Type I and II Diabetic Adipose-Derived Stem Cells Respond In Vitro to Dehydrated Human Amnion/Chorion Membrane Allograft Treatment by Increasing Proliferation, Migration, and Altering Cytokine Secretion

Abstract

Objective: Human amniotic membranes have been shown to be effective for healing diabetic foot ulcers clinically and to regulate stem cell activity in vitro and in vivo; however, diabetic stem cells may be impaired as a sequela of the disease. In this study, dehydrated human amnion/chorion membrane (dHACM) allografts (EpiFix^®; MiMedx Group) were evaluated for their ability to regulate diabetic stem cells in vitro. Approach: Human adipose-derived stem cells (ADSCs) from normal, type I diabetic, and type II diabetic donors were treated with soluble extracts of dHACM and evaluated for proliferation after 3 days by DNA assay, chemotactic migration after 1 day by transwell assay, cytokine secretion after 3 days by multiplex ELISA, and gene expression after 5 days by reverse transcription-polymerase chain reaction. Results: Although diabetic ADSCs demonstrated decreased responses compared to normal ADSCs, dHACM treatment stimulated diabetic ADSCs to proliferate after 3 days and enhanced migration over 24 h, similar to normal ADSCs. dHACM-treated diabetic ADSCs modulated secretion of soluble signals, including regulators of inflammation, angiogenesis, and healing. All ADSCs evaluated also responded to dHACM treatment with altered expression of immunomodulatory genes, including interleukins (IL)-1α, IL-1β, and IL-1RA. Innovation: This is the first reported case demonstrating that diabetic ADSCs respond to novel amniotic membrane therapies, specifically treatment with dHACM. Conclusion: dHACM stimulated diabetic ADSCs to migrate, proliferate, and alter cytokine expression suggesting that, despite their diabetic origin, ADSCs may respond to dHACM to accelerate diabetic wound healing.

Michelle Massee, BS

Figure 1.

Response of normal ADSCs to MMC treatment after 24 h. Each bar represents the average number of cells per treatment (n=5). The dashed line indicates the original seeding density. Error bars indicate standard deviation from mean values. ADSCs, adipose-derived stem cells; MMC, mitomycin C.

Figure 2.

Proliferation of normal, type I diabetic, and type II diabetic ADSCs over 72 h in response to treatment with soluble extracts of dHACM, as well as negative and positive controls. Each bar represents the average number of cells per treatment for each cell type (n=5). Error bars indicate standard deviation from mean values. *p≤0.05, where indicated versus basal media control. dHACM, dehydrated human amnion/chorion membrane.

Figure 3.

Migration of MMC pretreated normal, type I diabetic, and type II diabetic ADSCs over 24 h in response to treatment with soluble extracts of dHACM extracts embedded in a 0.5% agarose gel, as well as basal medium and complete medium controls. The negative control wells contained basal medium, while the positive control wells were supplemented with newborn calf serum. Each bar represents the relative fluorescent units (RFUs) representing the average number of cells per treatment for each cell type normalized to basal medium controls, which is represented by a black line in the graph (n=5). Error bars indicate standard deviations from mean values. *p≤0.05, where indicated versus negative control. Separate wells were designated for DAPI staining as a visual confirmation of the migration response. (a) Representative image of migration of normal ADSCs in response to basal medium in agarose. (b) Representative image of migration of normal ADSCs in response to 10 mg/mL dHACM extract in agarose. (c) Representative image of migration in response to serum in agarose.

Figure 4.

Growth factor secretion by type I and type II diabetic ADSCs, normalized to normal ADSCs, in complete medium. Data for type I and II diabetic ADSCs were normalized to quantities of each factor measured for normal ADSCs, the latter of which is represented by a black line in the graph.

Figure 5.

Cytokine secretion by dHACM-treated ADSCs. The data are presented as a heat map with upregulation relative to basal media represented as a green color gradient and downregulation represented as a red color gradient. N, normal ADSCs; TI, type 1 diabetic ADSCs; TII, type 2 diabetic ADSCs.

Figure 6.

Gene expression of dHACM-treated normal, type I diabetic, and type II diabetic ADSCs. Fold regulation of (a) IL-1α, (b) IL-1β, (c) IL-1RA, (d) TNF-α, (e) PTGES, (f) TGF-β1, and (g) HGF is expressed relative to ADSCs cultured in basal medium (fold regulation=1). *Significant upregulation from basal medium (p≤0.05). ^#Significant downregulation from basal medium (p≤0.05). HGF, hepatocyte growth factor; IL, interleukins; PTGES, prostaglandin E synthase; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-α.