The Earliest Stages of Loss of Stem Cell Self-Renewal in-Vivo Is Linked to Upregulated Biosynthesis of “Quiet” Mitochondria and Is Influenced by CXCR4 Activation and STAT3 Gene Deletion.

Abstract 2546

Poster Board II-523

One attribute of all stem cells is self-renewal (SR). Progress has been made in identifying genes/proteins involved in stem cell SR mechanisms, especially in embryonic stem cells, but very little is known in adult stem cells such as hematopoietic stem cells (HSCs). Mitochondria (Mt) have recently been considered more than an ATP generator and mediator of apoptosis. They act as a kind of scaffold for integrating numerous signals to and from the nucleus and from the extracellular environment, and signals involved in differentiation and cell cycle. They also stabilize microtubules of the mitotic spindle and suppress rotation of the mitotic division plane, potentially influencing asymmetric/symmetric stem cell divisions. Populations of primitive hematopoietic cells with low Mt activity (i.e. Rho123low) are highly enriched in long-term repopulating (LTR)-HSC. Because Mt can suppress mitotic spindle rotation by stabilizing spindle microtubules, potentially influencing asymmetric divisions, we hypothesized that Mt biogenesis/function could be important for mechanisms of SR loss and early-stage differentiation of mouse HSC. We examined Mt mass and Mt membrane potential in primitive mouse CD34lo/neg LINnegSca-1posc-kitpos (CD34-LSK) cells, CD34+LSK cells, and related populations. We note that as the small CD34-LSK cells begin to express CD34, Mt mass significantly increases (2×), while Mt membrane potential does not change in these CD34+LSK cells. The CD34+LSK cells then increase in size without further increases in Mt mass, but with upregulated Mt membrane potential. Because CD34 expression in LSK cells is associated with loss of LTR-HSC (i.e. self-renewal) and retention of only short-term repopulating ability, we propose this previously uncharacterized population of LSK cells with increased numbers/mass of “quiet” Mt may be an important component during initial stages of loss of SR and asymmetric HSC division. We found that primitive LSK cells from mice constitutively expressing an SDF-1α/CXCL12 transgene (TG mice) have shifted ratios of these various mitochondrially distinct populations that may be related to CXCR4 signaling and its known influence on glycolysis and STAT3 activity, both of which can influence Mt metabolism. LSK cells in TG mouse total bone marrow significantly increased from .039+/_.01% to .085+/_.02% (n=6), but they were predominantly CD34+LSK cells. These CD34+LSK cells were composed of two distinct types of cells; small/high-Mt mass and large/high-Mt mass cells. The small LSK cells have lower Mt membrane potential while the large LSK cells had increased Mt membrane potential. Because CXCR4 can signal through STAT3, and because STAT3 can bind to Mt and influence their metabolic activities (i.e. membrane potential), we hypothesized that mice with a tissue-specific targeted gene deletion of STAT3 in hematopoietic cells might result in LSK cells that are defective in upregulating Mt membrane potential, which could shift the proportions of the three mitochondrially distinct populations of LSK cells toward the smaller, Mt mass-low, Mt membrane potential-low type of LSK cell. Bone marrow from targeted STAT3-deleted had LSK numbers increased 50%, but they were composed almost entirely of small, Mt mass-high, membrane potential-low, CD34+ type of LSK cells. This is consistent with our hypothesis and suggests that Mt membrane potential may be important for latter stages of CD34-LSK cell differentiation to more lineage-committed progenitor cells but may not be required for initial stages of SR loss. We propose a model where Mt biogenesis occurs before/during loss of SR in CD34-LSK cells. These Mt are metabolically quiescent (“quiet”), attach to spindle microtubules of dividing cells, and influence spindle orientation resulting in loss of SR (small, Mt mass high, Mt potential low, CD34+LSK cells). Before further differentiation, these cells upregulate Mt metabolism/membrane potential and increase in size to facilitate further expansion and differentiation (large, Mt mass high, Mt potential high, CD34+LSK cells). This model could help explain the observed phenotypes of SDF-1 α/CXCL12 transgenic mice and hematopoietic cell-targeted STAT3 deleted mice. This information may also be useful in the search for strategies to prevent loss of SR capacity of HSCs in culture and expansion of HSCs in-vitro.