Lineage Fate Decisions Research Articles

Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging

Neural stem/progenitor cell (NSPC) proliferation and self-renewal, as well as insult-induced differentiation, decrease markedly with age. The molecular mechanisms responsible for these declines remain unclear. Here, we show that levels of NAD(+) and nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in mammalian NAD(+) biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD(+) levels. Nampt is the main source of NSPC NAD(+) levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical in oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC-mediated oligodendrogenesis upon insult. These phenotypes recapitulate defects in NSPCs during aging, giving rise to the possibility that Nampt-mediated NAD(+) biosynthesis is a mediator of age-associated functional declines in NSPCs.

Adult, but not neonatal, human lymphoid progenitors respond to TLR9 ligation by producing functional NK-like cells

Remarkable progress has been made in characterizing factors controlling lineage fate decisions of primitive progenitors that initiate the lymphoid program in bone marrow. However, the understanding of neonatal/adult differences in environmental signals that influence differentiation pathway stability is still incomplete. Our recent findings suggest that Toll-like receptors provide a mechanism for producing cells of the innate immune system from early stages of lymphoid development in mice. We now show that both human early multilymphoid progenitors and more differentiated lymphoid progenitors from normal adult bone marrow express TLR9. Furthermore, they respond to its ligation by upregulating the expression of IL-15Rβ (CD122) and accelerating the production of functional natural killer (NK)-like cells. Proliferation of the presumed equivalent progenitor cells from umbilical cord blood was stimulated by CpG-containing oligonucleotides or herpes simplex virus, but the already robust NK-cell formation was unchanged. This new information adds to other known differences between neonatal and adult lymphoid progenitors and suggests only the latter replenish innate NK-like cells in response to Toll-like receptor agonists.

The Bright Side of Hematopoiesis: Regulatory Roles of ARID3a/Bright in Human and Mouse Hematopoiesis

ARID3a/Bright is a DNA-binding protein that was originally discovered for its ability to increase immunoglobulin transcription in antigen-activated B cells. It interacts with DNA as a dimer through its ARID, or A/T-rich interacting domain. In association with other proteins, ARID3a increased transcription of the immunoglobulin heavy chain and led to improved chromatin accessibility of the heavy chain enhancer. Constitutive expression of ARID3a in B lineage cells resulted in autoantibody production, suggesting its regulation is important. Abnormal ARID3a expression has also been associated with increased proliferative capacity and malignancy. Roles for ARID3a in addition to interactions with the immunoglobulin locus were suggested by transgenic and knockout mouse models. Over-expression of ARID3a resulted in skewing of mature B cell subsets and altered gene expression patterns of follicular B cells, whereas loss of function resulted in loss of B1 lineage B cells and defects in hematopoiesis. More recent studies showed that loss of ARID3a in adult somatic cells promoted developmental plasticity, alterations in gene expression patterns, and lineage fate decisions. Together, these data suggest new regulatory roles for ARID3a. The genes influenced by ARID3a are likely to play pivotal roles in lineage decisions, highlighting the importance of this understudied transcription factor.

INKT cells require TSC1 for terminal maturation and effector lineage fate decisions

Terminal maturation of invariant NKT (iNKT) cells from stage 2 (CD44+NK1.1-) to stage 3 (CD44+NK1.1+) is accompanied by a functional acquisition of a predominant IFN-γ-producing (iNKT-1) phenotype; however, some cells develop into IL-17-producing iNKT (iNKT-17) cells. iNKT-17 cells are rare and restricted to a CD44+NK1.1- lineage. It is unclear how iNKT terminal maturation is regulated and what factors mediate the predominance of iNKT-1 compared with iNKT-17. The tumor suppressor tuberous sclerosis 1 (TSC1) is an important negative regulator of mTOR signaling, which regulates T cell differentiation, function, and trafficking. Here, we determined that mice lacking TSC1 exhibit a developmental block of iNKT differentiation at stage 2 and skew from a predominantly iNKT-1 population toward a predominantly iNKT-17 population, leading to enhanced airway hypersensitivity. Evaluation of purified iNKT cells revealed that TSC1 promotes T-bet, which regulates iNKT maturation, but downregulates ICOS expression in iNKT cells by inhibiting mTOR complex 1 (mTORC1). Furthermore, mice lacking T-bet exhibited both a terminal maturation defect of iNKT cells and a predominance of iNKT-17 cells, and increased ICOS expression was required for the predominance of iNKT-17 cells in the population of TSC1-deficient iNKT cells. Our data indicate that TSC1-dependent control of mTORC1 is crucial for terminal iNKT maturation and effector lineage decisions, resulting in the predominance of iNKT-1 cells.

Snail1-dependent control of embryonic stem cell pluripotency and lineage commitment

Embryonic stem cells (ESCs) exhibit the dual properties of self-renewal and pluripotency as well as the ability to undergo differentiation that gives rise to all three germ layers. Wnt family members can both promote ESC maintenance and trigger differentiation while also controlling the expression of Snail1, a zinc-finger transcriptional repressor. Snail1 has been linked to events ranging from cell cycle regulation and cell survival to epithelial-mesenchymal transition (EMT) and gastrulation, but its role in self-renewal, pluripotency or lineage commitment in ESCs remains undefined. Here we demonstrate using isogenic pairs of conditional knockout mouse ESCs, that Snail1 exerts Wnt- and EMT independent control over the stem cell transcriptome without affecting self-renewal or pluripotency-associated functions. By contrast, during ESC differentiation, an endogenous Wnt-mediated burst in Snail1 expression regulates neuroectodermal fate while playing a required role in epiblast stem cell exit and the consequent lineage fate decisions that define mesoderm commitment.

Measuring microRNA expression in mouse hematopoietic stem cells.

MicroRNAs (miRNAs) are important regulators of diverse biologic processes. In the hematopoietic system, miRNAs have been shown to regulate lineage fate decisions, mature immune effector cell function, apoptosis, and cell cycling, and a more limited number of miRNAs has been shown to regulate hematopoietic stem cell (HSC) self-renewal. Many of these miRNAs were initially identified as candidate regulators of HSC function by comparing miRNA expression in hematopoietic stem and progenitors cells (HSPCs) to their mature progeny. While the measurement of miRNA expression in rare cell populations such as HSCs poses practical challenges due to the low amount of RNA present, a number of techniques have been developed to measure miRNAs in small numbers of cells. Here, we describe our protocol for measuring miRNAs in purified mouse HSCs using a highly sensitive real-time quantitative PCR strategy that utilizes microfluidic array cards containing pre-spotted TaqMan probes that allows the detection of mature miRNAs in small reaction volumes. We also describe a simple data analysis method to evaluate miRNA expression profiling data using an open-source software package (HTqPCR) using mouse HSC miRNA profiling data generated in our lab.

Identification Of Bipotential Lin-CD34+CD38- Integrin α2- Erythrocyte-Megakaryocyte Progenitors In Human Bone Marrow

Human hematopoietic progenitor cells with megakaryocyte and erythroid commitment have been defined as the Lin-CD34+CD38+CD123-CD45RA- and in addition, as CD110+ (Manz et al. 2002; Edvardsson et al. 2006). However, previous colony assays have shown that bipotential megakaryocyte-erythroid progenitors (MEPs) also reside in the stem cell-enriched CD34+CD38- fraction in bone marrow (BM) (Debili et al., 1996). So far, the phenotype of this MEP population has remained obscure.We have here studied the expression of integrin α2 chain in normal human bone marrow CD34+CD38- stem and progenitors. Integrin α2 chain was expressed in most BM CD34+CD38- cells (96.3±3.8; mean±SD), in contrast to a more restricted expression in cord blood CD34+CD38- cells (Wong, et al. 2013). In order to investigate the potential functional differences associated with the expression of integrin α2 on BM CD34+CD38- cells, we characterized the integrin α2+ and α2- cell populations by in vitro LTC-IC, single cell colony assays and gene expression analysis. The LTC-IC progenitors were exclusively integrin α2+. In contrast, the integrin α2- fraction was highly enriched in erythroid BFU-E progenitors. In single cell colony assay in methylcellulose, 21.96% of single Lin-CD34+CD38- integrin α2- cells gave rise to BFU-E, as compared to 0.65% of integrin α2+ cells. Likewise, the frequency of megakaryocyte colony forming cells, analyzed by a serum free collagen based culture system, was significantly higher in the integrin α2- fraction, suggesting an existence of the bipotential MEP in the integrin α2- fraction.To confirm the existence of bipotential MEPs in the Lin-CD34+CD38-CD45RA- integrin α2- cell population, we performed single cell clonogenic assay using serum free medium with cytokines (SCF, TPO, IL-3 and EPO) in Terasaki plates. At day 12-14 of culture 30-40% of the cells showed clonal growth. The erythroid and megakaryocytic differentiation of the single cell derived clones was assessed by their immunophenotype and morphology. Interestingly, FACS analysis of the 32 clones indicated that all of them contained erythroid cells (CD235+CD41-). Most importantly, 22 of the 32 clones contained both erythroid and megakaryocytic cells (CD235-CD41+), showing a high degree of bipotential MEP activity in the integrin α2- population. Minimal myeloid differentiation (CD15/CD33/CD66b+ cells <0.5%) was seen in only 3 of the 32 clones. During the differentiation the integrin α2 receptor was upregulated in CD41+ megakaryocytic cells but not in the CD235+ erythroid cells. Furthermore, 83 clones derived from single cells, including clones with very limited number of cells, were individually transferred to cytospin slides for evaluation of their morphology. The small clones invariably consisted of megakaryocytic cells. 48.19% of all clones analyzed were identified as bipotent MEPs. This finding was further supported by upregulation of HBD and downregulation of GATA3, HLF in the integrin α2- cells, revealed by gene expression analysis. Taken together, our data demonstrate a high frequency of bipotential MEP progenitors in adult BM Lin-CD34+CD38-CD45RA- integrin α2- cell fraction and suggest that BM Lin-CD34+CD38- integrin α2- cells are transcriptionally primed towards development into erythroid and megakaryocytic cells. The identification of this novel bipotential MEP population provides a means for further analysis on lineage fate decisions of primitive erythroid and megakaryocytic cells and may facilitate studies aiming to expand and differentiate these lineages for clinical transplantation and transfusion trials. Disclosures:Ekblom:Novartis and BMS: Honoraria.

Transcriptional fine-tuning of microRNA-223 levels directs lineage choice of human hematopoietic progenitors

MicroRNAs (miRNAs) regulate cell proliferation, differentiation and death during development and postnatal life. The expression level of mature miRNAs results from complex molecular mechanisms, including the transcriptional regulation of their genes. MiR-223 is a hematopoietic-specific miRNA participating in regulatory signaling networks involving lineage-specific transcription factors (TFs). However, the transcriptional mechanisms governing its expression levels and its functional role in lineage fate decision of human hematopoietic progenitors (HPCs) have not yet been clarified. We found that in CD34(+)HPCs undergoing unilineage differentiation/maturation, miR-223 is upregulated more than 10-fold during granulopoiesis, 3-fold during monocytopoiesis and maintained at low levels during erythropoiesis. Chromatin immunoprecipitation and promoter luciferase assays showed that the lineage-specific expression level of mature miR-223 is controlled by the coordinated binding of TFs to their DNA-responsive elements located in 'distal' and 'proximal' regulatory regions of the miR-223 gene, differentially regulating the transcription of two primary transcripts (pri-miRs). All this drives myeloid progenitor maturation into specific lineages. Accordingly, modulation of miR-223 activity in CD34(+)HPCs and myeloid cell lines significantly affects their differentiation/maturation into erythroid, granulocytic and monocytic/macrophagic lineages. MiR-223 overexpression increases granulopoiesis and impairs erythroid and monocytic/macrophagic differentiation. Its knockdown, meanwhile, impairs granulopoiesis and facilitates erythropoiesis and monocytic/macrophagic differentiation. Overall, our data reveal that transcriptional pathways acting on the differential regulation of two pri-miR transcripts results in the fine-tuning of a single mature miRNA expression level, which dictates the lineage fate decision of hematopoietic myeloid progenitors.

Gli3-mediated hedgehog inhibition in human pluripotent stem cells initiates and augments developmental programming of adult hematopoiesis

AbstractPrograms that control early lineage fate decisions and transitions from embryonic to adult human cell types during development are poorly understood. Using human pluripotent stem cells (hPSCs), in the present study, we reveal reduction of Hedgehog (Hh) signaling correlates to developmental progression of hematopoiesis throughout human ontogeny. Both chemical- and gene-targeting–mediated inactivation of Hh signaling augmented hematopoietic fate and initiated transitions from embryonic to adult hematopoiesis, as measured by globin regulation in hPSCs. Inhibition of the Hh pathway resulted in truncation of Gli3 to its repressor, Gli3R, and was shown to be necessary and sufficient for initiating this transition. Our results reveal an unprecedented role for Hh signaling in the regulation of adult hematopoietic specification, thereby demonstrating the ability to modulate the default embryonic programs of hPSCs.

Stem Cell Dynamics and Heterogeneity: Implications for Epidermal Regeneration and Skin Cancer

The skin epithelium undergoes constant renewal, a process that is driven by stem cells (SCs) localising to the interfollicular epidermis and different regions of the hair follicle. Over the last years, tremendous progress has been made to unravel the physiological function of distinct stem and progenitor cell populations by using genetic lineage tracing in vivo, transplantation, clonogenicity approaches and live cell imaging. It turned out that these cell compartments constitute heterogeneous SC pools and that individual SCs respond differently to various signals sent by the microenvironment. Recent genetic manipulation experiments and elegant mouse models have shed light on the signalling pathways being crucial for self-renewal and lineage fate decisions during tissue homeostasis. Here, we summarise current concepts of SC function in mammalian skin and focus on the dynamic behaviour of SCs during morphogenesis and tissue regeneration of the skin epithelium. Clearly, understanding the cellular and molecular mechanisms of SC regulation and function during tissue homeostasis has enormous impact on our view of the pathogenesis of various skin diseases and will be beneficial for regenerative medicine. Recent experiments suggest an important role of tissue SCs in the process of skin tumour initiation and progression. For the future, the genuine challenge is to further dissect SC function in pathophysiological settings and to translate our knowledge to design novel efficient therapeutic strategies for treatment of cutaneous cancer.

Stem Cell Dynamics and Heterogeneity: Implications for Epidermal Regeneration and Skin Cancer

The skin epithelium undergoes constant renewal, a process that is driven by stem cells (SCs) localising to the interfollicular epidermis and different regions of the hair follicle. Over the last years, tremendous progress has been made to unravel the physiological function of distinct stem and progenitor cell populations by using genetic lineage tracing in vivo, transplantation, clonogenicity approaches and live cell imaging. It turned out that these cell compartments constitute heterogeneous SC pools and that individual SCs respond differently to various signals sent by the microenvironment. Recent genetic manipulation experiments and elegant mouse models have shed light on the signalling pathways being crucial for self-renewal and lineage fate decisions during tissue homeostasis. Here, we summarise current concepts of SC function in mammalian skin and focus on the dynamic behaviour of SCs during morphogenesis and tissue regeneration of the skin epithelium. Clearly, understanding the cellular and molecular mechanisms of SC regulation and function during tissue homeostasis has enormous impact on our view of the pathogenesis of various skin diseases and will be beneficial for regenerative medicine. Recent experiments suggest an important role of tissue SCs in the process of skin tumour initiation and progression. For the future, the genuine challenge is to further dissect SC function in pathophysiological settings and to translate our knowledge to design novel efficient therapeutic strategies for treatment of cutaneous cancer.

Prospective Isolation of the Earliest Common Myeloid Progenitor in Adult Hematopoiesis

Abstract 768In murine hematopoiesis, hematopoietic stem cells (HSCs) and multi-potent progenitors (MPPs) have been identified within the LSK (Lin− Sca-1+ c-kit+) fraction of bone marrow cells (Morrison SJ and Weissman IL Immunity 1994). We have proposed that the first commitment step at the myeloid versus lymphoid bifurcation occurred outside the LSK fraction, where myeloid or lymphoid lineage-committed progenitors such as common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) are prospectively isolated (Akashi et al Nature 2000). However, recent studies revealed that the initial commitment step might occur within LSK fraction. Adolfsson et al showed that a fraction of LSK cells expressing Flt3 at a high level have lost megakaryocyte/erythroid (MegE) potential and were largely primed for granulocyte/monocyte (GM) and lymphoid cells. This population was named lymphoid-primed multipotent progenitors (LMPPs) (Adolfsson et al Cell 2005). In our hands, by utilizing mice harboring a fluorescent reporter for GATA-1 transcription factor, we demonstrated that the initial upregulation of GATA-1 transcription factor was observed within MPP population, which was defined as CD34+ LSK fraction, and GATA-1+ MPPs were capable of generating only myelo-erythroid cells but lacked lymphoid potential (Arinobu et al Cell Stem Cell 2007). This result strongly suggested that the initial myelo-erythroid commitment occurs at the MPP stage.To isolate the earliest myelo-erythroid LSK progenitors in normal mice without utilizing the GATA-1 reporter system, we conducted expression profiling of GATA-1+ MPP population by cDNA microarray analyses and following validation by FACS analyses. We found that a cell-surface antigen CD41 was specifically expressed at a high level in GATA-1+ MPP cells, indicating that CD41hi MPP cells might be the corresponding population to GATA-1+ MPP cells.CD41hi MPPs gave rise exclusively to GM and MegE colonies in vitro, but no lymphoid colonies were observed even under lymphoid-inducible conditions such as co-culture with OP9 or OP9-DL1 stromal cell layer. In vivo, CD41hi MPPs showed differentiation potential into GM cells, erythroid cells and platelets, lacking B or T lymphoid cell-producing potential in congenic transplantation assay. While the GM cell-reconstitution potential of LMPPs and CMPs peaked around day10 and day4 after transplantation, respectively, CD41hi MPPs had their peak around 3 weeks and relatively long-lasting reconstitution potential, presumably reflecting their immaturity. To further evaluate differentiation potential more directly and quantitatively, CD41hi MPPs with LMPPs or CMPs were injected together into one individual recipient mouse. In this head-to-head competitive assay system, CD41hi MPPs generated a plenty of mature GM cells, whose numbers were nearly ten times of those produced from the original CMPs or LMPPs in a reconstitution setting. Thus CD41hi MPPs possess potent restricted-lineage differentiation potential into GM and MegE lineages.In addition, the gene expression analysis by single-cell quantitative real-time PCR and cDNA microarray revealed that the CD41hi MPP population expresses both GM- and MegE-affiliated genes, but not lymphoid genes, reflecting their lineage restriction. More importantly, each single CD41hi MPP cell expresses either one or both of GATA-1 and PU.1 at a low level, suggesting that the promiscuous expression of these transcription factors might play a critical role in myelo-erythroid lineage commitment (Miyamoto et al Developmental Cell 2002).Furthermore, to evaluate the functional importance of CD41hi MPP population, we created the systemic infectious model mice, mimicking the severe peritonitis. CD41hi MPP pool as well as GMP expands dramatically in response to an increased demand for GM cells, while LMPP does not; thus, CD41hi MPPs compose physiologically important pathway in myelo-erythropoiesis.Accordingly, CD41hi MPP population might represent the earliest branch point for myelo-erythroid development, which resides upstream of the conventional CMP. Based on these data, we propose to redefine the true CMP as CD41hi MPP population. This CD41hi earliest myelo-erythroid progenitor should be useful to investigate the molecular mechanisms for hematopoietic lineage fate decision. Disclosures:No relevant conflicts of interest to declare.

The role of ID3 in iNKT cell development (115.12)

Abstract Invariant NKT cells comprise an unconventional T cell population with innate characteristics. Like the conventional αβ T cell lineage, they develop in the thymus from DP precursors after receiving TCR-dependent positive selection signals, although utilizing different pathways. iNKT cells further mature through a series of differentiation stages and acquire their effector properties intrathymically. The ID3 protein is required to reduce E protein activity for proper selection of DP progenitors towards conventional thymocyte lineages. Here, we investigate the requirement for ID3 in the thymic development of iNKT lineage, in vivo. We show that the frequency of iNKT cells in the Id3-/- thymus is higher than in WT littermates, in a cell-intrinsic manner. Id3-deficient post-selected DP cells express higher levels of the canonical Vα14-Jα18 TCRα chain transcripts. Additionally, the transcription factor PLZF, a molecular determinant of the NKT lineage, is expressed in a subset of post-selected DP cells in the Id3-/- mice. However, further thymic iNKT differentiation is blocked at an immature stage. Taken together these results suggest that ID3 restricts the innate T cell potential of DP precursors thus contributing to the lineage fate decisions at the DP stage. However, ID3 is necessary for the completion of the innate T cell program during iNKT differentiation. Ongoing gene expression analysis will identify potent E protein target genes essential for NKT development

Evidence for the divergence of innate and adaptive T-cell precursors before commitment to the αβ and γδ lineages

AbstractIn addition to adaptive T cells, the thymus supports the development of unconventional T cells such as natural killer T (NKT) and CD8αα intraepithelial lymphocytes (IELs), which have innate functional properties, particular antigenic specificities, and tissue localization. Both conventional and innate T cells are believed to develop from common precursors undergoing instructive, TCR-mediated lineage fate decisions, but innate T cells are proposed to undergo positive instead of negative selection in response to agonistic TCR signals. In the present study, we show that, in contrast to conventional αβT cells, innate αβT cells are not selected against functional TCRγ rearrangements and express TCRγ mRNA. Likewise, in contrast to the majority of γδT cells, thymic innate γδT cells are not efficiently selected against functional TCRβ chains. In precursors of conventional T cells, autonomous TCR signals emanating from the pre-TCR or γδTCR in the absence of ligand mediate selection against the TCR of the opposite isotype and αβ/γδ lineage commitment. Our data suggest that developing innate T cells ignore such signals and rely solely on agonistic TCR interactions. Consistently, most innate T cells reacted strongly against autologous thymocytes. These results suggest that innate and adaptive T-cell lineages do not develop from the same pool of precursors and potentially diverge before αβ/γδ lineage commitment.

MEIS1 Regulates Early Erythroid and Megakaryocytic Lineage Decision

Abstract 2365The transcription factor MEIS1 is essential for definitive hematopoiesis in the mouse embryo and during zebrafish development. MEIS1 deficient mice die at mid gestation, mainly due to a poorly developed HSC compartment and a reduction in myeloerythroid colony forming cells. We have previously shown by comparative transcriptional profiling of all human adult blood cell lineages that MEIS1 is specifically expressed in megakaryocytes (MK). It is however not yet known if and where MEIS1 participates in the human hematopoietic transcription factor hierarchy.We established that primary hematopoietic stem and progenitor (CD34+) cells in human bone marrow had high expression of MEIS1, which was comparable to CD34+ cells in cord blood (CB) and even higher (p<0.01) in mobilized peripheral blood (MPB). Compared to other bone marrow progenitor subsets, MEIS1 expression was highest in hematopoietic stem cells (HSC:CD34+/CD38−) and significantly reduced (p<0.01) in downstream granulocyte monocyte progenitors (GMP:CD34+/CD38+/CD110−/CD45RA+) and megakaryocyte-erythrocyte progenitors (MEP:CD34+/CD38+/CD110+/CD45RA−). We then analyzed the hematopoietic potential of CD34+ cells derived from CB or MPB, in which MEIS1 expression was silenced with short hairpins or overexpressed using lentiviral vectors. Cells were sorted for GFP expression at 48 hours after transduction and evaluated for colony forming potential. MEIS1 knockdown in human CD34+ cells resulted in a near absence of BFU-E, a 70% reduction in CFU-GM and a 25% reduction in CFU-MEG (p<0.01). When transduced CD34+ cells were cultured in suspension with EPO, SCF and IL-3 promoting differentiation towards erythroblasts or with TPO and IL-1β towards megakaryocytes, knockdown of MEIS1 significantly inhibited the proliferation of both erythroblasts and megakaryocytes (p<0.01). In addition, the expression of CD235a on erythroblasts was reduced by 60% (p<0.01) upon MEIS1 knockdown, whereas no changes were detected in the expression of CD41 on MKs. On the other hand, overexpression of MEIS1 altered lineage differentiation of human CD34+ cells without influencing the total outgrowth of colonies, inducing a 3-fold increase of BFU-E and a 2.5-fold increase in CFU-MEG at the expense of a 3-fold reduction in CFU-GM. Furthermore, since established erythroblast cultures were not affected by knockdown of MEIS1, we hypothesized that MEIS1 might regulate the erythroid/megakaryocytic lineage decision in the common myeloid progenitor (CMP). To test this hypothesis, we sorted CMPs (CD34+/CD38+/CD110−/CD45RA−), transduced the cells with MEIS1 and evaluated their colony forming potential. Indeed, overexpression of MEIS1 in CMP induced an increase in CFU-MEG and BFU-E and a striking absence of CFU-GM. When MEIS1 was overexpressed in GMP no differences were observed compared to the empty vector.In summary, MEIS1 expression in primary human progenitor cells is essential for the formation of myeloid cells, erythroblasts and megakaryocytes. However, a surplus of MEIS1 inhibits myeloid differentiation and enhances erythroid and megakaryocytic development. We therefore conclude that MEIS1 is a novel regulator for differentiation of human CMPs and that gene dosage of MEIS1 determines its lineage fate decisions. Disclosures:No relevant conflicts of interest to declare.

Satb1 Determines Hematopoietic Stem Cell Differentiation Toward the Lymphoid Lineages

Abstract 388There is accumulating evidence that combinations of transcription factors coordinately and sequentially regulate lymphopoiesis. Five transcription factors, PU.1, Ikaros, E2A, EBF, and Pax5 are known to be hierarchically involved in early steps in B-lineage differentiation. However, it remains unclear whether the initiation of lymphoid differentiation is regulated entirely by transcription factors in a hierarchical manner. A major goal of our study was to find key genes involved in specification of lymphoid fates. For this purpose, we compared gene expression profiles between hematopoietic stem cells (HSC) and early lymphoid progenitors (ELP), which were sorted from E14.5 fetal liver of Rag1/GFP knock-in heterozygous embryos. As a result, we found that the expression of Satb1, a global chromatin regulator, was significantly increased along the differentiation of HSC to ELP. To explore roles of Satb1 in early lymphoid specification, we performed transplantation experiments, injecting HSC isolated from bone marrow of 2 weeks old Satb1-null mice into wild-type (WT) mice. We observed that Satb1-null HSC could not reconstitute CD3+ T cells in lethally irradiated WT recipients. Indeed, CD3+ T lineage recoveries from Satb1-null HSC were decreased approximately 10-fold compared with WT HSC. On the other hand, we observed varied levels of reconstitution of the B lineage and no reduction in reconstitution of the myeloid lineage resulted from Satb1 ablation. These results demonstrate that expression of Satb1 in HSC is indispensable for lymphopoiesis, but not for myelopoiesis. Furthermore, our data indicate that abnormalities of lymphoid development observed in Satb1-null mice are intrinsic to Satb1-deficient HSC. Next we conducted over-expression experiments to define the role of Satb1 in lineage fate decisions of HSC. Limiting dilution assays in MS5 co-culture condition revealed 1 in 41 Satb1-transduced Flt3−Lineage−Sca1+c-Kit+ (LSK) cells produced B cells. However, only 1 in 143 control ones were lymphopoietic under these conditions. These results suggest that Satb1 affects the lineage fate of HSC and promotes their commitment to lymphoid cells. Next we examined if the exogenous expression of Satb1 promotes B lymphocyte growth from ES cells in the OP9 co-culture system. We established ES cell clones, which can be induced to express Satb1/GFP on removal of tetracycline (Tet) from the culture medium. Eight days after Tet deprivation, 22 % of GFP+ cells expressed CD45 and CD19. On the other hand, only 1% of GFP− cells expressed same cell-surface markers. A majority of the CD19+ cells in Satb1/GFP+ ES-derived cells were positive for CD11b/Mac1 and/or CD5, suggesting that B1-B lineage cells were produced. In addition, Igh rearrangement assay revealed that DH-JH recombination occurred in the Satb1/GFP+ ES-derived cells. These results indicate that Satb1 over-expression directs even ES cells to differentiate toward the lymphoid lineage.Lymphopoietic activity becomes compromised during aging. Accumulating evidence suggests that the earliest lymphoid progenitor pools proximal to HSC are deficient in aged bone marrow. It is likely that the down-regulation of genes mediating lymphoid specification and function is involved as a major cause. Satb1 has been listed in microarray panels as one of the genes that are down-regulated in aged HSC (Rossi et al., 2005). To confirm this observation, we sorted CD150+ LSK cells from bone marrow of 6-weeks or 2-years old mice and examined expressions of Satb1. As a result, the aged CD150+LSK cells showed reduced expression (about 6-fold) of Satb1 compared with these cells from young mice. We then examined whether Satb1 expression could restore the lymphopoietic activity of progenitors derived from aged mice. Satb1-transduced Rag1/GFP− LSK cells produced significantly higher percentages of B220+ cells than control cells. With respect to the recovered B-lineage cell counts, about 3-fold more B220+ Rag1/GFP+ Mac1−cells were obtained by Satb1transduction than mock transduction. These results demonstrate that Satb1 can at least partially restore the lymphopoietic activity of aged hematopoietic progenitors. In conclusion, our results indicate that Satb1 plays critical roles in producing lymphoid lineage from primitive stem/progenitor cells. Such activity in generating lymphoid cells may be of clinical significance and useful to overcome immuno-senescence. Disclosures:No relevant conflicts of interest to declare.

All Hematopoietic Cells Develop from Hematopoietic Stem Cells through Flk2/Flt3-Positive Progenitor Cells

While it is clear that a single hematopoietic stem cell(HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports onthe lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways invivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.

The WTX Tumor Suppressor Regulates Mesenchymal Progenitor Cell Fate Specification

WTX is an X-linked tumor suppressor targeted by somatic mutations in Wilms tumor, a pediatric kidney cancer, and by germline inactivation in osteopathia striata with cranial sclerosis, a bone overgrowth syndrome. Here, we show that Wtx deletion in mice causes neonatal lethality, somatic overgrowth, and malformation of multiple mesenchyme-derived tissues, including bone, fat, kidney, heart, and spleen. Inactivation of Wtx at different developmental stages and in primary mesenchymal progenitor cells (MPCs) reveals that bone mass increase and adipose tissue deficiency are due to altered lineage fate decisions coupled with delayed terminal differentiation. Specification defects in MPCs result from aberrant β-catenin activation, whereas alternative pathways contribute to the subsequently delayed differentiation of lineage-restricted cells. Thus, Wtx is a regulator of MPC commitment and differentiation with stage-specific functions in inhibiting canonical Wnt signaling. Furthermore, the constellation of anomalies in Wtx null mice suggests that this tumor suppressor broadly regulates MPCs in multiple tissues.

54 Terminal effector memory T cell expansion: Biomarker for lenalidomide resistance in myelodysplastic syndrome

A hierarchical hematopoietic development with myeloid versus lymphoid bifurcation has been proposed downstream of the multipotent progenitor (MPP) stage, based on prospective isolation of progenitors capable of generating only myeloerythroid cells (common myeloid progenitor, CMP) or only lymphocytes (common lymphoid progenitor, CLP). By utilizing GATA-1 and PU.1 transcription factor reporters, here we identified progenitor populations that are precursors for either CMPs or CLPs. Two independent populations expressing either GATA-1 or PU.1 resided within the CD34+Sca-1+c-Kit+ MPP fraction. The GATA-1+ MPP displayed potent myeloerythroid potential without giving rise to lymphocytes, whereas the PU.1+ MPP showed granulocyte/monocyte/lymphoid-restricted progenitor activity without megakaryocyte/erythroid differentiation. Furthermore, GATA-1+ and PU.1+ MPPs possessed huge expansion potential and differentiated into the original CMPs and CLPs, respectively. Thus, the reciprocal activation of GATA-1 and PU.1 primarily organizes the hematopoietic lineage fate decision to form the earliest hematopoietic branchpoint that comprises isolatable myeloerythroid and myelolymphoid progenitor populations.

The erythroid/myeloid lineage fate paradigm takes a new player

This issue of The EMBO Journal features a new paradigm for cell lineage commitment. By studying early haematopoiesis in zebrafish, Roger Patient and colleagues characterize the Tif1 gamma-ligase as a context-specific regulator of Gata1/PU1-driven fate decisions.