Hematopoietic Stem and Progenitor Cell Fate Decisions Are Regulated By a Cell-Autonomous p190-B/TGF-β Signaling Pathway

Hematopoietic stem cells (HSC) are undifferentiated cells that self-renew and produce all mature blood and immune cells in order to sustain life-long hematopoiesis. The molecular mechanisms that determine HSC fate decisions still remain poorly understood. Identifying these factors is of great biological and clinical importance, as alteration in HSC fate decisions will eventually lead to HSC depletion or malignant HSC expansion.

We previously showed that p190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, is a critical regulator of HSC self-renewal. P190-B loss enhanced long-term engraftment during serial transplantation; but, surprisingly, without altering their survival, blood lineage differentiation and the balance between quiescence and proliferation (Xu et al, Blood). Therefore, we hypothesize that p190-B regulates HSC self-renewal by controlling a HSC fate decision to self-renew or to differentiate, so-called asymmetric versus symmetric self-renewal divisions. To test this, we compared patterns of single WT and p190-B^-/- HSC (i.e. Lin^-c-kit⁺Sca-1⁺CD150⁺CD48^- [LSK-SLAM]) divisions over serial transplantation. First, we confirmed that the division rate of single LSK-SLAM was similar between the genotypes. Then, we performed the pair daughter cell assay to examine the ability of LSK-SLAM to generate daughter cells that retain multipotent lineage differentiation potential. We found that non-transplanted (CTL) HSC gave rise to 93% symmetric self-renewal divisions (i.e. both daughter cells are multipotent, neutrophil, erythroid, macrophage and megakaryocyte (nemM)), and 7% were asymmetric (only one daughter cell is multipotent). Interestingly, after 2 rounds of transplantation (2T), WT HSCs produced only 50% symmetric self-renewal divisions whereas p190-B-deficient HSCs maintained 92% symmetric self-renewal divisions. Hence, p190-B modulates asymmetric/symmetric self-renewal divisions to control HSC regeneration over serial transplantation.

Using transcriptional profiling and chemical screening, we identified, quite unexpectedly, the transforming growth factor beta (TGFβ) pathway as a regulatory pathway mediating p190-B functions. Expression of TGFβ responsive genes (mRNA expression of tgif2, smurf2) were higher in 2T-WT than in 2T-p190-B^–/–-HSCs and CTL HSC. To assess if increased TGFβ signaling affected HSC self-renewing decisions, we used the TGF-β inhibitor, SB431542, ex vivo. TGFβ inhibitor treatment of 2T-WT LSK-SLAM in pair daughter cell assay converted their fate decision to produce 90% symmetric self-renewal divisions; TGFβ inhibitor treatment ex vivo for 48h of WT LT-HSCs isolated from primary transplanted mice enhanced engraftment into secondary mice. Importantly, TGFβ inhibitor-treatment of mice over serial transplantation reversed transplant-related WT HSC decline as it enhanced HSC pool regeneration in BM and long-term HSC engraftment. Interestingly, it did not alter HSC cell cycle parameters or the relative heterogeneity of the HSC pool or blood lineage maturation. Mechanistically, p190-B appeared to regulate TGF-β signaling in a cell-autonomous manner by controlling bioactive TGF-β protein within HSCs. Finally we observed that TGFβ mediated its effect via non-canonical p38^MAPK signaling.

This study identifies a previously unknown p190-B/TGFβ signaling network that governs HSC fate decisions to self-renew or to differentiate, without altering HSC quiescence or development of the progeny. This also reveals an unexpected role for TGFβ as a regulator of HSC fate beyond its well-defined role in HSC hibernation. The clinical implications are important for the field of HSC expansion and bone marrow transplantation.