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Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5-azacytidine [AZA], 5-aza-2'-deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease progression and how DNA methylation contributes to MDS remain unclear. We analyzed global DNA methylation in purified CD34+ hematopoietic progenitors from MDS patients undergoing multiple rounds of AZA treatment. Differential methylation between MDS phenotypes was observed primarily at developmental regulators not expressed within the hematopoietic compartment and was distinct from that observed between healthy hematopoietic cell types. After AZA treatment, we observed only limited DNA demethylation at sites that varied between patients. This suggests that a subset of the stem cell population is resistant to AZA and provides a basis for disease relapse. Using gene expression data from patient samples and an in vitro AZA treatment study, we identified differentially methylated genes that can be activated following treatment and that remain silent in the CD34+ stem cell compartment of high-risk MDS patients. Haploinsufficiency in mice of one of these genes (NR4A2) has been shown to lead to excessive HSC proliferation, and our data suggest that suppression of NR4A2 by DNA methylation may be involved in MDS progression., (©AlphaMed Press.)

Hematopoietic cells (HPCs) develop from hemogenic endothelial cells (ECs), a specialized type of ECs undergoing hematopoietic transition. However, the mesoderm origin for hemogenic ECs or HPCs has not been clarified. To examine the origin for hemogenic mesoderm, we inactivated Etv2, a master regulator for EC/HPC commitment, in specific regions. Region-specific Etv2 ablation in early mesoderm caused local EC differentiation block, resulting in the loss of specific vascular beds without compensatory migration of residual ECs into avascular area. This feature of local EC/HPC differentiation block was correlated to the hemogenic potential of each mesoderm subset. We found that caudal-lateral mesoderm of E7.5-8.5 embryos represent the pre-committed population critical for generating hemogenic ECs. Etv2 ablation in caudal-lateral mesoderm by Hoxb6 Cre or Hoxb6CreER transgene affected vitelline plexus formation and intra-aortic hematopoietic clusters. In differentiated embryonic stem cells, this mesoderm subset marked by Hoxb6-lateral mesoderm promoter showed enriched T lymphopoietic potential among Flk-1(+) cells, which could be regarded as a characteristic for definitive HPCs. These findings indicate that critical mesoderm precursors possibly for definitive type hemogenic ECs are regionally specified in primitive mesoderm, suggesting that Hoxb6(+) caudal-lateral mesoderm represents the critical source of HPCs, which are potentially useful to enrich definitive HPCs from embryonic stem cells., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Early mesoderm can be classified into Flk-1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk-1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato-endothelial lineages has not been proved in mouse embryos., Results: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin-Kit+Sca-1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk-1+ population that exists until E8.5 may be the source for hemato-endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin-Kit+Sca-1+ (KSL) cells, respectively., Conclusions: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos., (Copyright © 2013 Wiley Periodicals, Inc.)

Ets family protein Etv2 (also called ER71 or Etsrp) is a key factor for initiation of vascular and blood development from mesodermal cells. However, regulatory mechanisms and inducing signals for Etv2 expression have been largely unknown. Previously, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors into endothelial cells (ECs) and hematopoietic cells (HPCs) using an embryonic stem cell (ESC) differentiation system. Here, we show that PKA activation in an earlier differentiation stage can trigger EC/HPC differentiation through Etv2 induction. We found Etv2 was markedly upregulated by PKA activation preceding EC and HPC differentiation. We identified two cAMP response element (CRE) sequences in the Etv2 promoter and 5'-untranslated region and confirmed that CRE-binding protein (CREB) directly binds to the CRE sites and activates Etv2 transcription. Expression of a dominant negative form of CREB completely inhibited PKA-elicited Etv2 expression and induction of EC/HPCs from ESCs. Furthermore, blockade of PKA significantly inhibited Etv2 expression in ex vivo whole-embryo culture using Etv2-Venus knockin mice. These data indicated that PKA/CREB pathway is a critical regulator for the initiation of EC/HPC differentiation via Etv2 transcription. This early-stage molecular linkage between a triggering signal and transcriptional cascades for differentiation would provide novel insights in vascular and blood development and cell fate determination., (Copyright © 2012 AlphaMed Press.)

Several lines of evidence suggest that the adult hematopoietic system has multiple developmental origins, but the ontogenic relationship between nascent hematopoietic populations under this scheme is poorly understood. In an alternative theory, the earliest definitive blood precursors arise from a single anatomical location, which constitutes the cellular source for subsequent hematopoietic populations. To deconvolute hematopoietic ontogeny, we designed an embryo-rescue system in which the key hematopoietic factor Runx1 is reactivated in Runx1-null conceptuses at specific developmental stages. Using complementary in vivo and ex vivo approaches, we provide evidence that definitive hematopoiesis and adult-type hematopoietic stem cells originate predominantly in the nascent extraembryonic mesoderm. Our data also suggest that other anatomical sites that have been proposed to be sources of the definitive hematopoietic hierarchy are unlikely to play a substantial role in de novo blood generation.

The mouse Flk1 gene is expressed in various mesodermal progenitor cells of developing embryos. Recent studies have shown that Flk1 expression marks multipotent mesodermal progenitors, giving rise to various hemato-cardiovascular cell lineages during development. Flk1 expression also marks hemato-cardiovascular cell lineages in differentiating embryonic stem (ES) cells, which may be used in transplantation decisions to treat cardiovascular diseases. Despite its developmental and clinical importance in cardiovascular tissues, the transcriptional regulatory system of Flk1 has remained unclear. Here, we report a novel enhancer of the mouse Flk1 gene directing early mesodermal expression during development as well as ES differentiation. The enhancer enriches various mesodermal progenitors, such as primitive erythropoietic progenitors, hemangioblast (BL-CFC) and cardiovascular progenitors (CV-CFC). The enhancer is activated by Bmp, Wnt and Fgf, and it contains Gata-, Cdx-, Tcf/Lef-, ER71/Etv2- and Fox-binding sites, some of which are bound specifically by each of these transcription factors. As these transcription factors are known to act under the control of the Bmp, Wnt and Fgf families, early Flk1 expression may be induced by cooperative interactions between Gata, Tcf/Lef, Cdx and ER71/Etv2 under the control of Bmp, Wnt and Fgf signaling. The enhancer is required for early Flk1 expression and for hemangioblast development during ES differentiation.

Lnk is an intracellular adaptor protein reported as a negative regulator of proliferation in c-Kit positive, Sca-1 positive, lineage marker-negative (KSL) bone marrow cells. The KSL fraction in mouse bone marrow is believed to represent a population of hematopoietic and endothelial progenitor cells (EPCs). We report here that, in vitro, Lnk(-/-) KSL cells form more EPC colonies than Lnk(+/+) KSL cells and show higher expression levels of endothelial marker genes, including CD105, CD144, Tie-1, and Tie2, than their wild-type counterparts. In vivo, the administration of Lnk(+/+) KSL cells to a mouse spinal cord injury model promoted angiogenesis, astrogliosis, axon growth, and functional recovery following injury, with Lnk(-/-) KSL being significantly more effective in inducing and promoting these regenerative events. At day 3 following injury, large vessels could be observed in spinal cords treated with KSL cells, and reactive astrocytes were found to have migrated along these large vessels. We could further show that the enhancement of astrogliosis appears to be caused in conjunction with the acceleration of angiogenesis. These findings suggest that Lnk deletion reinforces the commitment of KSL cells to EPCs, promoting subsequent repair of injured spinal cord through the acceleration of angiogenesis and astrogliosis.

Hematopoietic stem cell (HSC) proliferation is tightly regulated by a poorly understood complex of positive and negative cell-cycle regulatory mechanisms. Necdin (Ndn) is an evolutionally conserved multifunctional protein that has been implicated in cell-cycle regulation of neuronal cells. Here, we provide evidence that necdin plays an important role in restricting excessive HSC proliferation during hematopoietic regeneration. We identify Ndn as being preferentially expressed in the HSC population on the basis of gene expression profiling and demonstrate that mice deficient in Ndn show accelerated recovery of the hematopoietic system after myelosuppressive injury, whereas no overt abnormality is seen in steady-state hematopoiesis. In parallel, after myelosuppression, Ndn-deficient mice exhibit an enhanced number of proliferating HSCs. Based on these findings, we propose that necdin functions in a negative feedback loop that prevents excessive proliferation of HSCs during hematopoietic regeneration. These data suggest that the inhibition of necdin after clinical myelosuppressive treatment (eg, chemotherapy, HSC transplantation) may provide therapeutic benefits by accelerating hematologic recovery.

In the developing mouse, vascular endothelial cell (EC) and hematopoietic cell (HPC) lineages are two initial cell lineages that diverge from mesodermal cells, which have been roughly subdivided into three subtypes according to their geographical location: the organizer, embryonic mesoderm in the primitive streak, and extraembryonic mesoderm during gastrulation. Although the initial progenitors that become the two lineages appear in both vascular endothelial growth factor receptor 2(+) (VEGFR2(+)) lateral and extraembryonic mesoderm, little is known about the underlying molecular events that regulate the derivation of ECs and HPCs. Here, we describe an experimental system consisting of two types of embryonic stem cell lines capable of distinguishing between organizer and the middle section of the primitive streak region. Using this system, we were able to establish a defined culture condition that can separately induce distinct types of mesoderm. Although we were able to differentiate ECs from all mesoderm subsets, however, the potential of HPCs was restricted to the VEGFR2(+) cells derived from primitive streak-type mesodermal cells. We also show that the culture condition for the progenitors of primitive erythrocytes is separated from that for the progenitors of definitive erythrocytes. These results suggest the dominant role of extrinsic regulation during diversification of mesoderm.

Nanog is a key molecule involved in the maintenance of the self-renewal of undifferentiated embryonic stem (ES) cells. In this work we investigate whether Nanog can enhance self-renewal in hematopoietic stem cells. Contrary to our expectation, no positive effect of Nanog transduction was detected in bone marrow reconstitution assays. However, recipients of Nanog-transduced (Nanog) hematopoietic stem cells (HSCs) invariantly develop a unique disorder typified by an atrophic thymus occupied by Nanog-expressing gammadeltaT-cell receptor-positive (TCR(+)) cells (Nanog T cells). All thymi are eventually occupied by Nanog T cells with CD25(+)CD44(+) surface phenotype that home selectively to the thymus on transfer and suppress normal thymocyte development, which is partly ascribed to destruction of the microenvironment in the thymus cortex. Moreover, this initial disorder invariantly develops to a lymphoproliferative disorder, in which Nanog T cells undergo unlimited proliferation in the peripheral lymphoid tissues and eventually kill the host. This invariable end result suggests that Nanog is a candidate oncogene for gammadeltaT-cell malignancy.

The first haematopoietic stem cells (HSCs) appear in the aorta-gonad-mesonephros (AGM) region, major vitelline and umbilical vessels, and placenta; however, whether they arise locally or from immigrant yolk sac precursor cells remains unclear. This issue is best addressed by measuring cell-lineage relationships rather than cell potentials. To undertake long-term in vivo tracing of yolk sac cells, we designed a non-invasive pulse-labelling system based on Cre/loxP recombination. Here we show that in Runx1(+/-) (runt-related transcription factor 1) heterozygous mice, yolk sac cells expressing Runx1 at embryonic day 7.5 develop into fetal lymphoid progenitors and adult HSCs. During mid-gestation the labelled (embryonic day 7.5) yolk sac cells colonize the umbilical cord, the AGM region and subsequently the embryonic liver. This raises the possibility that some HSCs associated with major embryonic vasculature are derived from yolk sac precursors. We observed virtually no contribution of the labelled cells towards the yolk sac vasculature, indicating early segregation of endothelial and haematopoietic lineages.

Hemangioblasts are thought to be one of the sources of hematopoietic progenitors, yet little is known about their localization and fate in the mouse embryo. We show here that a subset of cells co-expressing the hematopoietic marker GATA-1 and the endothelial marker VE-cadherin localize on the yolk sac blood islands at embryonic day 7.5. Clonal analysis demonstrated that GATA-1(+) cells isolated from E7.0-7.5 embryos include a common precursor for hematopoietic and endothelial cells. Moreover, this precursor possesses primitive and definitive hematopoietic bipotential. By using a transgenic complementation rescue approach, GATA-1(+) cell-derived progenitors were selectively restored in Runx1-deficient mice. In the rescued mice, definitive erythropoiesis was recovered but the rescued progenitors did not display multilineage hematopoiesis or intra-aortic hematopoietic clusters. These results provide evidence of the presence of GATA-1(+) hemangioblastic cells in the extra-embryonic region and also their functional contribution to hematopoiesis in the embryo.

Definitive hematopoiesis has been proposed to arise from hemogenic endothelial cells during mouse embryogenesis. The c-myb proto-oncogene is essential for the development of definitive hematopoiesis and was reported to be activated in hemogenic endothelial cells. To investigate whether c-Myb is involved in regulating the development of hemogenic endothelial cells, we conditionally induced c-myb over-expression during the in vitro differentiation of embryonic stem cells. VE-cadherin+ CD45- cells inducibly expressing c-Myb showed an increase in multilineage colony formation as well as an augmented capacity of the colony forming cells to self-renew in vitro under the condition that only the endogenous c-myb gene was expressed during differentiation of hematopoietic cells. Over-expression of c-Myb in the endothelial population led to activation of genes associated with definitive hematopoiesis such as Runx1, Hoxb4, Mll and Etv6. Our data provide evidence that c-Myb is able to exert an effect in endothelial cells which fosters the establishment of their hemogenic potential.

We describe here a unique case of therapy-related myelodysplastic syndrome (t-MDS) with inv(16)(p13q22) after autologous stem cell transplantation for lymphoma. The rare and smallest I type CBFbeta/MYH11 fusion transcript with a breakpoint at nucleotide 399 of CBFbeta and at nucleotide 2134 of MYH11 was detected in the bone marrow cells by reverse transcription polymerase chain reaction analysis. However, the fusion transcript was undetectable in the pretransplant peripheral blood stem cells. These results suggest that the stem cell damage leading to t-MDS may be induced mainly by the conditioning regimen for transplantation. Taken together with previous reports, the I type fusion transcript is preferentially induced with chemotherapy.

Here, we show a functional role of casein kinase I (CKI) epsilon in hematopoietic cell survival through the modification of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Introduction of wild-type (WT)-CKIepsilon into interleukin-3 (IL-3)-dependent 32D cells increased the sensitivity to genotoxic stresses, such as gamma-irradiation, etoposide, and IL-3 deprivation, whereas kinase-negative (KN)-CKIepsilon suppressed it. Contrary to KN-CKIepsilon, WT-CKIepsilon attenuated the IL-3-induced activation of Akt with the increase of PTEN activity. Similarly, the increase of Akt activation, as well as PTEN inactivation, was accompanied both by a decrease of CKIepsilon expression induced by all-trans retinoic acid and by the addition of a specific inhibitor for CKIepsilon in HL-60 cells. CKIepsilon seems to activate PTEN by physical interaction. These results suggest that the CKIepsilon-induced down-regulation of PI3K/Akt signaling through PTEN lead to amplified sensitivity to apoptosis. Thus, the suppression of CKIepsilon in many human leukemia cell lines may play a role in the cell immortalization.

During early mouse embryogenesis, fetal liver kinase-1 (Flk-1), a receptor for vascular endothelial growth factor, and Runx1, a runt domain transcription factor, have prerequisite roles in the generation of hematopoietic lineages. Flk-1 expression is maintained in successive stages from mesodermal to endothelial cells and is down-regulated in nascent hematopoietic cells, whereas Runx1 (Runt-related transcription factor 1) is expressed in embryonic sites of hematopoietic cell de novo generation and in practically all hematopoietic organs. Here we show that Runx1 represses Flk-1 during the development of hemogenic endothelial cells into hematopoietic cells. We established embryonic stem cell clones carrying the Venus gene, a modified version of yellow fluorescence protein, in the Runx1 locus and cultured them on OP9 cells. Flk-1+ cells appeared on day 3.5, and Runx1+ cells first appeared from the Flk-1+ fraction on day 4.5. The Flk-1+Runx1+ cells rapidly stopped expressing Flk-1 with further incubation and eventually gave rise to CD45+ or TER119+ cells. Runx1 repressed Flk-1 promoter transcriptional activity in an endothelial cell line, and this repression required intact DNA-binding and transactivating domains of Runx1 protein. The repressor activity of Runx1 endogenous Flk-1 was also confirmed overexpressing Runx1 in embryonic stem cell differentiation cultures. These results provide novel insight into the role Runx1 during the development of hematopoietic cell lineages.

The aorta-gonads-mesonephros (AGM) region of the mouse embryo has been assigned as the origin of definitive hematopoiesis. The transcription factor GATA-2 has specific but unclarified roles in early hematopoiesis. To elucidate the expression profile of GATA-2, we prepared transgenic mouse lines containing the green fluorescent protein (GFP) gene driven by GATA-2 gene regulatory elements. We also prepared a mouse line in which GFP reporter sequences were inserted into the endogenous GATA-2 gene. Both mouse mutants expressed GFP in the early hematopoietic tissues. The CD45 antigen, a marker of hematopoietic cells, was expressed in a small fraction of transgene (TG)-derived GFP+ cells. The remaining TG-GFP+/CD45- cells were adherent to plastic and produced CD45+ hematopoietic cells abundantly when cultured in vitro. Exogenous expression of GATA-2 in TG-GFP+/CD45- cells from the AGM region inhibited their differentiation into CD45+ cells. Loss of GATA-2 function through the disruption of the GATA-2 locus enhanced the earlier emergence of CD45+ cells in the yolk sac of the 9.5-day conceptus. These results demonstrated that GATA-2 is expressed in the precursor of hematopoietic cells and works as a gatekeeper to preserve their immaturity. A reduction of GATA-2 expression or activity is required for the differentiation of precursors to hematopoietic cells.

Objective: The aim of this study was to assess the potential of FLK1(+) and vascular endothelial (VE)-cadherin(+) populations from different stages of embryonic development to generate hematopoietic cells ex vivo and to contribute to the hematopoietic systems of recipient mice., Materials and Methods: FLK1(+) of VE-cadherin(+) cells were isolated from 7.5- to 9.5-dpc concepti and cultured ex vivo on OP9 stromal cells and hematopoietic development examined. VE-cadherin(+)CD45(-) cells from 8.5- and 9.5-dpc concepti were injected intrahepatically into newborn busulfan-treated SCID recipients and engraftment monitored., Results: VE-cadherin(+) cells from 7.5- and 8.5-dpc concepti can readily generate hematopoi-etic cells ex vivo compared to FLK1(+) VE-cadherin(-) cells. Similar hematopoietic potential can be found in the VE-cadherin(+) cells from the 8.5-dpc yolk sac. When VE-cadherin(+)CD45(-) cells were injected into SCID recipients, long-term engraftment, particularly within the lymphoid system, was observed. This potential was observed in VE-cadherin(+)CD45(-) cells from 9.5-dpc embryo or yolk sac but from tissues from younger concepti., Conclusions: FLK1(+)VE-cadherin(-) cells, possibly representing the lateral plate mesoderm, are not as effective at generating hematopoietic cells compared to similarly staged VE-cadherin(+) cells. VE-cadherin(+)CD45(-) cells can also contribute to the hematolymphoid system of intrahepatically injected newborn SCID recipients, suggesting that cells bearing an endothelial phenotype are capable of generating long-term hematopoietic precursors.

Background: The developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage., Results: We found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA-1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA-1- cells give rise to VE-cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE-cadherin+ cells., Conclusions: These observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE-cadherin+ endothelial cells in the development of haematopoietic and endothelial cells.

Mice deficient in lymphotoxin beta receptor (LTbetaR) or interleukin 7 receptor alpha (IL-7Ralpha) lack Peyer's patches (PPs). Deficiency in CXC chemokine receptor 5 (CXCR5) also severely affects the development of PPs. A molecular network involving these three signaling pathways has been implicated in PP organogenesis, but it remains unclear how they are connected during this process. We have shown that PP organogenesis is initiated at sites containing IL-7Ralpha(+) lymphoid cells and vascular cell adhesion molecule (VCAM)-1/intercellular adhesion molecule (ICAM)-1 expressing nonlymphoid elements. Here we characterize these lymphoid and nonlymphoid components in terms of chemokine signals. The lymphoid population expresses CXCR5 and has a strong chemotactic response to B lymphocyte chemoattractant (BLC). Importantly, chemokines produced by VCAM-1(+)ICAM-1(+) nonlymphoid cells mediate the recruitment of lymphoid cells. Furthermore, we show that these VCAM-1(+)ICAM-1(+) cells are mesenchymal cells that are activated by lymphoid cells through the LTbetaR to express adhesion molecules and chemokines. Thus, promotion of PP development relies on mutual interaction between mesenchymal and lymphoid cells.

It has been widely accepted that hematopoietic and endothelial cell lineages diverge from a common progenitor referred to as the hemangioblast. Recently, analyses of the potential of progenitor cells purified from mouse embryos as well as embryonic stem cells differentiating in vitro resolved intermediate stages between mesodermal cells and committed precursors for hematopoietic and endothelial cell lineages. There are two distinct hematopoietic cell lineages which have different origins, i.e., primitive hematopoietic lineage derived from mesoderm or hemangioblasts and definitive hematopoietic lineage derived from endothelial cells. The endothelium is suggested to provide a milieu in which the definitive hematopoietic lineage acquires multiple potentials.

Osteoclasts are hematopoietic cells essential for bone resorption. To understand the process of osteoclastogenesis, we have developed a culture system that employs a stromal cell line, in which differentiation of osteoclasts from single embryonic stem (ES) cells occurs. This culture, which did not require any cell passaging or other manipulations, enabled us to investigate the temporal and spatial localization of the osteoclast lineage in the colonies formed from ES cells. Cells expressing tartrate-resistant acid phosphatase, a specific marker of the osteoclast lineage, were first detected on day 8, and subsequently became localized at the periphery of colonies and matured into multinucleated cells to resorb bone. Addition of macrophage colony-stimulating factor and osteoprotegerin-ligand, which are produced by stromal cells, promoted osteoclastogenesis in whole colonies, indicating that the location and maintenance of mature osteoclasts as well as the growth and differentiation of osteoclast precursors are regulated by these two factors., (Copyright 2001 Academic Press.)

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

Embryonic stem cells can differentiate in vitro into hematopoietic cells through two intermediate stages; the first being FLK1(+) E-cadherin- proximal lateral mesoderm and the second being CD45(-) VE-cadherin+ endothelial cells. To further dissect the CD45(-) VE-cadherin+ cells, we have examined distribution of alpha4-integrin on this cell population, because alpha4-integrin is the molecule expressed on hematopoietic stem cells. During culture of FLK1(+) E-cadherin- cells, CD45(-) VE-cadherin+ alpha4-integrin- cells differentiate first, followed by alpha4-integrin+ cells appearing in both CD45(-) VE-cadherin+ and CD45(-) VE-cadherin- cell populations. In the CD45(-) VE-cadherin+ cell population, alpha4-integrin+ subset but not alpha4-integrin- subset had the potential to differentiate to hematopoietic lineage cells, whereas endothelial cell progenitors were present in both subsets. The CD45(-) VE-cadherin- alpha4-integrin+ cells also showed hematopoietic potential. Reverse transcription-polymerase chain reaction analyses showed that differential expression of the Gata2 and Myb genes correlated with the potential of the alpha4-integrin+ cells to give rise to hematopoietic cell differentiation. Hematopoietic CD45(-) VE-cadherin+ alpha4-integrin+ cells were also present in the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our results suggest that the expression of alpha4-integrin is a marker of the earliest precursor of hematopoietic cell lineage that was diverged from endothelial progenitors.

We have investigated the lymphohematopoietic potentials of endothelial cells (EC) and hematopoietic cells (HPC) sorted from embryos. Expression of VE-cadherin, CD45, and Ter119 was used to distinguish EC (VE-cadherin+CD45-Ter119-) from HPC (VE-cadherin-CD45+). Thus defined, EC population takes up acetylated LDL and coexpresses CD31, Flk1, and CD34. In E9.5 embryos, EC from yolk sac (YS) and the embryo proper generate blood cells, including lymphocytes. Thus, lymphohematopoietic EC do exist in the embryo, and they are generated both in YS and the embryo proper. On the other hand, HPC with lymphopoietic potency appear first in the embryo proper. These findings implicate involvement of multiple environmental cues for acquiring lymphopoietic competency during differentiation of HPC.

The receptor tyrosine kinase Flk-2/Flt3 was originally cloned from hematopoietic stem cell-enriched fetal liver and placenta and is believed by some investigators to play a role in the regulation of the hematopoietic stem cell. However, targeted disruption of the flt3 gene results in a specific deficiency in early B cell progenitors. Using an antagonistic monoclonal antibody developed against the extracellular domain of Flt3, we investigated the expression and function of the molecule on B lymphoid lineage cells in the bone marrow (BM) of adult mice. Approximately 10% of B220+ cells in the BM expressed Flt3 on the cell surface, and most of the cells belonged to a pro-B cell fraction when judged by an expression pattern of CD43, heat-stable antigen, and BP-1. However, B lymphoid precursor cells that are clonable in vitro could not be enriched in the B220+/Flt3+ cell fraction sorted by flow cytometry. Furthermore, proliferation of B lymphoid precursor cells in the adult BM was not blocked by administration of the antagonistic monoclonal antibodies against Flt3 and c-Kit, suggesting that signalings mediated by Flt3 and c-Kit receptors are not essential for the proliferation of B cell progenitors in adult mouse BM.

During mammalian gastrulation, posterior part of primitive ectoderm differentiates into subsets of mesoderm cells, the most proximal portion of which subsequently gives rise to vascular endothelial cells and hematopoietic cells. To analyze this process in which the potency of multipotent epiblast cells is restricted progressively into hematopoietic cell lineages, we have established a culture system that allows differentiation of epiblast cells as well as embryonic stem cells into hematopoietic cells. Using this culture system, we showed that all parts of epiblast tissues at early streak and late bud stages preserved hematogenic potency, while it was lost from anterior region at early head fold stages. However, this loss of hematogenic potency in the anterior region of the head fold stage embryo was reinduced by addition of activin in the culture. Moreover, in order to detect transitory stages of mesoderm cells that are the direct progeny of multipotent epiblast cells, we developed three monoclonal antibodies that are able to define distinct subsets of mesoderm cells in the gastrulating embryo. These results are discussed from a view of cell-mass based commitment.

The establishment of culture conditions that selectively support hematopoietic stem cells is an important goal of hematology. In this study, we investigated the possibility of using for this purpose a defined medium, mSFO2, which was developed for stromal cell-dependent bone marrow cultures. We found that a combination of epidermal growth factor (EGF), the OP9 stromal cell line, which lacks macrophage colony-stimulating factor, recombinant stem cell factor, and the chemically defined medium mSFO2 provides a microenvironment where c-Kit+ Thy-1+/lo Mac-1+/lo B220- TER119- common beta + IL-2R gamma + gp130+ cells are selectively propagated from normal, unfractionated bone marrow cells. This cell population produced an in vitro colony at a very high efficiency (50%), whereas it has only limited proliferative ability in the irradiated recipient. Thus, the cells selected in this culture condition might represent colony-forming units in culture (CFU-c) with short-term reconstituting ability. Transferring this cell population into medium containing differentiation signals resulted in the rapid production of mature myelomonocytic and B cell lineages in vitro and in vivo. The fact that a similar culture condition was created by erb-B2-transduced OP9 in the absence of EGF indicated that EGF exerts its effect by acting on OP9 rather than directly on CFU-c. These results suggested that the balance between self-renewal and differentiation of CFU-c can be regulated by extra-cellular signals.

We analyzed the role of Fyn tyrosine kinase in cell cycle progression of B lymphocyte progenitor (pro B cell). Whereas there were no substantial defects in the intramarrow B cell genesis in the fyn(-) mouse, and long-term proliferation of fyn(-) pro B cells was maintained in vitro under a serum containing culture condition, the cell cycle was arrested at G2/M upon serum deprivation. Morphological analyses demonstrated that the cytokinesis of fyn(-) pro B cells was retarded in the presence of serum and that the entry of fyn(-) pro B cells into late telophase was completely blocked under the serum-free condition. In contrast, the earlier phases of mitosis of fyn(-) pro B cells proceeded normally without FCS. This failure to initiate late telophase resulted in the accumulation of elliptical binucleated cells that might be the outcome of the nuclear division without cytokinesis. Consistent with this defect in the progression of cytokinesis, Fyn was localized in the midspace of dividing pro B cells at anaphase. These results suggested that Fyn localizes at the midspace of dividing pro B cells and regulates the progression of cytokinesis.

Whereas the molecular natures of M-CSF/CSF-1 and its receptor c-fms are well characterized, its actual role in the intramarrow hematopoiesis remains obscure. This is because disruption of this signaling pathway results in the osteopetrosis mouse that lacks the bone cavity for hematopoiesis. To elucidate the role of c-fms in intramarrow hematopoiesis, we produced an antagonistic monoclonal antibody to murine c-fms and investigated its expression and function in the normal bone marrow. c-fms+ cells were detected both in mature and immature hematopoietic cells. Morphologically, c-kit+c-fms-, c-kit+c-fms+ and c-kit-c-fms+ cells were medium sized blasts, large promyelocytes with azurophilic granules and mature monocytes respectively. CFU-M was 10-fold more enriched in the c-kit+c-fms- than c-kit+c-fms+ fraction. Moreover, injection of the anti c-fms antibody had no effect on the production of CFU-M in the bone marrow, while anti-c-kit mAb could deplete them. As c-kit+c-fms+ cells were readily generated in the culture of c-kit+c-fms- cells, most of the CFU-M in the bone marrow are, in fact, c-fms- cells that differentiate into c-fms+ upon culture. These observations indicate a clear functional hierarchy of c-kit and c-fms in the bone marrow. Namely, c-kit plays the primary role in the production and maintenance of CFU-M, while c-fms, though it co-expressed with c-kit and functions as the growth receptor for M-CSF in the culture, has only a minimum role in the proliferation of c-fms+ cells in the bone marrow.

Recently, two genes, AML1 and ETO have been isolated from the chromosomal breakpoint of t(8;21). In this study, we isolated and identified fusion transcripts from a leukemic cell line carrying t(8;21). We demonstrated by PCR analysis that these transcripts are consistently expressed in fresh leukemic cells with t(8;21). On the other hand, the wild type of ETO is expressed in several hematopoietic cells from different lineage, while the expression of AML1 was present in all hematopoietic cells investigated. These widespread expression suggests these molecules play an essential role in hematopoiesis.

The t(8;21) is a common chromosomal abnormality, preferentially associated with acute leukemia showing features of myeloid differentiation. Recently, two genes--AML1, which has a unique runt domain, and ETO (MTG8)--have been isolated from the chromosomal breakpoint. In this study, we isolated and identified two fused transcripts from a leukemic cell line carrying t(8;21). AML1 and ETO were fused at the same position in these transcripts. One of the transcripts codes a unique domain, including two zinc finger domains and three proline- and one leucine-rich region. The other transcript codes only for one proline- and leucine-rich region but lacks zinc finger domains. We demonstrated by polymerase chain reaction (PCR) analysis that 1) these two transcripts are consistently expressed in leukemic cells with t(8;21) obtained from patients and 2) expression of AML1 was not restricted to the particular stage of hematopoietic differentiation but was present in all hematopoietic cells investigated. We also provide evidence that two wild types of ETO transcripts containing the region of the ETO gene in fused transcripts are expressed in hematopoietic cells from different lineages. The widespread expression of AML1 and ETO in hematopoietic cells suggests a fundamental role of these proteins in hematopoiesis. Furthermore, the differences in the carboxy termini of ETO may modulate the activity of fused proteins resulting from the chromosomal translocation t(8;21).

The proto-oncogene c-kit, encoding a receptor-type tyrosine kinase, is allelic with the W locus of the mouse. The stromal cell line OP9, capable of supporting long-term hematopoiesis, was newly established from a newborn B6C3F1-op/op mouse calvaria. When bone marrow cells of WBB6F1-W/Wv mice were cocultured with the OP9 cells in liquid medium, hematopoiesis declined to a level one-thousandth of that in the cocultures of bone marrow cells of WBB6F1-+/+ mice and stromal cells by day 21. In contrast, when bone marrow cells of W/Wv mice were cocultured with OP9 cells in semisolid medium, at least 61% of the number of colonies were detected until the end of our observation period of 42 days when compared with that in control cocultures, although colonies formed by hematopoietic stem cells of W/Wv mice were significantly smaller than those of normal stem cells. After a 24-hour incubation with OP9 cells, fewer stem cells of W/Wv mice than normal ones adhered to the stromal cells. Adhesion of normal stem cells to stromal cells was inhibited by the addition of an antagonists anti-c-kit monoclonal antibody, ACK2. These results demonstrate that the c-kit receptor plays an important role not only in the proliferative response of hematopoietic stem cells but also in their adhesion to stromal cells.

To examine the in vivo role of c-kit receptor in B lymphopoiesis we have evaluated precursor B cell populations expressing c-kit in mouse bone marrow and the effects on B cell genesis of administering a neutralizing anti-c-kit mAb, ACK2. Double immunofluorescence labeling and mitotic arrest were used to examine bone marrow cells from BALB/c mice. Almost one-half of TdT+ cells and one-quarter of B220+ cells coexpressed c-kit, mainly at low intensities, and were actively proliferating in vivo. c-kit+ cells that lacked B lineage markers expressed c-kit in high intensities and entered mitosis at an exceptionally rapid rate. In ACK2-treated mice, erythroid and granulocytic cells were almost completely absent from the bone marrow, whereas, in contrast, B lymphopoiesis was stimulated. Pre-B cells expressing cytoplasmic mu-chains; as well as TdT+B220+ cells before mu expression, were increased two- to fourfold in number and production rate. IgM-bearing B lymphocytes were increased in bone marrow and spleen. The results demonstrate that many early precursor B cells in mouse bone marrow constitutively express c-kit receptor. The failure of ACK2 treatment to block B lymphopoiesis, however, suggests that c-kit receptor function is not essential for precursor B cell development in vivo, but can be replaced by alternative signaling systems. The stimulation of B cell genesis by ACK2 treatment may reflect a conferred advantage in the competition for microenvironmental factors which underlies the balance between B lymphopoiesis and other hemopoietic lineages in vivo.

We have analyzed c-kit expression by hematopoietic progenitors from normal and 5-fluorouracil (5-FU)-treated mice by staining with monoclonal anti-c-kit antibody ACK-4. Marrow cells that were enriched for progenitors by a combination of metrizamide density separation and negative immunomagnetic selection with lineage-specific monoclonal antibodies (MoAbs) were separated into three populations based on the level of c-kit expression, c-kit(high), c-kit(low), and c-kit-. The majority of colony-forming cells from normal mice were in c-kit(high) population, whereas most of the progenitors from 5-FU-treated mice were in the c-kit(low) population. Optimal colony formation from c-kit(low) cells from 5-FU-treated mice required the interactions of at least two factors among interleukin-3 (IL-3), IL-11 and steel factor (SF) whereas colony formation from c-kit(high) cells of normal mice was supported well by IL-3 alone. Blast cells that were derived from 5-day culture of c-kit(low) post 5-FU cells were c-kit(high). These observations suggest that the primitive hematopoietic progenitors in cell cycle dormancy are c-kit(low) whereas actively cell cycling maturer progenitors are c-kit(high). Mature cells, with the exception of mast cells, derived from secondary culture of the c-kit(high) blast cells expressed little, if any, c-kit. These results are consistent with a model in which c-kit expression progresses from low levels on primitive, dormant multipotent progenitors to high levels on later, actively cycling progenitors, and finally, decreases to very low or undetectable levels on most mature blood cells, with the exception of mast cells.

We found that c-kit-positive, lineage marker-negative, Thy-1lo cells are present in both bone marrow and thymus ("BM c-kit" and "thymus c-kit" cells). Although the two cell types are phenotypically similar, only BM c-kit cells showed the potential to form colonies in vitro as well as in vivo. However, both of them revealed extensive growth and differentiation potential to T cells after direct transfer into an irradiated adult thymus, or a deoxyguanosine-treated fetal thymus. Time course analysis showed that thymus c-kit cells differentiated into CD4CD8 double-positive cells approximately 4 d earlier than BM c-kit cells did. In addition, anti-c-kit antibody blocked T cell generation of BM c-kit cells but not of thymus c-kit cells. Intravenous injection of thymus c-kit resulted in the generation of not only T cells, but B as well as NK1.1+ cells. These data provide evidence that thymus c-kit cells represent common lymphoid progenitors with the differentiation potential to T, B, and possibly NK cells. The c-kit-mediated signaling appears to be essential in the transition from BM c-kit to thymus c-kit cells.

Mouse pluripotent hematopoietic stem cells (PHSC) were fractionated based on size and density using counterflow centrifugal elutriation (CCE). These heterogeneous PHSC populations were further enriched by subtraction of cells with lineage-specific markers (Lin-) followed by positive sorting for c-kit expression. The cells were characterized for their functional and biochemical properties. We defined a subpopulation of c-kit-positive cells that expressed high numbers of c-kit receptors (c-kitBR). One hundred c-kitBR cells from either low- or higher-density fractions were sufficient to repopulate the lymphohematopoietic system in WBB6F1-W/Wv (W/Wv) recipients, whereas no PHSC were found in cells with low (c-kitDULL) or no (c-kitNEG) c-kit expression. Lin- c-kitBR cells were separated into RhoDULL and RhoBR subsets based on their ability to efflux rhodamine 123 (Rho). The PHSC were concentrated in Lin- c-kitBR RhoDULL cells and the number of Lin- c-kitBR RhoBR cells correlated directly with the number of day 12 colony-forming unit-spleen (CFU-S12) in each fraction. We were not able to enrich further for PHSC using monoclonal antibodies to the cell-surface markers AA4.1 or CD4, which have been used by others to isolate PHSC. The small, low-density Lin- c-kitBR subset contained PHSC and few CFU-S12. This enabled us to assay PHSC for expression of the flk-2 gene, which encodes a tyrosine kinase receptor present on fetal liver PHSC. Purified RNA from the low-density Lin- c-kitBR subset did not contain flk-2 mRNA. We suggest that AA4.1, CD4 and flk-2 are expressed as stage-specific markers on PHSC in cell cycle.

The SV40 large T gene under the control of immunoglobulin enhancer induced hyperproliferation of multilineage hematopoiesis in transgenic mice. Huge splenomegaly was the major gross abnormality; mice were rather anemic, and neither leukoerythroblastosis nor invasion into tissues such as liver, kidneys or lymph nodes was common. In the latter phases of the disease, the proliferating cell type tended to shift to a variety of single-lineage hematopoiesis, but the majority of mice still showed the presence of multilineage hematopoiesis; such cells were somewhat dysplastic but low in neoplastic potential. A long-term observation by transplantation of the hematopoietic cells into lethally irradiated C57BL/6 mice resulted in a variety of neoplastic growths in the recipients; not only was myelodysplastic hypercellularity seen, but also single-lineage hematopoietic malignancies such as B cell lymphomas/leukemias, histiocytic malignancies and even myeloid leukemias. The disease bore the proliferative feature solely in the spleen and bone marrow, and the transition from multilineage myelodysplasia into single-lineage hematopoiesis at some frequency is reminiscent of myelodysplastic syndromes (MDS) in humans. The results that the SV40 large T antigen was expressed in every proliferating cell, and that there was no apparent increase in any colony-stimulating cytokine(s), together with the results of the transplantation assays, suggested that the hyperproliferation of the hematopoietic cells was a direct consequence of the expression of SV40 large T antigen in these cells themselves.

The protooncogene c-kit encodes a receptor type tyrosine kinase and is allelic with the W locus of mice. SLF, the c-Kit ligand which is encoded by the Sl locus, has growth promoting activity for hemopoietic stem cells. Previous studies demonstrated that c-Kit is functionally required for the proliferation of hemopoietic progenitor cells at various differentiation stages in adult bone marrow. However, the absence of functional SLF and c-Kit in fetuses with mutant alleles of Sl and W loci produces only minor effects on the myeloid and early erythroid progenitor cells in the fetal liver, although the level of the late erythroid progenitor cells is significantly affected. We used an anti-c-Kit monoclonal antibody to investigate the expression and function of c-Kit in murine fetal hemopoietic progenitor cells. Flow-cytometric analysis showed that hemopoiesis in the yolk sac and fetal liver started from cells that express c-Kit. The c-Kit expression decreased upon maturation into erythrocytes in each organ. By fluorescence activated cell sorting, the c-Kit+ cell population was enriched with the hemopoietic progenitor cells clonable in vitro (CFU-E, BFU-E and GM-CFC). To elucidate whether c-Kit functions in these progenitor cells in vivo, we took advantage of the antagonistic anti-c-Kit monoclonal antibody, ACK2, which can block the function of c-Kit. Administration of ACK2 after 12.5 days of gestation rapidly eliminated BFU-E and GM-CFC as well as CFU-E from the fetal liver. However, the number of these progenitor cells in the yolk sac and fetal liver was less affected when the fetuses were given ACK2 before 12.5 days of gestation. Our results provide evidence that there are two waves of hemopoiesis in murine embryos relative to c-Kit dependency. The c-Kit has an essential role on the growth of hemopoietic progenitor cells in the fetal liver after 12.5 days of gestation, whereas the progenitor cells in the liver and yolk sac of the earlier embryo do not depend on c-Kit and its ligand SLF.

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker-negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c-kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c-kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.

The preadipose cell line, PA6, can support long-term hemopoiesis. Frequency of the hemopoietic stem cells capable of sustaining hemopoiesis in cocultures of bone marrow cells and PA6 cells for 6 wk was 1/5.3 x 10(4) bone marrow cells. In the group of dishes into which bone marrow cells had been inoculated at 2.5 x 10(4) cells/dish, 3 of 19 dishes (16%) contained stem cells capable of reconstituting erythropoiesis of WBB6F1-W/Wv mice, indicating that PA6 cells can support the proliferation of primitive hemopoietic stem cells. When the cocultures were treated with an antagonistic anti-c-kit monoclonal antibody, ACK2, only a small number of day 12 spleen colony-forming units survived; and hemopoiesis was severely reduced. However, when the cocultures were continued with antibody-free medium, hemopoiesis dramatically recovered. To examine the proliferative properties of the ACK2-resistant stem cells, we developed a colony assay system by modifying our coculture system. Sequential observations of the development of individual colonies and their disappearance demonstrated that the stem cells having higher proliferative capacity preferentially survive the ACK2 treatment. Furthermore, cells of subclones of the PA6 clone that were incapable of supporting long-term hemopoiesis expressed mRNA for the c-kit ligand. These results suggest that a mechanism(s) other than that involving c-kit receptor and its ligand plays an important role in the survival and proliferation of primitive hemopoietic stem cells.

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK-2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr-1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c-kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.

The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c-kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.

We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7-producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.