Definitive Hematopoietic Stem Cells Research Articles

Lymphoid, Long-Term Repopulating, and Endothelial Potential of the Embryonic Hemangioblast.

Hematopoietic and endothelial cells are thought to arise from a common progenitor termed the hemangioblast. Direct evidence for the hemangioblast was first obtained from embryonic stem cells differentiated in vitro as embryoid bodies (EBs). Flk-1+ cells from early EBs generate colonies in response to VEGF and SCF (the BL-CFC assay) which can be replated to give secondary hematopoietic or endothelial cells. Bipotent BL-CFCs have also recently been derived from the posterior primitive streak of neural plate stage mouse embryos. However, a direct relationship between the early embryonic hemangioblast defined by the BL-CFC assay and the hematopoietic stem cell remains unproven. Hemangioblast-derived hematopoiesis in vitro is transient and restricted to myelo-erythroid differentiation. Lymphoid potential and long-term repopulation, two hallmarks of the definitive hematopoietic stem cell, have eluded detection to date. Previous work has shown that the homeodomain transcription factor, HoxB4, by enhancing self-renewal in vitro, can reveal latent definitive HSC activity of transient embryonic hematopoietic progenitors. Using an ES cell line with doxycycline-inducible HoxB4 expression, we have investigated the definitive hematopoietic and endothelial potential of individual hemangioblast colonies. BL-CFC numbers were unaffected by HoxB4 expression during EB differentiation, however they were increased threefold by induction during the BL-CFC assay. By replating one half of the cells from an individual blast colony in endothelial medium and the other half on an OP9 monolayer with hematopoietic cytokines, we show that the majority (60%) of HoxB4-induced BL-CFCs are bipotent. HoxB4 expression was compatible with endothelial differentiation and allowed exponential expansion of hematopoietic progenitors on OP9 cocultures. When switched to OP9-DL1 with lymphoid cytokines, T-lymphopoiesis was observed characterized by CD25 expression followed by CD4, CD8, and CD3epsilon expression. To assay long-term repopulation, individual blast colonies were picked and divided into endothelial medium and OP9 monolayers. The hematopoietic arms of colonies defined retrospectively to have been bipotent (endothelial differentiation was observed in vitro) were transplanted into sublethally irradiated Rag2; gamma-c; CD45.1 immunodeficient mice. Mice with long-term hematopoietic engraftment were identified by the presence of CD45.2 cells in peripheral blood 3 months post-transplant. Lymphoid and myeloid contribution was evaluated by costaining with Gr-1, B220, CD19, CD4, and CD8. The donor-derived component of these hematopoietic chimeras, including their entire lymphoid arm (approximately 1/3 of engrafted mice showed lymphoid differentiation) is by definition clonally derived from a single hemangioblast. These results clearly show that the embryonic hemangioblast is not intrinsically limited in its hematopoietic potential. Under conditions that favor self-renewal, lymphoid differentiation and long-term repopulation become evident, revealing the link between endothelial development and definitive hematopoiesis at the clonal level.

Novel binding partners of Ldb1 are required for haematopoietic development

Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. We identify a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although we originally identified the complex in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 we postulate that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells.

The differentiation program of embryonic definitive hematopoietic stem cells is largely α4 integrin independent

Previous analyses of the roles of alpha4 integrins in hematopoiesis by other groups have led to conflicting evidence. alpha4 integrin mutant cells developing in [alpha4 integrin(-/-): wt] chimeric mice are not capable of completing lymphomyeloid differentiation, whereas conditional inactivation of alpha4 integrin in adult mice has only subtle effects. We show here that circumventing the fetal stage of hematopoietic stem cell (HSC) development by transplantation of embryonic alpha4 integrin(-/-) cells into the adult microenvironment results in robust and stable long-term generation of alpha4 integrin(-/-) lymphoid and myeloid cells, although colonization of Peyer patches and the peritoneal cavity is significantly impaired. We argue here that collectively, our data and the data from other groups suggest a specific requirement for alpha4 integrin during the fetal/neonatal stages of HSC development that is essential for normal execution of the lymphomyeloid differentiation program.

Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors

Although adult mouse hematopoietic stem cells (HSCs) have been purified to near homogeneity, it remains impossible to achieve this with fetal HSCs. Adult HSC purity recently has been enhanced using the SLAM family receptors CD150, CD244, and CD48. These markers are expressed at different stages of the hematopoiesis hierarchy, making it possible to highly purify adult HSCs as CD150(+)CD48(-)CD244(-) cells. We found that SLAM family receptors exhibited a similar expression pattern in fetal liver. Fetal liver HSCs were CD150(+)CD48(-)CD244(-), and the vast majority of colony-forming progenitors were CD48(+)CD244(-)CD150(-) or CD48(+)CD244(+)CD150(-), just as in adult bone marrow. SLAM family markers enhanced the purification of fetal liver HSCs. Whereas 1 (11%) of every 8.9 Thy(low)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution in irradiated mice, 1 (18%) of every 5.7 CD150(+)CD48(-)CD41(-) cells and 1 (37%) of every 2.7 CD150(+)CD48(-)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution. These data emphasize the robustness with which SLAM family markers distinguish progenitors at different stages of the hematopoiesis hierarchy and enhance the purification of definitive HSCs from diverse contexts. Nonetheless, CD150, CD244, and CD48 are not pan-stem cell markers, as they were not detectably expressed by stem cells in the fetal or adult nervous system.

Is the Fetal Heart a Hematopoietic Organ?

To the Editor: The article by Tomanek et al1 presents well the in vivo data on the requirement of VEGF family members for tubulogenesis and coronary artery formation. The authors observed that VEGF-Trap (a chimera of R1 and R2), apart from precluding formation of coronary arteries, causes a massive accumulation of erythrocytes in the subepicardium and in the interventricular septum. The important finding of this study was that erythrocytes that are components of blood islands derive from epicardial-derived precursors. The authors assume that proepicardial cells include a hemangioblast population. From this observation a conclusion can be drawn that either the proepicardium or the fetal heart exhibits a hematopoietic activity. Considering the topic of blood island derivation in the embryonic heart I would like to comment that although the derivation of the endothelial cell component in these structures has been proven, the origin of erythrocytes is …

Hematopoiesis from Human Embryonic Stem Cells: Overcoming the Immune Barrier in Stem Cell Therapies

The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC-derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self-renewal may improve in vitro production both quantitatively and qualitatively.

The caudal-related homeobox genes cdx1a and cdx4 act redundantly to regulate hox gene expression and the formation of putative hematopoietic stem cells during zebrafish embryogenesis

The hox genes play a central role in organogenesis and are implicated in the formation of hematopoietic stem cells (HSCs). The cdx genes encode homeodomain transcription factors that act as master regulators of the hox genes. In zebrafish, mutations in cdx4 cause a severe, but not complete, deficit in embryonic blood cells. Here, we report the expression and function of cdx1a, a zebrafish Cdx1 paralogue. Using morpholino-mediated knockdown of cdx1a in a cdx4 mutant background, we show that a deficiency in both cdx genes causes a severe perturbation of hox gene expression and a complete failure to specify blood. The hematopoietic defect in cdx-deficient embryos does not result from a general block in posterior mesoderm differentiation as endothelial cells and kidney progenitors are still formed in the doubly deficient embryos. In addition, cdx-deficient embryos display a significant reduction in runx1a+ putative HSCs in the zebrafish equivalent to the aorta–gonad–mesonephros (AGM) region. Overexpressing hoxa9a in cdx-deficient embryos rescues embryonic erythropoiesis in the posterior mesoderm as well as the formation of HSCs in the AGM region. Taken together, these results suggest that the cdx-hox pathway plays an essential role in the formation of both embryonic erythroid cells and definitive HSCs during vertebrate embryogenesis.

Definitive Hematopoiesis from Acetyl LDL Incorporating Endothelial Cells in the Mouse Embryo

Previously, we reported generation of erythropoiesis from acetyl low-density lipoprotein (Ac-LDL)- incorporating endothelial cells (ECs) in the mouse embryo. However, it is still unclear whether the other types of definitive hematopoietic cells (HCs) can be generated from these cells. In this study, ECs at 10 days post coitum (dpc) were tagged with Ac-LDL-DiI and were shown to release DiI+ HCs into the circulation after 12 h of whole embryo culture. The hematopoietic clusters in the main arteries were also stained with DiI. The circulating DiI+ HCs expressed c-Kit and half of these cells displayed blastic morphology. In vitro culture and in vivo reconstitution experiments demonstrated that the circulating DiI+ HCs contained definitive multipotent progenitors, including hematopoietic stem cells (HSCs). Furthermore, the sorted DiI+ HCs were able to colonize the fetal liver (FL) when introduced back into the blood stream of a secondary recipient embryo. These results suggest that Ac-LDL incorporating ECs can produce definitive HSCs and HCs colonizing FL in the mouse embryo.

BMP4 Favors Hematopoietic Differentiation from Murine Embryonic Stem Cells and Acts by Activation of the cdx-hox Pathway.

Cdx4 and cdx1, members of the caudal family of homeodomain-containing transcriptional regulators, are important for specifying the hematopoietic fate of mesoderm in the zebrafish. We have shown that the cdx4 gene plays a role in enhancing hematopoietic fate during in vitro differentiation of murine ESCs (Davidson et al., Nature 2003). Cdx4 induces hox genes, and genetic modification of mESCs with a combination of cdx4 and hoxb4 promotes long-term engraftment of ESC-derived HSCs in lethally irradiated primary and secondary mice (Wang et al, submitted). While cdx1 is known to be a direct target of signaling by the embryonic morphogens fgf, wnt3a, and retinoids, morphogens acting upstream of cdx4 have not yet been defined. Our goal is to determine optimal morphogen conditions for hematopoietic commitment from murine embryonic stem cells by evaluating activation of the cdx-hox pathway. We have developed quantitative RT-PCR assays for the cdx genes (cdx4, cdx1 and cdx2) and multiple hox genes as well as markers specific to hematopoietic stem cells and lineages. We have used these assays, together with a reporter line engineered to express GFP from the brachury locus (Fehling et al., Development 2003), to characterize the conditions for mesodermal induction and hematopoietic fate specification following addition of morphogens to differentiating cultures of ES cells under serum-free conditions. Among all morphogens tested (BMP4, activin, nodal, wnt3a, wnt5a, sonic hedgehog, indian hedgehog, retinoic acid), only BMP4 has been found to strongly induce CDX4 gene expression within the developing embryoid bodies, while addition of the BMP4 inhibitor noggin to serum suppressed CDX4 expression. Addition of BMP4 significantly increases the number of emerging CD41+ and CD45+ cells, the precursors of definitive hematopoietic stem cells. We are currently analyzing the functional changes following BMP4 exposure, and correlating hematopoietic maturation with changes in the Hox gene expression pattern. Analysis of the cdx-hox gene pathway provides a means of otpimizing induction of hematopoietic fate by application of embryonic morphogens.

The embryonic aorta‐gonad‐mesonephros region as a generator of haematopoietic stem cells

During mammalian embryonic development the definitive haematopoietic stem cells (HSCs) may arise either in the extra-embryonic mesoderm or in the aorta-gonad-mesonephros (AGM) region that forms in close proximity to the assembling urogenital system, generating the gonad, cortex of the adrenal gland and metanephros. Researchers have been attempting for a long time to define the region of importance for generating the definitive HSCs that colonize the fetal liver and bone marrow, the two major sites where haematopoiesis takes place in the adult. The fetal liver might gain HSCs from both of the primary haematopoietic sources, but the extra-embryonic HSCs seem not to be able to colonize adult bone marrow directly. It is known that the microenvironment around the HSCs is important for directing cell fates, but we do not yet have much idea about the cell-cell interactions, tissue interactions and molecules that regulate cell behaviour in the AGM. We will here discuss the contribution of the AGM to definitive haematopoiesis in mammals and review some of the cell-cell interactions and associated signalling systems involved in the development of AGM stem cells.

Placenta as a site for hematopoietic stem cell development

The discovery of a major hematopoietic stem cell (HSC) pool in mid-gestation mouse placenta has defined the placenta as yet another important anatomical site that participates in HSC development. Placental HSC activity starts in parallel with the AGM region, before HSCs are found in circulation or have colonized the fetal liver. Moreover, placental hematopoietic activity culminates in a rapid expansion of the definitive HSC pool, which occurs during the time when the fetal liver HSC reservoir begins to grow. Furthermore, hematopoietic cells in mid-gestation mouse placenta are not instructed for differentiation along the myeloerythroid lineage, as in the fetal liver. These findings suggest that the placenta provides a supportive niche where the definitive hematopoietic stem cell pool can be temporarily established during development. Future studies are needed to characterize the developmental events that lead to the establishment of placental HSC pool, and to define the microenvironmental signals that support this process. Furthermore, if the stem cell-promoting properties of the placental niche can be harnessed in vitro to support HSC formation, maturation, and/or expansion in culture, these assets may greatly improve hematopoietic stem cell-based therapies in the future.

Patterning definitive hematopoietic stem cells from embryonic stem cells

Derived from the inner cell mass of blastocysts, embryonic stem cells (ESCs) retain the pluripotent features of early embryonic epiblast cells. In vitro, ESCs undergo spontaneous differentiation into a multitude of tissues, and thus are a powerful tool for the study of early developmental processes and a promising resource for cell-based therapies. We have pursued the derivation of functional, multipotent and engraftable hematopoietic stem cells (HSCs) from ESCs in order to investigate the genetic pathways specifying blood formation, as well as to lay the foundation for hematopoietic cell replacement therapies based on engineered ESCs. Theoretically, the generation of HSCs from patient-specific ESCs derived by nuclear transfer could provide for autologous hematopoietic therapies for the treatment of malignant and genetic bone marrow disorders. Although significant progress has been made in achieving hematopoietic differentiation from both murine and human ESCs, we have only a primitive understanding of the underlying mechanisms that specify hematopoietic cell fate, and a very limited capacity to direct the differentiation of the definitive HSC that would be suitable for clinical engraftment studies. Here we will review the progress to date and the significant problems that remain, and outline a strategy to achieve the directed differentiation of HSCs under conditions that might be appropriate for clinical scale-up and disease applications.

Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver

The yolk sac and the para-aortic splanchnopleura/aorta-genital ridges-mesonephros (P-Sp/AGM) region are the main sites of haematopoietic activity in the mouse embryo at the pre-liver stage of development. By day 11.5 of gestation, the AGM region is capable of autonomous initiation and expansion of definitive haematopoietic stem cells (HSCs). By day 12.5, HSC activity in the AGM region is reduced whilst a second wave of HSCs begins to emerge in the yolk sac. We show here that HSCs emerging in both locations are marked by co-expression of the endothelial-specific marker VE-cadherin and the pan-leukocyte antigen CD45. Phenotypic characterisation using CD31, TIE2, FLK1, Ac-LDL receptors, and CD34 markers demonstrated significant similarities between this VE-cadherin+CD45+ ;double-positive' population and endothelial cells suggesting a common origin for these cells. The double-positive fraction also expressed the stem cell markers Kit, Sca1 and AA4.1. Long-term transplantation experiments demonstrated that the double-positive population, which constituted less than 0.05% of the day 11.5 AGM region and the day 12.5 yolk sac, is highly enriched for HSCs. In vitro assays showed that this population is also enriched for myeloid progenitors. During foetal liver colonization, circulating HSCs remained within the VE-cadherin+ cell fraction, although their phenotypic similarity with endothelial cells became less prominent. Upon liver colonisation the majority of HSCs downregulated VE-cadherin, expression of which was completely lost in the adult bone marrow. Partial loss of VE-cadherin expression in HSCs can be observed extra hepatically in the advanced AGM region by E12.5. Similarly, the CD34+KIT+ population in the placenta, recently identified as a reservoir of HSCs, partly lose VE-cadherin expression by E12.5. By culturing isolated E11.5 AGM region and E12.5 yolk sac we show that the developmental switch from a ;primary' VE-cadherin+CD45+ to a more ;advanced' VE-cadherin-CD45+ phenotype does not require contact of HSCs with the liver and is probably a function of developmental time.

Spatial differences in hematopoiesis but not in stem cells indicate a lack of regional patterning in definitive hematopoietic stem cells

Most tissues are patterned so that progenitors in different locations are programmed to have different properties. Stem cells from different regions of the nervous system acquire intrinsic differences in their properties as they migrate through distinct environments. Hematopoietic stem cells (HSCs) also migrate through diverse environments throughout life, raising the question of whether HSCs also acquire at least transient changes in their properties as they are exposed to diverse environments. Although we observed significant differences in hematopoiesis between the fetal liver and fetal spleen, we were not able to detect phenotypic, functional, or gene expression differences between the HSCs in these organs. Regional differences in definitive hematopoiesis are therefore not determined by regional differences between HSCs. We were also not able to detect phenotypic, functional, or gene expression differences between HSCs in different adult bone marrow compartments. Our failure to detect differences among stem cells from different regions of the hematopoietic system at the same time during development suggests that the hematopoietic system has evolved mechanisms to prevent the spatial reprogramming of HSC properties as they migrate between distinct environments.

The emergence of definitive hematopoietic stem cells in the mammal

Hematopoietic stem cells (HSC) are the basis for blood formation during adult life. The amazing potency of HSCs has been exploited for over 30 years in regenerative therapies for patients with blood-related genetic disease and leukemia. As clinically important cells and also as the most widely studied cell differentiation system, they have been the focus of intense fundamental research. Indeed, HSC research has established many paradigms in the more general field of stem cells. Recently, the study of the embryonic origins of HSCs and their genetic program is beginning to provide unique insights into how these stem cells are formed, maintained, and expanded, and how they contribute to the complex adult hematopoietic system. Although many short-lived hematopoietic progenitors are present in early stage mammalian embryos, this review will focus on the events leading to emergence of the most potent cells of the hematopoietic system, HSCs and on their developmental lineage relationships. Developmental and genetic studies further our understanding of the fate determination events occurring in several embryonic tissues leading to the generation of potent HSCs--those cells with the ability to long-term, high-level repopulate all hematopoietic lineages of the adult. Several mammalian embryonic tissues contribute to the growth and/or generation of potent HSCs that are the source of blood cells throughout the lifespan of the individual. Insight into how mammalian HSC fate is determined has been provided through functional, phenotypic, and genetic studies at early developmental stages.

The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo

Homeodomain proteins of the Meis subfamily are expressed dynamically in several organs during embryogenesis and exert potent regulatory activity through their interaction with Hox proteins and other transcription factors. Here we show that Meis1 is expressed in the hematopoietic stem cell (HSC) compartment in the fetal liver, and in the primary sites of definitive hematopoiesis, including the aorta–gonad–mesonephros (AGM) mesenchyme, the hemogenic embryonic arterial endothelium, and hematopoietic clusters within the aorta, vitelline, and umbilical arteries. We inactivated the Meis1 gene in mice and found that Meis1 mutant mice die between embryonic days 11.5 and 14.5, showing internal hemorrhage, liver hypoplasia, and anemia. In Meis1 mutant mouse fetal liver and AGM, HSC compartments are severely underdeveloped and colony-forming potential is profoundly impaired. AGM mesenchymal cells expressing Runx1, an essential factor for definitive HSC specification, are almost absent in mutant mice. In addition, hematopoietic clusters in the dorsal aorta, vitelline, and umbilical arteries are reduced in size and number. These results show a requirement for Meis1 in the establishment of definitive hematopoiesis in the mouse embryo. Meis1 mutant mice also displayed complete agenesis of the megakaryocyte lineage and localized defects in vascular patterning, which may cause the hemorrhagic phenotype.

Early Fetal Liver Readily Repopulates B Lymphopoiesis in Adult Bone Marrow

Fetal liver (FL) becomes a major organ of hematopoiesis at mouse embryonic day (E) 11 and E12, when definitive hematopoietic stem cells, originating from the aorta-gonads-mesonephros region, colonize the hepatic tissue. Unipotent B-cell progenitors are very rare in FL by day 12, whereas erythropoiesis prevails. We have studied hematopoiesis in FL from different gestational ages, with special emphasis on B lymphopoiesis. The mRNA levels of selected liver-specific genes, hematopoietic lineage-specific genes, and genes for selected cytokines/hormones as well as for their receptors were evaluated by real-time polymerase chain reaction in FL from E12.5, E14.5, and E17.5, adult liver and adult bone marrow (BM). The level of B lineage-related gene expression in FL was very low at E12.5. There was also a significantly lower fraction of B220+ and CD19+ B cells in E12.5 FL compared with E17.5 FL. To analyze whether these differences reflect different stem cell potentials occurring during FL development, 10(6) or 5 x 10(6) of FL cells collected from embryos at E12.5 or E17.5 and those from adult BM were transplanted into sublethally irradiated (3- or 6-Gy) congenic mice. Short-term and long-term repopulation of B and T cells and granulocyte/macrophage lineages from donor FL or adult BM cells were evaluated in competition to adult hematopoiesis of sublethally irradiated recipients. In short-term repopulation, the transplantation of E12.5 FL cells resulted in a lower blood chimerism compared with that of E17.5 FL cells. However, the proportion of B lymphopoiesis exerted by E12.5 FL cells was not different from that of E17.5 FL or adult BM. This study demonstrates that E12.5 FL contains hematopoietic stem cells with fully developed B-cell repopulating capacity and that the developmental period of fetal hematopoiesis between E12.5 and E17.5 is not an obligatory phase for the adult B lymphopoiesis.

Human embryonic stem cell–derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential

AbstractEmbryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell–derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase–positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.

O-18 MEF regulates the quiescence of primitive hematopoietic progenitor cells and their susceptibility to myelosuppression

The AML1/CBFβ transcription factor complex, a frequent target of chromosomal translocations in leukemia, is essential for the generation of definitive hematopoietic stem cells. Paradoxically, expression of the acute myeloid leukemia-associated AML1-ETO fusion protein in mice results not in leukemia, but in embryonic lethality due to an absence of normal hematopoiesis. To bypass the embryonic lethality, we generated a mouse strain with a conditional AML1-ETO knockin allele that contains a loxP bracketed transcriptional stop cassette 5′ to the AML1-ETO fusion site. Activation of this allele in vivo by Cre-mediated recombination resulted in an enhanced replating efficiency of myeloid progenitors, but it did not block their differentiation, nor was it sufficient to induce leukemia. However, induction of cooperating mutations resulted in the development of an acute myeloid disease that mimicked many of the features of human AML1-ETO-expressing leukemia.

Haploinsufficiency of AML1 results in a decrease in the number of LTR-HSCs while simultaneously inducing an increase in more mature progenitors

The AML1/CBFbeta transcriptional complex is essential for the formation of definitive hematopoietic stem cells (HSCs). Moreover, development of the hematopoietic system is exquisitely sensitive to the level of this complex. To investigate the effect of AML1 dosage on adult hematopoiesis, we compared the hematopoietic systems of AML1+/- and AML1+/+ mice. Surprisingly, loss of a single AML1 allele resulted in a 50% reduction in long-term repopulating hematopoietic stem cells (LTR-HSCs). This decrease did not, however, extend to the next level of hematopoietic differentiation. Instead, AML1+/- mice had an increase in multilineage progenitors, an expansion that resulted in enhanced engraftment following transplantation. The expanded pool of AML1+/- progenitors remained responsive to homeostatic mechanisms and thus the number of mature cells in most lineages remained within normal limits. Two notable exceptions were a decrease in CD4(+) T cells, leading to an inversion of the CD4(+) to CD8(+) T-cell ratio and a decrease in circulating platelets. These data demonstrate a dosage-dependent role for AML1/CBFbeta in regulating the quantity of HSCs and their downstream committed progenitors, as well as a more restricted role in T cells and platelets. The latter defect mimics one of the key abnormalities in human patients with the familial platelet disorder resulting from AML1 haploinsufficiency.