Schlafen2 is a regulator of quiescence in adult murine hematopoietic stem cells.

Even though hematopoietic stem cells (HSC) are characterized by their ability to self-renew and differentiate, they primarily reside in quiescence. Despite the immense importance of this quiescent state, its maintenance and regulation is still incompletely understood. Schlafen2 (Slfn2) is a cytoplasmic protein known to be involved in cell proliferation, differentiation, quiescence, interferon response, and regulation of the immune system. Interestingly, Slfn2 is highly expressed in primitive hematopoietic cells. In order to investigate the role of Slfn2 in the regulation of HSC we have studied HSC function in the elektra mouse model, where the elektra allele of the Slfn2 gene contains a point mutation causing loss of function of the Slfn2 protein. We found that homozygosity for the elektra allele caused a decrease of primitive hematopoietic compartments in murine bone marrow. We further found that transplantation of elektra bone marrow and purified HSC resulted in a significantly reduced regenerative capacity of HSC in competitive transplantation settings. Importantly, we found that a significantly higher fraction of elektra HSC (as compared to wild-type HSC) were actively cycling, suggesting that the mutation in Slfn2 increases HSC proliferation. This additionally caused an increased amount of apoptotic stem and progenitor cells. Taken together, our findings demonstrate that dysregulation of Slfn2 results in a functional deficiency of primitive hematopoietic cells, which is particularly reflected by a drastically impaired ability to reconstitute the hematopoietic system following transplantation and an increase in HSC proliferation. This study thus identifies Slfn2 as a novel and critical regulator of adult HSC and HSC quiescence.

BMP signaling is required for postnatal murine hematopoietic stem cell self-renewal.

Life-long production of blood from hematopoietic stem cells (HSCs) is a process of strict modulation. Intrinsic and extrinsic signals govern fate options like self-renewal - a cardinal feature of HSCs. Bone morphogenetic proteins (BMP) have an established role in embryonic hematopoiesis, but less is known about its functions in adulthood. Previously, SMAD-mediated BMP signaling has been proven dispensable for HSCs. However, the BMP Type II receptor (BMPR-II) is highly expressed in HSCs, leaving the possibility that BMPs function via alternative pathways. Here, we establish that BMP signaling is required for self-renewal of adult HSCs. Through conditional knockout we show that BMPR-II deficient HSCs have impaired self-renewal and regenerative capacity. BMPR-II deficient cells have reduced p38 activation, implying that non-SMAD pathways operate downstream of BMPs in HSCs. Indeed, a majority of primitive hematopoietic cells do not engage in SMAD-mediated responses downstream of BMPs in vivo. Furthermore, deficiency of BMPR-II results in increased expression of TJP1, a known regulator of self-renewal in other stem cells, and knockdown of TJP1 in primitive hematopoietic cells partly rescues the BMPR-II null phenotype. This suggests TJP1 may be a universal stem cell regulator. In conclusion, BMP signaling, in part mediated through TJP1, is required endogenously by adult HSCs to maintain self-renewal capacity and proper resilience of the hematopoietic system during regeneration.

TGF-ß signaling in the control of hematopoietic stem cells.

Blood is a tissue with high cellular turnover, and its production is a tightly orchestrated process that requires constant replenishment. All mature blood cells are generated from hematopoietic stem cells (HSCs), which are the self-renewing units that sustain lifelong hematopoiesis. HSC behavior, such as self-renewal and quiescence, is regulated by a wide array of factors, including external signaling cues present in the bone marrow. The transforming growth factor-ß (TGF-ß) family of cytokines constitutes a multifunctional signaling circuitry, which regulates pivotal functions related to cell fate and behavior in virtually all tissues of the body. In the hematopoietic system, TGF-ß signaling controls a wide spectrum of biological processes, from homeostasis of the immune system to quiescence and self-renewal of HSCs. Here, we review key features and emerging concepts pertaining to TGF-ß and downstream signaling pathways in normal HSC biology, featuring aspects of aging, hematologic disease, and how this circuitry may be exploited for clinical purposes in the future.

Human hematopoietic stem/progenitor cells overexpressing Smad4 exhibit impaired reconstitution potential in vivo.

Hematopoietic stem cells (HSCs) constitute a rare population of tissue-specific cells that can self-renew and differentiate into all lineages of the blood cell system. These properties are critical for tissue regeneration and clinical applications of HSCs. Cord blood is an easily accessible source of HSCs. However, the number of HSCs from one unit is too low to effectively transplant most adult patients, and expansion of HSCs in vitro has met with limited success because of incomplete knowledge regarding mechanisms regulating self-renewal. Members of the TGF-ß superfamily have been shown to regulate HSCs through the Smad signaling pathway; however, its role in human HSCs has remained relatively uncharted in vivo. Therefore, we asked whether enforced expression of the common-Smad, Smad4, could reveal a role for TGF-ß in human hematopoietic stem/progenitor cells (HSPCs) from cord blood. Using a lentiviral overexpression approach, we demonstrate that Smad4 overexpression sensitizes HSPCs to TGF-ß, resulting in growth arrest and apoptosis in vitro. This phenotype translates in vivo into reduced HSPC reconstitution capacity yet intact lineage distribution. This suggests that the Smad pathway regulates self-renewal independently of differentiation. These findings demonstrate that the Smad signaling circuitry negatively regulates the regeneration capacity of human HSPCs in vivo.

Smad4 is critical for self-renewal of hematopoietic stem cells.

Members of the transforming growth factor beta (TGF-beta) 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-beta 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.

BMP7 promotes proliferation of nephron progenitor cells via a JNK-dependent mechanism.

The iterative formation of nephrons during embryonic development relies on continual replenishment of progenitor cells throughout nephrogenesis. Defining molecular mechanisms that maintain and regulate this progenitor pool is essential to understanding nephrogenesis in developmental and regenerative contexts. Maintenance of nephron progenitors is absolutely dependent on BMP7 signaling, and Bmp7-null mice exhibit rapid loss of progenitors. However, the signal transduction machinery operating downstream of BMP7 as well as the precise target cell remain undefined. Using a novel primary progenitor isolation system, we have investigated signal transduction and biological outcomes elicited by BMP7. We find that BMP7 directly and rapidly activates JNK signaling in nephron progenitors resulting in phosphorylation of Jun and ATF2 transcription factors. This signaling results in the accumulation of cyclin D3 and subsequent proliferation of PAX2(+) progenitors, inversely correlating with the loss of nephron progenitors seen in the Bmp7-null kidney. Activation of Jun and ATF2 is severely diminished in Bmp7-null kidneys, providing an important in vivo correlate. BMP7 thus promotes proliferation directly in nephron progenitors by activating the JNK signaling circuitry.

Angptl4 maintains in vivo repopulation capacity of CD34+ human cord blood cells.

OBJECTIVES: Methods to expand hematopoietic stem cells (HSCs) ex vivo encompass an attractive approach that would substantially broaden the clinical applicability of HSCs derived from cord blood (CB). Recently, members of the angiopoietin-like (Angptl) family of growth factors were shown to expand both murine and human HSCs. Specifically, Angptl5 has been implicated in the expansion of human NOD/SCID-repopulating cells (SRCs) ex vivo. Here, we sought to evaluate the potential of additional Angptls to expand human SRCs from CB. Additionally, the purpose of this study was to evaluate the reproducibility of Angptl-mediated expansion of SRCs across independent experiments. METHODS: Human CD34(+) cells from CB were cultured in vitro for eleven or 8 d in the presence or absence of Angptls. The reconstitution capacity of expanded cells was subsequently measured in vivo by transplantation into NOD/SCID or NSG mice and compared with that of uncultured cells. RESULTS: We report here that Angptl4 functions to maintain SRC activity of CD34(+) CB-derived cells ex vivo as assayed in NOD/SCID and NSG mice. However, all Angptls tested, including Angptl1, Angptl4, and Angptl5, were associated with variation between experiments. CONCLUSION: Our findings indicate that Angptl4 and Angptl5 can lead to increased engraftment capacity of SRCs, but more frequently, these factors are associated with maintenance of SRC activity during ex vivo culture. Thus, Angptl-mediated expansion of SRCs ex vivo is associated with more interexperimental variation than previously thought. We conclude that Angptls would be useful in instances where there is a need to maintain HSCs ex vivo, such as during transduction for gene therapy applications.

An in vivo reporter of BMP signaling in organogenesis reveals targets in the developing kidney.

BACKGROUND: Bone morphogenetic proteins (BMPs) regulate essential processes during organogenesis, and a functional understanding of these secreted proteins depends on identification of their target cells. In this study, we generate a transgenic reporter for organogenesis studies that we use to define BMP pathway activation in the developing kidney. RESULTS: Mouse strains reporting on BMP pathway activation were generated by transgenically expressing beta-galactosidase under the control of BMP responsive elements from Id1. Reporter expression corresponds well with immunoassays for pathway activation in all organs studied, validating the model. Using these reporters we have generated a detailed map of cellular targets of BMP signaling in the developing kidney. We find that SMAD dependent BMP signaling is active in collecting duct trunks, but not tips. Furthermore, glomerular endothelial cells, and proximal nephron tubules from the renal vesicle stage onward show pathway activation. Surprisingly, little activation is detected in the nephrogenic zone of the kidney, and in organ culture BMP treatment fails to activate SMAD dependent BMP signaling in nephron progenitor cells. In contrast, signaling is efficiently induced in collecting duct tips. CONCLUSION: Transgenic reporters driven by control elements from BMP responsive genes such as Id1 offer significant advantages in sensitivity and consistency over immunostaining for studies of BMP pathway activation. They also provide opportunities for analysis of BMP signaling in organ and primary cell cultures subjected to experimental manipulation. Using such a reporter, we made the surprising finding that SMAD dependent BMP signaling is inactive in nephron progenitors, and that these cells are refractory to activation by applied growth factors. Furthermore, we find that the BMP pathway is not normally active in collecting duct tips, but that it can be ectopically activated by BMP treatment, offering a possible explanation for the inhibitory effects of BMP treatment on collecting duct growth and branching.

Endoglin is not critical for hematopoietic stem cell engraftment and reconstitution but regulates adult erythroid development.

Endoglin is a transforming growth factor-beta (TGF-beta) accessory receptor recently identified as being highly expressed on long-term repopulating hematopoietic stem cells (HSC). However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglin's role in HSC biology: one that efficiently knocks down expression via lentiviral-driven short hairpin RNA and another that uses retroviral-mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin-suppressed myeloid progenitors (colony-forming unit-granulocyte macrophage) displayed a higher degree of sensitivity to TGF-beta-mediated growth inhibition, whereas endoglin-overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short-term or long-term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast-forming unit-erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.

Signaling via Smad2 and Smad3 is dispensable for adult murine hematopoietic stem cell function in vivo.

Transforming growth factor-ß (TGFß) is a member of a large family of polypeptide growth factors. TGFß signals mainly through the intracellular proteins Smad2 and Smad3, which are highly similar in amino acid sequence identity. A number of studies have shown that these proteins, dependent on context, have distinct roles in the TGFß signaling pathway. TGFß is one of the most potent inhibitors of hematopoietic stem and progenitor cell proliferation in vitro, but its role in hematopoiesis in vivo is still being determined. To circumvent possible redundancies at the receptor level and to address specifically the role of the Smad circuitry downstream of TGFß and activin in hematopoiesis, we studied the effect of genetically deleting both Smad2 and Smad3 in adult murine hematopoietic cells. Indeed, TGFß signaling is impaired in vitro in primitive bone marrow (BM) cells of Smad2 and Smad3 single knockout models. However, blood parameters appear normal under steady state and in the transplantation setting. Interestingly, upon deletion of both Smad2 and Smad3 in vivo, mice quickly develop a lethal inflammatory disease, suggesting that activin/TGFß signaling is crucial for immune cell homeostasis in the adult context. Furthermore, concurrent deletion of Smad2 and Smad3 in BM cells in immune-deficient nude mice did not result in any significant alterations of the hematopoietic system. Our findings suggest that Smad2 and Smad3 function to mediate crucial aspects of the immunoregulatory properties of TGFß, but are dispensable for any effect that TGFß has on primitive hematopoietic cells in vivo.

The stem cell regulator PEDF is dispensable for maintenance and function of hematopoietic stem cells.

Pigment epithelium derived factor (PEDF), a ubiquitously expressed 50 kDa secreted glycoprotein, was recently discovered to regulate self-renewal of neural stem cells and have a supportive effect on human embryonic stem cell growth. Here, we analyzed expression of PEDF in the murine hematopoietic stem cell (HSC) compartments and found that PEDF is highly expressed in primary long-term HSCs. Therefore, we characterized the hematopoietic system in a knockout mouse model for PEDF and using this model we surprisingly found that PEDF is dispensable for HSC regulation. PEDF knockout mice exhibit normal hematopoiesis in steady state conditions and the absence of PEDF lead to normal regeneration capacity in a serial competitive transplantation setting. Additionally, PEDF-deficient cells exhibit unaltered lineage distribution upon serial transplantations. When human cord blood stem and progenitor cells were cultured in media supplemented with recombinant PEDF they did not show changes in growth potential. Taken together, we report that PEDF is not a critical regulatory factor for HSC function during regeneration in vivo or growth of human stem/progenitor cells in vitro.

Quiescence of hematopoietic stem cells and maintenance of the stem cell pool is not dependent on TGF-beta signaling in vivo.

OBJECTIVE: Maintained quiescence of hematopoietic stem cells (HSCs) is of critical importance to prevent premature exhaustion of the stem cell pool under conditions of hematopoietic stress. The growth inhibitory cytokine transforming growth factor beta (TGF-beta) has been shown to play a critical role in maintaining quiescence of HSCs in vitro. Here, we have used conditional knockout mice for the TGF-beta type I receptor (TbetaRI) to ask whether the naturally quiescent state of HSCs in vivo is dependent on TGF-beta signaling and thus whether TGF-beta serves as a protective factor for the stem cell pool during conditions of stress. METHODS: TbetaRI null and control bone marrow chimeras were subjected to repeated treatments with the cell cycle-specific cytotoxic drug 5-fluorouracil (5-FU) and surviving HSCs were assayed by competitive transplantation experiments. Exhaustion of stem cells was provoked by serially transplanting TGF-beta signaling-deficient as well as normal BM cells into lethally irradiated recipients, which were monitored for survival. RESULTS: Surprisingly, we found that TGF-beta receptor-deficient HSCs have similar susceptibility, compared to controls, to repeated 5-FU treatments, indicative of normally maintained quiescence in these cells. Likewise, hematopoietic failure occurred at similar stages in serially transplanted recipients of TbetaRI null and control BM, respectively, demonstrating normal consumption of the stem cell pool during hematopoietic stress. CONCLUSIONS: These findings clearly demonstrate that, despite a key role in vitro, TGF-beta does not provide the necessary signal that induces the quiescent state of HSCs and maintains the stem cell pool in vivo.

Isolation and culture of cells from the nephrogenic zone of the embryonic mouse kidney.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering naïve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated http://www.gudmap.org/, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

Signaling pathways governing stem-cell fate.

Hematopoietic stem cells (HSCs) are historically the most thoroughly characterized type of adult stem cell, and the hematopoietic system has served as a principal model structure of stem-cell biology for several decades. However, paradoxically, although HSCs can be defined by function and even purified to near-homogeneity, the intricate molecular machinery and the signaling mechanisms regulating fate events, such as self-renewal and differentiation, have remained elusive. Recently, several developmentally conserved signaling pathways have emerged as important control devices of HSC fate, including Notch, Wingless-type (Wnt), Sonic hedgehog (Shh), and Smad pathways. HSCs reside in a complex environment in the bone marrow, providing a niche that optimally balances signals that control self-renewal and differentiation. These signaling circuits provide a valuable structure for our understanding of how HSC regulation occurs, concomitantly with providing information of how the bone marrow microenvironment couples and integrates extrinsic with intrinsic HSC fate determinants. It is the focus of this review to highlight some of the most recent developments concerning signaling pathways governing HSC fate.

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.

Smad7 promotes self-renewal of hematopoietic stem cells.

The Smad-signaling pathway downstream of the transforming growth factor-beta superfamily of ligands is an evolutionarily conserved signaling circuitry with critical functions in a wide variety of biologic processes. To investigate the role of this pathway in the regulation of hematopoietic stem cells (HSCs), we have blocked Smad signaling by retroviral gene transfer of the inhibitory Smad7 to murine HSCs. We report here that the self-renewal capacity of HSCs is promoted in vivo upon blocking of the entire Smad pathway, as shown by both primary and secondary bone marrow (BM) transplantations. Importantly, HSCs overexpressing Smad7 have an unperturbed differentiation capacity as evidenced by normal contribution to both lymphoid and myeloid cell lineages, suggesting that the Smad pathway regulates self-renewal independently of differentiation. Moreover, phosphorylation of Smads was inhibited in response to ligand stimulation in BM cells, thus verifying impairment of the Smad-signaling cascade in Smad7-overexpressing cells. Taken together, these data reveal an important and previously unappreciated role for the Smad-signaling pathway in the regulation of self-renewal of HSCs in vivo.

TGF-beta signaling-deficient hematopoietic stem cells have normal self-renewal and regenerative ability in vivo despite increased proliferative capacity in vitro.

Studies in vitro implicate transforming growth factor beta (TGF-beta) as a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. In vivo studies have been hampered by early lethality of knock-out mice for TGF-beta isoforms and the receptors. To directly assess the role of TGF-beta signaling for hematopoiesis and hematopoietic stem cell (HSC) function in vivo, we generated a conditional knock-out model in which a disruption of the TGF-beta type I receptor (T beta RI) gene was induced in adult mice. HSCs from induced mice showed increased proliferation recruitment when cultured as single cells under low stimulatory conditions in vitro, consistent with an inhibitory role of TGF-beta in HSC proliferation. However, induced T beta RI null mice show normal in vivo hematopoiesis with normal numbers and differentiation ability of hematopoietic progenitor cells. Furthermore HSCs from T beta RI null mice exhibit a normal cell cycle distribution and do not differ in their ability long term to repopulate primary and secondary recipient mice following bone marrow transplantation. These findings challenge the classical view that TGF-beta is an essential negative regulator of hematopoietic stem cells under physiologic conditions in vivo.