Mitochondrial fusion agonists improve expanded cord blood hematopoietic stem and progenitor cells function in vivo

Umbilical cord blood (UCB) is a resource for hematopoietic stem cell transplantation (HSCT) with many advantages. However, a barrier to using UCB for HSCT is insufficient cell dosage for adult patients. Expansion of UCB cells in vitro could overcome dose limitations, however methods to expand potent HSCs have not been fully realized. Thus, discovery of novel molecular targets to expand HSCs ex vivo are needed. Studies in mice have shown Mitofusin 2 (Mfn2), a protein involved in mitochondrial fusion and tethering, maintains potent HSCs in vitro. Recently, small molecule Mfn2 agonists with high efficacy have been reported. Therefore, we hypothesized that addition of Mfn2 agonists in UCB ex vivo cultures could improve expanded HSC function. Our study showed MFN2 is highly expressed in UCB HSCs. Cultures of phenotypic HSCs (CD34+,CD38-,CD45RA-,CD90+,CD201+) expanded in StemSpan with Cmpd A or B (5nM) for 7d showed no difference in HSC count or frequency compared to vehicle. Nevertheless, 30-wk primary xenografts showed ≥5-fold increased long-term chimerism in recipients of cultures expanded with Cmpd B. Secondary xenografts showed persistent chimerism in recipients of CmpB culture, but not vehicle. Resorted CD90+ HSCs after Mfn2 agonist treatment showed upregulation of genes involved in ribosomes, stress granules and autophagy. OP-Puro assays showed significant reduction in ribosome translation in Mfn2 agonist HSCs. ATF4 and phos-eIF2a, involved in ER stress response and stress granule formation, were upregulated in Mfn2 agonist HSCs. Further, increased acidic lysosome content and more LC3B autophagosomes were found in Mfn2 agonist HSCs. Thus, our data strongly shown Mfn2 agonists maintain long-term repopulation potential of UCBs during ex vivo expansion. Our findings suggest that Mfn2 agonists are viable candidates towards improving the expansion of UCB cells for HSCT. Umbilical cord blood (UCB) is a resource for hematopoietic stem cell transplantation (HSCT) with many advantages. However, a barrier to using UCB for HSCT is insufficient cell dosage for adult patients. Expansion of UCB cells in vitro could overcome dose limitations, however methods to expand potent HSCs have not been fully realized. Thus, discovery of novel molecular targets to expand HSCs ex vivo are needed. Studies in mice have shown Mitofusin 2 (Mfn2), a protein involved in mitochondrial fusion and tethering, maintains potent HSCs in vitro. Recently, small molecule Mfn2 agonists with high efficacy have been reported. Therefore, we hypothesized that addition of Mfn2 agonists in UCB ex vivo cultures could improve expanded HSC function. Our study showed MFN2 is highly expressed in UCB HSCs. Cultures of phenotypic HSCs (CD34+,CD38-,CD45RA-,CD90+,CD201+) expanded in StemSpan with Cmpd A or B (5nM) for 7d showed no difference in HSC count or frequency compared to vehicle. Nevertheless, 30-wk primary xenografts showed ≥5-fold increased long-term chimerism in recipients of cultures expanded with Cmpd B. Secondary xenografts showed persistent chimerism in recipients of CmpB culture, but not vehicle. Resorted CD90+ HSCs after Mfn2 agonist treatment showed upregulation of genes involved in ribosomes, stress granules and autophagy. OP-Puro assays showed significant reduction in ribosome translation in Mfn2 agonist HSCs. ATF4 and phos-eIF2a, involved in ER stress response and stress granule formation, were upregulated in Mfn2 agonist HSCs. Further, increased acidic lysosome content and more LC3B autophagosomes were found in Mfn2 agonist HSCs. Thus, our data strongly shown Mfn2 agonists maintain long-term repopulation potential of UCBs during ex vivo expansion. Our findings suggest that Mfn2 agonists are viable candidates towards improving the expansion of UCB cells for HSCT.