P190-B RhoGAP and intracellular cytokine signals balance hematopoietic stem and progenitor cell self-renewal and differentiation.
ABSTRACT: The mechanisms regulating hematopoietic stem and progenitor cell (HSPC) fate choices remain ill-defined. Here, we show that a signalling network of p190-B RhoGAP-ROS-TGF-?-p38MAPK balances HSPC self-renewal and differentiation. Upon transplantation, HSPCs express high amounts of bioactive TGF-?1 protein, which is associated with high levels of p38MAPK activity and loss of HSC self-renewal in vivo. Elevated levels of bioactive TGF-?1 are associated with asymmetric fate choice in vitro in single HSPCs via p38MAPK activity and this is correlated with the asymmetric distribution of activated p38MAPK. In contrast, loss of p190-B, a RhoGTPase inhibitor, normalizes TGF-? levels and p38MAPK activity in HSPCs and is correlated with increased HSC self-renewal in vivo. Loss of p190-B also promotes symmetric retention of multi-lineage capacity in single HSPC myeloid cell cultures, further suggesting a link between p190-B-RhoGAP and non-canonical TGF-? signalling in HSPC differentiation. Thus, intracellular cytokine signalling may serve as 'fate determinants' used by HSPCs to modulate their activity.
Project description:Pluripotent stem cells (PSCs) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had the CD34(+)CD38(-/lo)CD90(+)CD45(+)GPI-80(+) fetal liver (FL) HSPC immunophenotype, but exhibited poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, medial HOXA gene expression induced by retinoic acid signalling marks the establishment of the definitive HSPC fate and controls HSPC identity and function.
Project description:Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow, self-renewal, proliferation, and differentiation to mature blood cells. Here, we show that loss of p190-B RhoGTPase activating protein, a negative regulator of Rho GTPases, results in enhanced long-term engraftment during serial transplantation. This effect is associated with maintenance of functional HSC-enriched cells. Furthermore, loss of p190-B led to marked improvement of HSC in vivo repopulation capacity during ex vivo culture without altering proliferation and multilineage differentiation of HSC and progeny. Transcriptional analysis revealed that p190-B deficiency represses the up-regulation of p16(Ink4a) in HSCs in primary and secondary transplantation recipients, providing a possible mechanism of p190-B-mediated HSC functions. Our study defines p190-B as a critical transducer element of HSC self-renewal activity and long-term engraftment, thus suggesting that p190-B is a target for HSC-based therapies requiring maintenance of engraftment phenotype.
Project description:Umbilical cord blood-derived haematopoietic stem cells (HSCs) are essential for many life-saving regenerative therapies. However, despite their advantages for transplantation, their clinical use is restricted because HSCs in cord blood are found only in small numbers. Small molecules that enhance haematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified, but in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular circuitry that underpins the self-renewal of human HSCs will facilitate the development of targeted strategies that expand HSCs for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs, the post-transcriptional mechanisms that guide HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2-RNA interactions, we show that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signalling through post-transcriptional downregulation of canonical AHR pathway components in cord blood HSPCs. Our study gives mechanistic insight into RNA networks controlled by RNA-binding proteins that underlie self-renewal and provides evidence that manipulating such networks ex vivo can enhance the regenerative potential of human HSCs.
Project description:Advances in pluripotent stem cell and reprogramming technologies have given us the hope of generating hematopoietic stem cells (HSCs) in culture. To succeed, greater understanding of the self-renewing HSC during human development is required. We discovered that the glycophosphatidylinositol-anchored surface protein GPI-80 defines a subpopulation of human fetal liver hematopoietic stem/progenitor cells (HSPCs) with self-renewal ability. CD34(+)CD38(lo/-)CD90(+)GPI-80(+) HSPCs were the sole population that maintained proliferative potential and an undifferentiated state in stroma coculture and engrafted in immunodeficient mice. GPI-80 expression also enabled tracking of HSPCs once they emerged from endothelium and migrated between human fetal hematopoietic niches. GPI-80 colocalized on the surface of HSPCs with Integrin alpha-M (ITGAM), which in leukocytes cooperates with GPI-80 to support migration. Knockdown of GPI-80 or ITGAM was sufficient to compromise HSPC expansion in culture and engraftment in vivo. These findings indicate that human fetal HSCs employ mechanisms used in leukocyte adhesion and migration to mediate HSC self-renewal.
Project description:Hematopoietic stem and progenitor cells (HSPCs) undergo self-renewal and differentiation to guarantee a constant supply of short-lived blood cells. Both intrinsic and extrinsic factors determine HSPC fate, but the underlying mechanisms remain elusive. Here, we report that Proteinase 3 (PR3), a serine protease mainly confined to granulocytes, is also expressed in HSPCs. PR3 deficiency intrinsically suppressed cleavage and activation of caspase-3, leading to expansion of the bone marrow (BM) HSPC population due to decreased apoptosis. PR3-deficient HSPCs outcompete the long-term reconstitution potential of wild-type counterparts. Collectively, our results establish PR3 as a physiological regulator of HSPC numbers. PR3 inhibition is a potential therapeutic target to accelerate and increase the efficiency of BM reconstitution during transplantation.
Project description:Steady hematopoiesis is essential for lifelong production of all mature blood cells. Hematopoietic stem and progenitor cells (HSPCs) found in the bone marrow ensure hematopoietic homeostasis in an organism. Failure of this complex process, which involves a fine balance of self-renewal and differentiation fates, often result in severe hematological conditions such as leukemia and lymphoma. Several molecular and metabolic programs, internal or in close interaction with the bone marrow niche, have been identified as important regulators of HSPC function. More recently, nutrient sensing pathways have emerged as important modulators of HSC homing, dormancy, and function in the bone marrow. Here we describe a method for reliable measurement of various amino acids and minerals in different rare bone marrow (BM) populations, namely HSPCs. We found that the amino acid profile of the most primitive hematopoietic compartments (KLS) did not differ significantly from the one of their direct progenies (common myeloid progenitor CMP), while granulocyte-monocyte progenitors (GMPs), on the opposite of megakaryocyte-erythroid progenitors (MEPs), have higher content of the majority of amino acids analyzed. Additionally, we identified intermediates of the urea cycle to be differentially expressed in the KLS population and were found to lower mitochondrial membrane potential, an established readout on self-renewal capability. Moreover, we were able to profile for the first time 12 different minerals and detect differences in elemental contents between different HSPC compartments. Importantly, essential dietary trace elements, such as iron and molybdenum, were found to be enriched in granulocyte-monocyte progenitors (GMPs). We envision this amino acid and mineral profiling will allow identification of novel metabolic and nutrient sensing pathways important in HSPC fate regulation.
Project description:Hematopoietic stem cell (HSC) self-renewal is tightly controlled by cytokines and other signals in the microenvironment. While stem cell factor (SCF) is an early acting cytokine that activates the receptor tyrosine kinase KIT and promotes HSC maintenance, how SCF/KIT signaling is regulated in HSCs is poorly understood. The protein tyrosine phosphatase 4A (PTP4A) family (aka PRL [phosphatase of regenerating liver] phosphatases), consisting of PTP4A1/PRL1, PTP4A2/PRL2, and PTP4A3/PRL3, represents an intriguing group of phosphatases implicated in cell proliferation and tumorigenesis. However, the role of PTP4A in hematopoiesis remains elusive. To define the role of PTP4A in hematopoiesis, we analyzed HSC behavior in Ptp4a2 (Prl2) deficient mice. We found that Ptp4a2 deficiency impairs HSC self-renewal as revealed by serial bone marrow transplantation assays. Moreover, we observed that Ptp4a2 null hematopoietic stem and progenitor cells (HSPCs) are more quiescent and show reduced activation of the AKT and ERK signaling. Importantly, we discovered that the ability of PTP4A2 to enhance HSPC proliferation and activation of AKT and ERK signaling depends on its phosphatase activity. Furthermore, we found that PTP4A2 is important for SCF-mediated HSPC proliferation and loss of Ptp4a2 decreased the ability of oncogenic KIT/D814V mutant in promoting hematopoietic progenitor cell proliferation. Thus, PTP4A2 plays critical roles in regulating HSC self-renewal and mediating SCF/KIT signaling.
Project description:Expression of miR-143 and miR-145 is reduced in hematopoietic stem/progenitor cells (HSPCs) of myelodysplastic syndrome patients with a deletion in the long arm of chromosome 5. Here we show that mice lacking miR-143/145 have impaired HSPC activity with depletion of functional hematopoietic stem cells (HSCs), but activation of progenitor cells (HPCs). We identify components of the transforming growth factor ? (TGF?) pathway as key targets of miR-143/145. Enforced expression of the TGF? adaptor protein and miR-145 target, Disabled-2 (DAB2), recapitulates the HSC defect seen in miR-143/145<sup>-/-</sup> mice. Despite reduced HSC activity, older miR-143/145<sup>-/-</sup> and DAB2-expressing mice show elevated leukocyte counts associated with increased HPC activity. A subset of mice develop a serially transplantable myeloid malignancy, associated with expansion of HPC. Thus, miR-143/145 play a cell context-dependent role in HSPC function through regulation of TGF?/DAB2 activation, and loss of these miRNAs creates a preleukemic state.
Project description:While there are numerous small molecule inhibitory drugs available for a wide range of signalling pathways, at present, they are generally not used in combination in clinical settings. Previous reports have reported that the effects of glycogen synthase kinase (GSK)3?, p38MAPK, mTOR and histone deacetylase signaling combined together to suppress the stem?like nature of hematopoietic stem cells (HSCs), driving these cells to differentiate, cease proliferating and thereby impairing normal hematopoietic functionality. The present study aimed to determine the effect of HDACs, mTOR, GSK?3? and p38MAPK inhibitor combinations on the efficient expansion of HSCs using flow cytometry. Moreover, it specifically aimed to determine how inhibitors of the GSK3? signaling pathway, in combination with inhibitors of P38MAPK and mTOR signaling or histone deacetylase (HDAC) inhibitors, could affect HSC expansion, with the goal of identifying novel combination strategies useful for the expansion of HSCs. The results indicated that p38MAPK and/or GSK3? inhibitors increased Lin? cell and Lin?Sca?1+c?kit+ (LSK) cell numbers <em>in vitro</em>. Taken together, these results suggested that a combination of p38MAPK and GSK3? signaling may regulate HSC differentiation <em>in vitro</em>. These findings further indicated that the suppression of p38MAPK and/or GSK3? signalling may modulate HSC differentiation and self?renewal to enhance HSC expansion.
Project description:Basal nuclear factor ?B (NF-?B) activation is required for hematopoietic stem cell (HSC) homeostasis in the absence of inflammation; however, the upstream mediators of basal NF-?B signaling are less well understood. Here, we describe TRAF6 as an essential regulator of HSC homeostasis through basal activation of NF-?B. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient hematopoietic stem/progenitor cells (HSPCs) revealed changes in adaptive immune signaling, innate immune signaling, and NF-?B signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection is required for HSC function. In addition, we established that loss of I?B kinase beta (IKK?)-mediated NF-?B activation is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKK? similarly resulted in impaired HSC self-renewal and fitness. Taken together, TRAF6 is required for HSC homeostasis by maintaining a minimal threshold level of IKK?/NF-?B signaling.