207 One injection, one calf: control of metritis using umbilical cord blood-derived mesenchymal stem cells in livestock

Mesenchymal stem cells (MSCs) herald a new path in regenerative medicine therapy. They can be isolated from adipose tissue, bone marrow, cord blood, and dental pulp, among other tissues. The main functions of MSCs are to secrete anti-inflammatory molecules, immunomodulatory and anti-microbial cytokines, participate inparacrine signaling, and modulate the inflammatory response that kills bacteria and regenerate injured tissues. Previously, we have treated mastitis and hoof wounds of cows and buffaloes using MSCs, and all the animals were cured within a month. The present study aims to cure the metritis of cattle using umbilical cord blood-derived MSCs. The method uses MSCs that were aseptically isolated from the umbilical cord blood of a cow during delivery. The separated MSCs were then cultured in vitro. The cells were characterized as per the guideline of the International Society for Cellular Therapy (ISCT) with alkaline phosphatase and molecular markers such as CD44-, CD73-, CD90-, and CD105-positive markers and CD14-, CD19-, CD34-, and CD45-negative markers. Immunohistochemistry of these markers was also performed in the MSCs, and positive expression of genes such as CD44, CD73, CD90, and CD105 was observed. The MSCs were also differentiated in in vitro conditions into adipogenic, chondrogenic, neurogenic, and osteogenic lineages, and the identity of the differentiated lineage cells were confirmed. The MSCs were cryopreserved in liquid nitrogen and later revived under in vitro conditions in a CO₂ incubator. We have previously treated metritis in buffalo cows using MSCs (Bhaskar et al. 2023 Sci. Rep. 13, 11280), and we reported observations on MSCs being one of the most potent treatments for mastitis compared with antibiotic treatment (Ghai et al. 2022 Theriogenology 187, 141–151). In a wound treatment study, we observed that wounds in the treatment group healed the fastest, growing muscle, skin, and hair within a month compared with the antibiotics control group. In the present study, conducted on farms, 20 cows that had metritis more than four times were treated with UCB-MSCs at 10⁶ to 10⁷ cells mL⁻¹ with one injection of 2 mL i.v. and 2 mL injected in the vaginal muscles (Bhaskar et al. 2023). All the cows showed natural heat and were inseminated in time with proven bull semen. Among the 20 cows that were treated with UCB-MSCs, 18 cows delivered healthy calves and no longer had symptoms. Cytokine gene expression study of the blood of the treated cows was also performed by real-time PCR before treatment and after treatment within 7 days. Expression was observed for anti-inflammatory cytokine gene (IL10) and antimicrobial peptide genes (cathelicidin, cystatin, lipocalin 2), angiopoietin gene was expressed more significantly, and pro-inflammatory cytokine gene (IL6) had decreased expression. In conclusion, the MSCs were aseptically isolated from umbilical cord blood during the delivery of a cow, characterized, and differentiated in lineages. The MSCs cured metritis in cows, and they can be used as a regenerative therapy for cows with metritis to enable the production of healthy calves.