Mitochondrial Morphology Controls Hematopoietic Stem Cell Self-Renewal and Confers Them Divisional Memory

Hematopoietic stem cells (HSC) have extensive regenerative potential but limited self-renewal ability. The repopulation potential is gradually lost with HSC divisions and during stress hematopoiesis. Hence, we hypothesize that activation or inactivation of signaling pathways or other mechanisms, inherently occurring during HSC activation into cycle, drives their progressive functional decline. To address this, we used single cell RNA-sequencing and compared the transcriptome of HSCs [Lin^-c-Kit⁺Sca-1⁺CD48^-CD150⁺] that is expressed upon exit from quiescence and the one that is inherited by daughter cells upon HSC division. We compared HSCs with full self-renewal activity to HSCs that have lost self-renewal activity [i.e. before (NT-HSC) and after transplantation (T-HSC), respectively]. Analysis of the data was performed using unsupervised methods, namely iterative clustering and guide-gene selection (ICGS), [Olsson et al, Nature, 2016] and principal component (PCA) analysis. Each revealed that genes categorized in mitochondrial organization were dramatically upregulated in activated NT-HSC and remained highly expressed in their daughter cells. Remarkably, ICGS revealed at least 2 distinct regulatory states within T-HSC; one that clustered within NT-HSC, and another one that was clearly separated from NT-HSC. The latter was exclusively comprised of daughter T-HSCs, and quite unexpectedly was demarcated by a lack of expression of mitochondrial genes along with changes in expression of genes related to cytoskeleton organization. Using EGFP-mitochondria reporter mice; which labels mitochondria regardless of their membrane potential and activated state, we found that NT-HSC had 4-fold higher mitochondrial content than progenitors. High resolution confocal imaging indicated that mitochondria were punctuated and the network was dispersed in the entire cell volume in NT-HSC. However, in T-HSCs, mitochondria formed one or a few larger aggregates. Mitochondrial membrane potential was also severely reduced in T-HSC in vivo, indicating that HSC accumulate dysfunctional mitochondria after replicative stress during stress hematopoiesis. During cell division, in vitro, mitochondria were unequally distributed to daughter cells of single T-HSC division [one daughter cell had more aggregated mitochondria], whereas mitochondria were equally partitioned during division of NT-HSC, suggesting that HSC lose mitochondrial quality control after replicative stress. Further, mitochondria aligned along the microtubule network in NT-HSC but not in T-HSC. Mitochondrial morphology and distribution are maintained through regulated cycles of fission and fusion, which is essential for maintaining a functional organelle and for equal mitochondrial inheritance during division. We found that the mitochondrial fission protein Drp1, which normally accumulated and wrapped around mitochondria in NT-HSC during cell activation, failed to do so in T-HSCs, perhaps due to impaired mitochondrial-cytoskeleton association. Genetic loss of Drp1 caused severe mitochondrial aggregation in HSCs, and impaired HSC self-renewal and repopulation ability in competitive transplant studies, indicating that accumulation of aggregated and dysfunctional mitochondria causes HSC functional defects. To understand whether HSCs accumulated abnormal mitochondrial morphology with divisional history during homeostatic conditions, we used the GFP label-retaining H2B mouse model. Remarkably, mitochondria were well dispersed and homogeneously distributed in GFP labeled-retaining HSCs (i.e. HSC that had not divided), while HSCs with history of cell division (no GFP) had larger and more compact aggregates. Hence, once HSCs exit from quiescence, they irreversibly accumulate dysfunctional mitochondria over the course of divisions due to lack of mitochondrial quality control mechanisms, which drives gradual heterogeneity and loss of self-renewal ability. Thus, HSCs use mitochondrial architecture to remember their divisions, in turn affecting their self-renewal ability. Therefore, drugs or pharmacological inhibitors targeting mitochondrial organization or its quality can be new therapeutic approach to improve HSC function following transplantation.