669 - Immune Modulatory Effects of Placental Mesenchymal Stem Cells-derived Extracellular Vesicles (plEVs) in Enhancing Wound Healing in diabetic conditions

Publication Number: 4655.669

Hady Omar, Michigan State University College of Human Medicine, East Lansing, MI, United States; Suraj Karanje, Michigan State University, Lansing, MI, United States; Amal Abdul-Hafez, Michigan State University, East Lansing, MI, United States; Ranga Prasanth Thiruvenkataramani, Michigan State University College of Human Medicine, Lansing, MI, United States; Alexandria Mayne, Michigan State University College of Human Medicine, Lansing, MI, United States; Sarah Mohammadi, Michigan State University College of Human Medicine, Okemos, MI, United States; Burra V. Madhukar, Michigan State University College of Human Medicine, E. Lansing, MI, United States; Said Omar, Michigan State University College of Human Medicine, Lansing, MI, United States

Background: Children with poorly controlled diabetes are at increased risk of poor wound healing. Multiple factors play a significant role in delayed diabetic wound healing, such as high blood glucose levels, modified immune response, neuropathy, impaired angiogenesis, and cytokine dysregulation. Placental mesenchymal stem cells (MSCs) and their derived extracellular vesicles (plEVs) play a major role in immune modulation and tissue regeneration. Placenta plEVs may have a novel therapeutic effect in enhancing diabetic wound healing.

Objective: To examine the effectiveness and immune modulatory effects of plEVs on wound healing in a diabetic skin model using human keratinocytes.

Design/Methods: Placentas were collected after IRBs approval and obtaining maternal informed consent from healthy full-term pregnancies. Wharton's jelly (WJ) was used to isolate MSCs and plEVs were isolated from passage 2-4 of WJ MSCs. Experiments were performed in vitro using human keratinocytes in high glucose medium to mimic diabetes. Effectiveness of plEVs was measured by wound/ scratch closure, cell proliferation (BrdU incorporation), and measuring pro inflammatory and immune modulatory cytokines by ELISA. The surface area (µm2) of the scratch assay was measured in the Control (1% FBS), scratch and plEVs treated groups. plEVs were added into the experimental groups (400 µg/ml) and observed every 12-hr period for 48 hours.

Results: Keratinocytes treated with plEVs significantly accelerated wound closure at 24 and 48 hrs as seen in Fig 1 (80 v 50%; p<.01) and the cells showed a 2.5-fold increase in the BrdU positive cells than controls. Pro-inflammatory cytokines; IL-1β (1.29 v 2.58 v 1.75 pg/ml; p<.05) and TNF-α (0.24 v 0.38 v 0.32 pg/ml) were elevated in control scratch/wound but not in plEVs group. Immune modulatory factors enhancing wound healing were significantly higher in plEVs than control/ wound groups; VEGF (463 v 237 v 234 pg/ml; p<.01), IL-4 (2.5 v 1.5 v 1.4 pg/ml; p<.01), PDGF (2893 v1697 v1643; p<.01) and GROα (1520 v 429 v 448; p<.01) as seen in Fig 2.

Conclusion(s): In diabetic conditions, Keratinocytes treated with plEVs showed enhancement of cell migration and proliferation, and wound healing. These findings suggest that plEVs promote wound healing by upregulating immune modulatory cytokines (VEGF, PDGF, GROα and IL-4) which play an important role in promoting fibroblast activation, recruitment of immune cells, wound contraction, angiogenesis, and tissue regeneration. Our study suggests that plEVs is a promising cell-free therapy for clinical use to provide immune modulatory benefits to enhance diabetic wound healing.

Scratch Assay of Control (not treated) and experimental (plEVs treated) Keratinocytes
Figure 1a: No plEVs treated Keratinocytes (HaCaT cells) images at 5 time points: 0h, 12h, 24h, 36h, and 48h. Scratch is not fully healed even at the 48-hour mark

Figure 1b: plEVs treated Keratinocytes (HaCaT cells) images at 5 time points: 0h, 12h, 24h, 36 h, and 48h. Scratch is fully healed at or before 36-hour mark

Expression of Pro-inflammatory and Immune Modulatory Cytokines with and without plEVs treated Keratinocytes
Figure 2: Isolated plEVs derived from placental MSCs were added into the experimental group (400 µg/ml). Cytokines were measured in keratinocytes supernatants after 24 hours. The pro-inflammatory cytokines: IL-1β, and TNFα were higher with scratch and not with plEVs treated cells. plEVs-treated group showed significantly higher Il-4, VEGF, PDGF and GROα levels, indicating that extracellular vesicles enhanced angiogenic signaling during wound closure. Total number of samples n=27 samples; each group has n=9 samples. ANOVA was used for statistical analysis followed by post Tukey test.