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Amniotic Fluid Stem Cells Ameliorate Wound Healing In A Diabetic Murine Model
Jennifer Kwong, B.A., Joseph Kuhn, M.D., Michael Chan, B.A., Arie Monas, Jasmine Lee, B.A., Alvaro Villarreal Ponce, Ph.D., Bruce Young, M.D., Piul S. Rabbani, Ph.D..
NYU Langone Health, New York, NY, USA.

Purpose: Adult multipotent stromal cells are being widely investigated for treatment of diabetic chronic ulcers that pose a $96billion annual healthcare burden. One less explored source of these cells is human amniotic fluid with ready availability and lack of immunogenicity in allogeneic use. We investigated the potential of human amniotic fluid-derived multipotent stromal cells (AFSCs) to promote wound healing in a preclinical type II diabetic model.
Methods: Amniotic fluid was collected with informed consent from full-term gestational patients at caesarian sections, approved by NYU Institutional Review Board. AFSCs were differentiated in adipogenic, osteogenic, chondrogenic media. For therapy, multiple donor AFSCs were cultured together and passage 3 cells used. Ten mm diameter full-thickness stented wounds were created on dorsum of adult LepRdb/db mice (blood glucose≥400mg/dL). 5x105 AFSCs in 10uL culture media were topically applied to the wound bed immediately after excision. Culture media, or no treatment were controls. For in vivo imaging, AFSCs were stained with DiI prior to topical application, and wounds were imaged using an In Vivo Imaging System at 560/620nm. Wounds were photographed to monitor wound closure. Wound tissues were harvested post-operatively for histological analysis. Statistical significance was determined by comparing experimental and control groups using a one-way ANOVA.
Results: Minimum 1x106 AFSCs were harvested per 1mL full-term amniotic fluid, determined by plastic adherence. AFSCs were multipotent and differentiated along adipogenic, osteogenic and chondrogenic lineages. DiI-labeled AFSCs were detectable in the diabetic wound bed up to 120hours post-treatment. AFSCs resulted in significantly accelerated diabetic wound closure by 191.6days, compared to vehicle or untreated wounds at 251.6 and 26.40.98days, respectively (p<0.05). Single topical application of AFSCs reduced pathologic wound healing time by 76% and 78.7% compared to vehicle and untreated diabetic wounds, respectively. Pathologic wound burden with AFSC treatment also significantly decreased by 56% and 44.2%, compared to vehicle and untreated diabetic wounds, respectively (p<0.05 for both). Vehicle and untreated diabetic wounds did not demonstrate any significant differences in any parameter, indicating that the cell culture media does not affect wound healing. AFSC administration also accelerated rate of wound healing for the diabetic wounds, compared to vehicle or untreated diabetic wounds, p<0.05 for both. There was no significant difference between wound healing rates of vehicle-treated and untreated diabetic wounds. Tissue sections from post-op-day 7 confirmed lower gap between epithelial wound edges with AFSC-treatment. Immunoreactivity showed similar quantities of F4/80-expressing macrophages in the wounds tissues at post-op days 1-3, in all AFSC-treated, vehicle and untreated diabetic wounds. By post-op day 7, higher numbers of arginase1-positive M2-macrophages are present in AFSC-treated diabetic wounds compared to vehicle-treated ones. The results indicate that AFSCs support the critical M2 macrophage subsets to contribute towards closure of diabetic cutaneous wounds.
Conclusions: With approximately 600mL per gestation, amniotic fluid provides a feasible and reliable source of multipotent stromal cells. Application of AFSCs successfully changed the immunophenotype of the diabetic wound and reduced wound healing time. Our results support further investigation to translate AFSCs into a readily available and viable therapy for chronic diabetic wounds.


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