Smad4 is critical for self-renewal of hematopoietic stem cells

Members of the transforming growth factor β (TGF-β) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-β superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4−/− primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal

The evolving view of the hematopoietic stem cell niche.

Hematopoietic stem cells (HSCs) reside in specialized microenvironments known as niches. The niche is essential to support HSC function and to maintain a correct balance between self-renewal and differentiation. Recent advances in defining different mesenchymal and endothelial bone marrow cell populations, as well as hematopoietic stem and progenitor cells, greatly enhanced our understanding of these niches and of the molecular mechanisms by which they regulate HSC function. In addition to the role in maintaining HSC homeostasis, the niche has also been implicated in the pathogenesis of blood disorders including hematological malignancies. Characterizing the extrinsic regulators and the cellular context in which the niches interact with HSCs will be crucial to define new strategies to enhance blood regeneration. Furthermore, a better understanding of the role of the niche in leukemia development will open new possibilities for the treatment of these disorders by using therapies aiming to target the leukemic niche specifically. To update on recent findings on this topic, the International Society for Experimental Hematology (ISEH) organized a webinar, presented by Prof. Sean J. Morrison and Dr. Simón Méndez-Ferrer and moderated by Dr. Cristina Lo Celso, entitled "The evolving view of the hematopoietic stem cell niche," which we summarize here

THE ROLE OF BMP SIGNALING AND ENDOGLIN IN REGULATION OF HEMATOPOIESIS

Hematopoiesis, the formation of blood cells, ultimately depends on a rare population of hematopoietic stem cells (HSCs), which can both self-renew to maintain the HSC pool, and differentiate into all mature blood lineages. HSC fate decisions are governed by a complex combination of numerous signals, although the molecular mechanisms are not fully understood. This thesis describes the role of two important regulatory candidates, Bone Morphogenetic Proteins (BMP) and Endoglin in adult hematopoiesis. Several publications have described the importance of BMP signaling in specification of hematopoietic tissue in developing embryos. In Article I and II, we investigate the full role of BMP signaling in adult mice, using single and double Cre-based conditional knockout mice for the BMP signaling mediators Smad1 and Smad5. We demonstrate that BMP signaling deficient bone marrow (BM) competes normally with wild-type cells and display unperturbed self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Thus, despite its crucial role in initial patterning of hematopoiesis, BMP signaling is not required to maintain adult hematopoiesis. Endoglin is a TGF-β accessory receptor recently demonstrated to identify functional long-term HSCs, although little is known regarding its function in these cells. We have investigated the role of endoglin in hematopoiesis by both knocking down, and over-expressing its levels using viral vectors. Interestingly, our study suggests a pivotal role for endoglin in adult erythropoietic development. However, transplantation of transduced BM stem and progenitor cells reveals that neither endoglin suppression nor over-expression affects the ability of HSCs to long-term repopulate recipient marrow

Decreased PGC1beta expression results in disrupted human erythroid differentiation, impaired hemoglobinization and cell cycle exit

Production of red blood cells relies on proper mitochondrial function, both for their increased energy demands during differentiation and for proper heme and iron homeostasis. Mutations in genes regulating mitochondrial function have been reported in patients with anemia, yet their pathophysiological role often remains unclear. PGC1β is a critical coactivator of mitochondrial biogenesis, with increased expression during terminal erythroid differentiation. The role of PGC1β has however mainly been studied in skeletal muscle, adipose and hepatic tissues, and its function in erythropoiesis remains largely unknown. Here we show that perturbed PGC1β expression in human hematopoietic stem/progenitor cells from both bone marrow and cord blood results in impaired formation of early erythroid progenitors and delayed terminal erythroid differentiation in vitro, with accumulations of polychromatic erythroblasts, similar to MDS-related refractory anemia. Reduced levels of PGC1β resulted in deregulated expression of iron, heme and globin related genes in polychromatic erythroblasts, and reduced hemoglobin content in the more mature bone marrow derived reticulocytes. Furthermore, PGC1β knock-down resulted in disturbed cell cycle exit with accumulation of erythroblasts in S-phase and enhanced expression of G1-S regulating genes, with smaller reticulocytes as a result. Taken together, we demonstrate that PGC1β is directly involved in production of hemoglobin and regulation of G1-S transition and is ultimately required for proper terminal erythroid differentiation

Defining the hematopoietic stem cell niche : The chicken and the egg conundrum

Understanding the in vivo regulation of hematopoietic stem cells (HSCs) will be critical to identifying key factors involved in the regulation of HSC self‐renewal and differentiation. The niche (microenvironment) in which HSCs reside has recently regained attention accompanied by a dramatic increase in the understanding of the cellular constituents of the bone marrow HSC niche. The use of sophisticated genetic models allowing modulation of specific lineages has demonstrated roles for mesenchymal‐derived elements such as osteoblasts and adipocytes, vasculature, nerves, and a range of hematopoietic progeny of the HSC as being participants in the regulation of the bone marrow microenvironment. Whilst providing significant insight into the cellular composition of the niche, is it possible to manipulate any given cell lineage in vivo without impacting, knowingly or unknowingly, on those that remain? J. Cell. Biochem. 112: 1486–1490, 2011. © 2011 Wiley‐Liss, Inc

Prospective isolation of radiation induced erythroid stress progenitors reveals unique transcriptomic and epigenetic signatures enabling increased erythroid output

Massive expansion of erythroid progenitor cells is essential for surviving anemic stress. Research towards understanding this critical process, referred to as stress-erythropoiesis, has been hampered due to the lack of specific marker-combinations enabling analysis of the distinct stress-progenitor cells capable of providing radioprotection and enhanced red blood cell production. Here we present a method for the precise identification and in vivo validation of progenitor cells contributing to both steady-state and stress-erythropoiesis, enabling for the first time in-depth molecular characterization of these cells. Differential expression of surface markers CD150, CD9 and Sca1 defines a hierarchy of splenic stress-progenitors during irradiation-induced stress recovery in mice, and provides high-purity isolation of the functional stress erythroid burst-forming-units (stress-BFU-E) with a 100-fold improved enrichment compared to the state-of-the-art. By transplanting purified stress-progenitors expressing the fluorescent protein Kusabira Orange, we determined their kinetics in vivo and demonstrated that CD150+CD9+Sca1 -stress-BFU-E provide a massive but transient radioprotective erythroid wave, followed by multi-lineage reconstitution from CD150+CD9+Sca1+ multi-potent stem/progenitor cells. Whole genome transcriptional analysis revealed that stress-BFU-E express gene signatures more associated with erythropoiesis and proliferation compared to steady-state BFU-E, and are bone morphogenetic protein 4-responsive. Evaluation of chromatin accessibility through ATAC sequencing reveals enhanced and differential accessibility to binding sites of the chromatin-looping transcription factor CTCF in stress-BFU-E compared to steady-state BFU-E. Our findings offer a molecular insight into the unique capacity of stress-BFU-E to rapidly form erythroid cells in response to anemia and constitute an important step towards identifying novel erythropoiesis stimulating agents

Gene expression profiling to define the cell intrinsic role of the SKI proto-oncogene in hematopoiesis and myeloid neoplasms

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced gene signatures associated with hematopoietic stem cells and myeloid differentiation. Here we provide detailed experimental methods and analysis for the gene expression profiling described in our recently published study of Singbrant et al. (2014) in Haematologica. Our data sets (available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39457) provide a resource for exploring the underlying molecular mechanisms of the involvement of the proto-oncogene SKI in hematopoietic stem cell function and development of myeloid neoplasms

Smad5 is dispensable for adult murine hematopoiesis.

Smad5 is known to transduce intracellular signals from bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-beta (TGF-beta) superfamily and are involved in the regulation of hematopoiesis. Recent findings suggest that BMP4 stimulates proliferation of human primitive hematopoietic progenitors in vitro, while early progenitors from mice deficient in Smad5 display increased self-renewal capacity in murine embryonic hematopoiesis. Here, we evaluate the role of Smad5 in the regulation of hematopoietic stem cell (HSC) fate decisions in adult mice by using an inducible MxCre-mediated conditional knockout model. Surprisingly, analysis of induced animals revealed unperturbed cell numbers and lineage distribution in peripheral blood (PB), bone marrow (BM), and the spleen. Furthermore, phenotypic characterization of the stem cell compartment revealed normal numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells in Smad5(-/-) BM. When transplanted in a competitive fashion into lethally irradiated primary and secondary recipients, Smad5-deficient BM cells competed normally with wild-type (wt) cells, were able to provide long-term reconstitution for the hosts, and displayed normal lineage distribution. Taken together, Smad5-deficient HSCs from adult mice show unaltered differentiation, proliferation, and repopulating capacity. Therefore, in contrast to its role in embryonic hematopoiesis, Smad5 is dispensable for hematopoiesis in the adult mouse