Research Paper Volume 13, Issue 21 pp 23981—24016

Restoration of aged hematopoietic cells by their young counterparts through instructive microvesicles release

Hematopoietic restoration with miRNAs. (A) Restoration protocol with NSG was similar to Figure 2H. Chimeric mice were given autologous CD3-depleted aged MPBs, transfected with miR-619, miR-combo, −619, −1303 and −4497, or control (RNA mimic) and then cultured for 7 days, (n = 18). (B–E) Mice were analyzed for huCD45 in BM and blood (B); huCD3, CD4 and CD8 in blood (C); HuCD19 in blood and BM (D); HuCD33 in BM (E). Results presented as % mean cells ± SD (F) Lymphoid:Myeloid ratio (CD3++CD19+/CD33+) in BM. (G) CFU-GM with huCD45+ cells from femurs, mean ± SD. (H) qPCR for PAX5 and PPM1F with RNA from huCD45+cells from femurs. Fold change ± SD used 1 for the lowest value. (I, J) RNA from `H’ were analyzed in qPCR human senescence and aging arrays. Normalized results used 1.5-fold cutoff to classify up- or down-regulation, or no change (I). SASF analyses with plasma. Semi-quantitative densitometry used 1.5-fold cutoff, similar to I (J). Differential gene and protein expressions as heatmaps (left), pie charts (top) and bar graph (bottom), mean ± SD. *p ≤ 0.05 vs. control. See also Supplementary Figure 5.

Figure 5. Hematopoietic restoration with miRNAs. (A) Restoration protocol with NSG was similar to Figure 2H. Chimeric mice were given autologous CD3-depleted aged MPBs, transfected with miR-619, miR-combo, −619, −1303 and −4497, or control (RNA mimic) and then cultured for 7 days, (n = 18). (BE) Mice were analyzed for huCD45 in BM and blood (B); huCD3, CD4 and CD8 in blood (C); HuCD19 in blood and BM (D); HuCD33 in BM (E). Results presented as % mean cells ± SD (F) Lymphoid:Myeloid ratio (CD3++CD19+/CD33+) in BM. (G) CFU-GM with huCD45+ cells from femurs, mean ± SD. (H) qPCR for PAX5 and PPM1F with RNA from huCD45+cells from femurs. Fold change ± SD used 1 for the lowest value. (I, J) RNA from `H’ were analyzed in qPCR human senescence and aging arrays. Normalized results used 1.5-fold cutoff to classify up- or down-regulation, or no change (I). SASF analyses with plasma. Semi-quantitative densitometry used 1.5-fold cutoff, similar to I (J). Differential gene and protein expressions as heatmaps (left), pie charts (top) and bar graph (bottom), mean ± SD. *p ≤ 0.05 vs. control. See also Supplementary Figure 5.