Figure 3.

H₂S enhances proliferation of HSPCs and differentiation of BMMNCs in-vitro. (a) BMMNCs were pulsed with NaHS/vehicle, washed and cultured in Methocult (3X10⁴/plate) for 14 days in a CO₂ incubator at 37ºC. On the 14^th day, the colonies were identified as shown and scored as (b) mean ± SEM number of CFUs/3X10⁴ cells. Treated cells displayed a significant increase in the CFU-GM and CFU-GEMM. The comparisons were made using student’s t-test. (n = 3, each assayed individually). (c) BMMNCs were treated with NaHS/vehicle, washed and plated in α-MEM+10% HI-FBS for 24 h. Thereafter, the cells were sorted, stained, fixed and permeabilized. Finally, the cells were stained with cell proliferation marker and the proportion of Ki67⁺ lin^neg and LSK cells were analyzed through flow cytometry. (d) The mean fluorescence intensity for Ki67 was also noted. Data are expressed as mean ± SEM and analyzed through two-way ANOVA. (e) Similarly, the treated cells were incubated for 6 and 24 h, thereafter sorted stained and fixed and the proportion of LSK population in cell cycle was quantified using 7-AAD viability dye through flow cytometry and represented as fold change in the S+ G₂M phase after NaHS exposure. Data are expressed as mean ± SEM and analyzed through two-way ANOVA. (n = 3,4 biological replicates, ns = non-significant *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001)