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L. Crisafulli et al.
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Figure 6. Forced miR-127 expression does not affect hematopoietic stem cell (HSC) maintenance. (A and B) FACS analysis of multi-lineage differentiation in periph- eral blood (PB) 18 weeks after transplant (A) and in bone marrow (BM) (B) of 127OE and empty vector (EV)-recipient mice. Histograms show absolute cell numbers and stacked columns show the relative abundance of each lineage determined by FACS analysis. (A) PB absolute cell number derives from hemocytometer analysis (127OE: n=6; EV: n=9; two independent experiments). (B) BM counts are relative to one femur and one tibia (127OE: n=5; EV: n=7; two independent experiments). (C) FACS analysis of 127OE and EV stem and progenitor cells in the BM of primary recipient mice. Cell numbers are relative to one femur and one tibia. (D-G) FACS analysis of 127OE and EV Lin– transduced cells transplanted in competition with non-transduced cells. Analyses are relative to primary (D and E) and secondary (F and G) recipient mice. (D) Percentage of OFP+ cells within donor CD11b+ cells in PB 18 weeks after transplant of transduced Lin–cells injected in competition with non-transduced cells in a 9:1 ratio (P=0.24). (E) Frequency of OFP+ cells within the indicated BM populations of primary recipients. (F) Frequency of OFP+ cells within the indicated BM populations of secondary recipients. (G) Percentage of OFP+ cells within donor CD11b+ and CD19+ BM cells of secondary recipients (****P<0.0001; Wilcoxon paired test).
to the next maturation step is the underlying cause of the observed HSC loss in vivo when miR-127-3p is not active.
Forced miR-127-3p expression does not affect hematopoietic stem cell maintenance
We next asked whether increased miR-127-3p levels affect HSC function in vivo. Lentiviral-mediated miR-127 overexpression was performed in wt CD45.2+ Lin– cells, followed by transplantation in CD45.1+ recipient mice (Online Supplementary Figure S5A). miR-127-overexpres- sion (127OE) vector carried the Orange Fluorescent Protein (OFP) as reporter gene and the corresponding empty vector (EV), carrying only the reporter protein, was used as control. We obtained very high transduction effi- ciency with both vectors (data not shown). Expression analysis through real-time PCR before and after transduc- tion confirmed that the progeny of infected cells expressed
miR-127-3p, reaching levels similar to those of miR-16, which is present in all hematopoietic cells (Online Supplementary Figure S5B). Periodic PB analysis revealed that 127OE Lin- cells were able to engraft and undergo multi-lineage differentiation, similar to Lin– cells trans- duced with the EV (data not shown). However, several weeks after transplant, peripheral B- and T-cell counts were significantly lower in mice transplanted with miR- 127 over-expressing cells, compared to mice transplanted with the EV (Figure 6A). These differences were present also in BM (Figure 6B) and spleen (data not shown), and are consistent with the reported suppression of germinal cen- ter regulators by human miR-127,21,27 likely reflecting the lack of miR-127 downregulation from the MPP stage onward rather than upregulation in HSC. In mice trans- planted with 127OE cells, the level of miR-127-3p expres- sion in the spleen several weeks after transplantation was
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haematologica | 2019; 104(9)