Abstract
Hematopoietic Stem Cells (HSC) have high regenerative potential to sustain the production of blood throughout life. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders. However, aging deregulates HSC functions. Aged HSC have decreased regenerative capacity and decreased production of lymphoid lineages. Mitochondria are critical regulators of HSC functions, but become defective with age, as shown by us and others. The mechanism behind mitochondrial defects are poorly understood. The lipid composition of mitochondrial membranes is important for their functions. Multi-omics analyses, including scRNA-seq and lipidomics, revealed significant differences in glycerophospholipids in young and mid-aged HSC with deregulated lipid remodeling, increased lipid storage and decreased free fatty acids. Using confocal microscopy in live cells, we identified that aged HSCs exhibited fewer but larger lipid droplets (LD), which are the storage units of lipids in a cell. In contrast, lipid trafficking assays consistently showed defective lipid incorporation into mitochondria. Lipids traffick between LD, which emanates from the endoplasmic reticulum (ER), and ER via mitochondrial-ER connections (MERC). High resolution imaging of mitochondria and ER in HSCs uncovered significant reduction in MERC in aged HSC, perhaps due to deregulated mitochondrial membrane lipids.
We next examined mitochondrial membrane lipids. Cardiolipin (CL) is a crucial glycerophospholipid present almost exclusively in mitochondrial membrane. CL is important for mitochondrial bioenergetics, dynamics, and for mitochondrial membrane architecture by stabilizing mitochondrial protein import and lipid transfer. We found the overall CL levels are reduced in aged HSC, as tested by flow cytometry using a CL marker non-acridine orange (NAO). Lipidomics revealed lower levels of remodeled mature CL. Tafazzin encoded by the gene TAZ catalyzes CL remodeling into its mature form which is required for maintaining mitochondrial functions. Mutations in TAZ causes reduction in mature CL, associated with mitochondrial defects. We found decreased expression of TAZ mRNA and protein in aged HSCs compared to young HSCs. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis showed significant decrease in HSC and multipotent progenitor frequency in 6-month-old TAZ KD mice compared to wild type (WT) mice. Following 5FU challenge, TAZ KD mice have abnormal peripheral blood recovery with decreased platelets but increased white blood counts. Progenitor and HSC frequencies were also decreased on day 10 and day 21 post 5FU. In serial competitive transplantation assays, while primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells, both in the peripheral blood and in the bone marrow. In contrast, Taz KD donor-derived HSC frequency was similar to that of WT cells. Hence, Taz-deficiency causes a defect in long-term HSC regenerative potential with amplification of the HSC pool as the expanse of differentiation.
Alcar (Acetyl-L-Carnitine) is known to promote CL production and restore mitochondrial defects due to loss of CL. We next evaluated whether Alcar could restore mid-aged HSC functions. For this, mid-aged HSCs were treated with Alcar for 3 days under conditions that maintain HSCs functions in vitro. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio of lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial.
This study indicates that HSC lipid composition becomes abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial CL in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.
