte serum (ALS) and tacrolimus. Rat ASCs and BM-MSCs were exposed to varying concentrations of tacrolimus and ALS in vitro. Serum from ALS-treated animals was added to cell cultures. Viability, susceptibility, and cytotoxicity parameters were evaluated. ALS inhibited ASC and BM-MSC viability and susceptibility in vitro in a dose-dependent manner. ASCs were more susceptible to both ALS and tacrolimus than BM-MSCs. Trypsinized and adherent ASCs were significantly smaller than BM-MSCs. This is the first report on the viability and susceptibility characteristics of BM-MSCs or ASCs to collateral effects of ALS and tacrolimus. These in vitro insights may impact choice of cell type as well as concomitant conditioning agents and the logistical coordination of the timing, dosing, and frequency of drug or cell therapy in solid organ transplantation or VCA protocols.

Single Implantable FK506 Disk Prevents Rejection in Vascularized Composite Allotransplantation

Background: In vascularized composite allotransplantation, medication nonadherence leads to increased acute rejections. Improving medication adherence would improve overall allograft survival. Regionally delivered immunosuppression, targeted to sites of allorecognition, may reduce or eliminate the need for daily systemic immunosuppression. Methods: The authors developed biodegradable FK disks containing FK506-loaded double-walled microspheres and tested their efficacy at preventing rejection in a Brown-Norway–to-Lewis rat hindlimb transplantation model. In some experimental group animals, one FK disk was implanted subcutaneously either in native nontransplanted leg or in a transplanted allograft. Regular blood FK506 levels were measured. The endpoint was 180-day allograft survival or grade 3 rejection. At the endpoint, tissue FK506 levels were measured and mixed lymphocytic reaction was performed. Results: A single FK disk maintained systemic blood FK506 levels between 5 and 15 ng/ml for 146 ± 11.1 days. After that, the levels declined to less than 5 ng/ml through the endpoint. There was significantly increased FK506 concentration in groin lymph nodes draining the implanted FK disk. Compared with other groups, animals with an FK disk in the transplanted allograft had 100 percent allograft survival to more than 180 days despite subtherapeutic levels below 5 ng/ml. In these animals, significant T-cell hyporesponsiveness was seen in groin lymph nodes draining the FK disk compared with robust splenic T-cell proliferation. Conclusions: Sustained regional immunosuppression (with a single FK506 disk) maintained the allograft by means of a high regional concentration of FK506. Notably, this was achieved at subtherapeutic blood concentrations of FK506, without any further systemic FK506 administration.

An Animal Model of Local Breast Cancer Recurrence in the Setting of Autologous Fat Grafting for Breast Reconstruction

Autologous fat grafting after breast cancer surgery is commonly performed, but concerns about oncologic risk remain. To model the interaction between fat grafting and breast cancer cells, two approaches were employed. In the first approach, graded numbers of viable MDA‐MB‐231 or BT‐474 cells were admixed directly into human fat grafts and injected subcutaneously into immune‐deficient mice to determine if the healing graft is a supportive environment for the tumor. In the second approach, graded doses of MDA‐MB‐231 cells were suspended in Matrigel and injected into the mammary fat pads of mice. Two weeks after the tumor cells engrafted, 100 μL of human adipose tissue was grafted into the same site. Histologically, MDA‐MB‐231 cells seeded within fat grafts were observed and stained positive for human‐specific pan‐cytokeratin and Ki67. The BT‐474 cells failed to survive when seeded within fat grafts at any dose. In the second approach, MDA‐MB‐231 cells had a strong trend toward lower Ki67 staining at all doses. Regression analysis on all groups with fat grafts and MDA‐MB‐231 revealed fat tissue was associated with lower cancer cell Ki67 staining. Healing fat grafts do not support the epithelial BT‐474 cell growth, and support the mesenchymal MDA‐MB‐231 cell growth only at doses ten times greater than in Matrigel controls. Moreover, fat grafts in association with MDA‐MB‐231 cancer cells already present in the wound resulted in decreased tumor proliferation and increased fibrosis. These findings suggest that clinical fat grafting does not induce breast cancer cell growth, and may even have a suppressive effect. Stem Cells Translational Medicine 2018;7:125–134