Branched chain amino acid transaminase 2 activity controls hsc generation-aging

Hematopoietic stem cells (HSC) are endowed with high regenerative potential but their self-renewal capacity is limited. Studies using the GFP-Histone 2B labeling system show HSC functions decline at each round of division (Stem Cell Reports 2014), also known as HSC generation-aging. We showed that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here, integrating HSC division tracking in vivo with mitochondrial membrane potential (MMP) reveals that label-retaining HSCs can be separated into 4 distinct populations: GFP(G)Hi;TMRE(T)lo, GHi;THi, GLo;TLo, GLo;THi, which are unexpectedly very heterogeneous in functional behavior. GHi;Tlo are slow-cycling cells that produce high, durable and multi-lineage cellular output. In contrast, GLo;TLo are fast-cycling and have low but myeloid-bias cellular ouput. GHi;THi and GLo;THi have the lowest repopulation capacity. Single cell RNA-sequencing analyses reveal that generation age alters HSC differentiation landscape with GLo;TLo producing distinct myeloid cell trajectory. Remarkably, each lineage cell derived from GLo;TLo donor cells had differential gene expression signatures compared to GHi;TLo donor cells, suggesting committed hematopoietic lineages retain memory of HSC division history. Analysis of differential gene expression and functional validation suggest that branched chain amino acid transaminase-2 is necessary for high, durable and multi-lineage HSC activity but is lost upon division. Exposure to branched chain amino acid catabolism product, alpha-Ketoisocaproic acid, in-vitro is sufficient to return generation-aged HSC to fully functioning HSC. Our results suggest that generation age drives HSC functional diversity via intrinsic metabolic reprograming and that it can be reversed through metabolite supplementation. Hematopoietic stem cells (HSC) are endowed with high regenerative potential but their self-renewal capacity is limited. Studies using the GFP-Histone 2B labeling system show HSC functions decline at each round of division (Stem Cell Reports 2014), also known as HSC generation-aging. We showed that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here, integrating HSC division tracking in vivo with mitochondrial membrane potential (MMP) reveals that label-retaining HSCs can be separated into 4 distinct populations: GFP(G)Hi;TMRE(T)lo, GHi;THi, GLo;TLo, GLo;THi, which are unexpectedly very heterogeneous in functional behavior. GHi;Tlo are slow-cycling cells that produce high, durable and multi-lineage cellular output. In contrast, GLo;TLo are fast-cycling and have low but myeloid-bias cellular ouput. GHi;THi and GLo;THi have the lowest repopulation capacity. Single cell RNA-sequencing analyses reveal that generation age alters HSC differentiation landscape with GLo;TLo producing distinct myeloid cell trajectory. Remarkably, each lineage cell derived from GLo;TLo donor cells had differential gene expression signatures compared to GHi;TLo donor cells, suggesting committed hematopoietic lineages retain memory of HSC division history. Analysis of differential gene expression and functional validation suggest that branched chain amino acid transaminase-2 is necessary for high, durable and multi-lineage HSC activity but is lost upon division. Exposure to branched chain amino acid catabolism product, alpha-Ketoisocaproic acid, in-vitro is sufficient to return generation-aged HSC to fully functioning HSC. Our results suggest that generation age drives HSC functional diversity via intrinsic metabolic reprograming and that it can be reversed through metabolite supplementation.