Allogenic adipose derived mesenchymal stem cells are effective than antibiotics in treating endometritis

Endometritis is a uterine inflammatory disease that causes reduced livestock fertility, milk production and lifespan leading to significant economic losses to the dairy industry. Mesenchymal stem cells (MSC) may act as an alternative for inefficacy of antibiotics and rising antibiotic resistance in endometritis. The present study aimed to cure the chronic endometritic buffaloes using allogenic adipose-derived MSCs (AD-MSC). AD-MSCs were isolated from buffalo adipose tissue and characterized by multilineage differentiation as well as MSC-specific markers. The in vivo safety and efficacy were assessed after infusion of AD-MSCs. In safety trial, cells were administered in healthy buffaloes via different routes (IV and IC) followed by examination of clinical and hematological parameters. In efficacy study, AD-MSCs treatments (IV and IC) and antibiotic therapy (ABT) in endometritic buffaloes were comparatively evaluated. AD-MSCs did not induced any immunological reaction in treated buffaloes. PMN count, CRP levels and VDS were significantly (p ≤ 0.05) reduced after AD-MSCs infusions in IV and IC groups and no significant difference was observed in antibiotic group. The IV group was marked with 50% absolute risk reduction in endometritis and 50% live calf births after artificial insemination in comparison with ABT group. Anti-inflammatory cytokines (IL4 and IL10) and anti-microbial peptides (PI3, CATHL4, LCN2 and CST3) expressions were significantly (p ≤ 0.05) upregulated in IV group. The calf delivery rate after the treatments in IV group was higher (50%, 3 calves) than the other groups (IC: 33.3%, 2 calves; ABT: 16.6%, 1 calf). In conclusion, the administration of AD-MSCs through IV route was found to be safe and efficacious for alleviating chronic endometritis in dairy buffaloes.

M1: Characterization of AD-MSCs. Figure S1: The buffaloes from IV group that calved after artificial insemination; Figure S2: The buffaloes from IC group that calved after artificial insemination; Figure S3: The buffalo from ABT group that calved after artificial insemination; Table S1: Details of primers used in the study; Table S2: Cervical vaginal discharge score allocated to each animal in IV, IC and ABT, on day 0 (D0) and day 15 (D15); Table S3: History of antibiotic treatments in animals before opting for the study.

Immunostaining of AD-MSCs
MSCs were cultured in 4-well dish and fixed with 4% paraformaldehyde in DPBS for 30 min and washed 3 times with DPBS. Cells were incubated with the 4% Bovine Serum Albumin (blocking solution) for 30 min at RT followed by an overnight incubation with primary antibodies (1:100) (Table S1)

Osteogenic differentiation of AD-MSCs and alizarin red staining
After the 3rd passage, AD-MSCs were seeded in multi-well plate at a density of 15,000 cells/cm 2 and incubated for 48-72 hrs to attain confluence, following replacement of standard medium with differentiation medium. The osteogenic medium was comprised of standard medium supplemented with 10% FBS, 100 nM dexamethasone, 200 mM ascorbic acid and 10 mM of b-glycerophosphate. After replacement with osteogenic differentiation medium, cells were incubated for 21 days at 38 °C, 5% CO2, and 95% relative humidity and the medium was replaced after every 72 hrs. Finally, cells were washed in DPBS, fixed in 95% methanol for 10 min, stained with 2% alizarin red S solution for 5 min, rinsed again with water and examined for Ca 2+ deposits using inverted microscope (IX51, Olympus, Japan).

Adipogenic differentiation of AD-MSCs and oil red O staining
The cells were cultured on fibronectin-coated multi-well plates until confluence as mentioned previously for osteogenic differentiation and standard culture medium was replaced by adipogenic differentiation medium prepared by supplementing 100 nM dexamethasone, 80 mM indomethacin, 0.5 mM 3-isobutyl-1-methylxantine, and 10 mg/ml insulin in standard culture medium. The cells were incubated with the differentiation medium at 38 °C, 5% CO2, and 95% relative humidity for 21 days with regular replacement of medium as described

S.No. Buffalo No.
Dates of antibiotic therapies prior to experiment