Kit regulates maintenance of quiescent hematopoietic stem cells

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (Kit(W41/W41)) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult Kit(W41/W41) mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin(-)Sca-1(+)Kit(high) (LSK)CD34(-)Flt3(-) long-term HSCs by 12 wk of age, whereas LSKCD34(+)Flt3(-) short-term HSCs and LSKCD34(+)Flt3(+) multipotent progenitors were less affected. Whereas homing and initial reconstitution of Kit(W41/W41) bone marrow cells in myeloablated recipients were close to normal, self-renewing Kit(W41/W41) HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation Kit(W41/W41) HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.     

c-Kit mutant mouse behavioral phenotype: altered meal patterns and CCK sensitivity but normal daily food intake and body weight

The mouse W/Wv mutation of the c-Kit receptor causes extensive loss of gastrointestinal interstitial cells of Cajal and vagal intramuscular arrays (IMAs; one of the two putative mechanoreceptors in gastrointestinal smooth muscle). To characterize the behavioral phenotype of the c-Kit mouse and to evaluate the roles of these mechanoreceptors in controlling food intake, meal patterns and daily intakes of W/Wv mice and controls were examined using solid (20-mg pellets) and liquid (Isocal) maintenance diets. After the meal pattern experiments, CCK (0.5, 1, 2, 4, 8, and 16 microg/kg ip) was administered to examine the role of the interstitial cells and vagal IMA mechanoreceptors in relaying peripheral signals of satiety activated by CCK-A receptors, whereas the specificity of the response was assessed with the antagonist devazepide (300 microg/kg ip). On both diets, the W/Wv mice ate smaller meals for shorter durations, with a compensatory increase in meal number, resulting in daily intakes and body weights similar to the controls. After CCK injections, the mutant mice consistently suppressed intake more ( approximately 2x) in 30-min tests, regardless of the test diet (12.5% glucose, chow, pellets, and Isocal). The increased sensitivity of W/Wv mice to CCK reflected an increased potency of the hormone (c-Kit mouse ED50 = 2.4 microg/kg; control ED50 = 6.4 microg/kg) and a shift of the dose-response curve to the left. Devazepide blocked the CCK suppression of ingestion. These results indicate that the selective loss of the interstitial cells and IMAs disrupts short-term feeding of the W/Wv mice by inducing an earlier satiety, possibly by altering gastric accommodation and/or emptying, without affecting the long-term mechanisms controlling overall intake or body weight. The results also suggest that the reduction of interstitial cells and IMAs augments the sensitivity to or increases the efficiency of exogenous CCK.     

Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment

The mechanism by which angiogenic factors recruit bone marrow (BM)-derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor-1 (VEGFR1) is expressed on human CD34(+) and mouse Lin(-)Sca-1(+)c-Kit(+) BM-repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1, but not VEGFR2, blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Placental growth factor (PlGF), which signals through VEGFR1, restored early and late phases of hematopoiesis following BM suppression. PlGF enhanced early phases of BM recovery directly through rapid chemotaxis of VEGFR1(+) BM-repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF-induced upregulation of matrix metalloproteinase-9, mediating the release of soluble Kit ligand. Thus, PlGF promotes recruitment of VEGFR1(+) HSCs from a quiescent to a proliferative BM microenvironment, favoring differentiation, mobilization and reconstitution of hematopoiesis.     

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.     

c-Kit-mediated functional positioning of stem cells to their niches is essential for maintenance and regeneration of adult hematopoiesis

The mechanism by which hematopoietic stem and progenitor cells (HSPCs) through interaction with their niches maintain and reconstitute adult hematopoietic cells is unknown. To functionally and genetically track localization of HSPCs with their niches, we employed novel mutant loxPs, lox66 and lox71 and Cre-recombinase technology to conditionally delete c-Kit in adult mice, while simultaneously enabling GFP expression in the c-Kit-deficient cells. Conditional deletion of c-Kit resulted in hematopoietic failure and splenic atrophy both at steady state and after marrow ablation leading to the demise of the treated adult mice. Within the marrow, the c-Kit-expressing GFP(+) cells were positioned to Kit ligand (KL)-expressing niche cells. This c-Kit-mediated cellular adhesion was essential for long-term maintenance and expansion of HSPCs. These results lay the foundation for delivering KL within specific niches to maintain and restore hematopoiesis.     

Innate immune adaptor MyD88 mediates neutrophil recruitment and myocardial injury after ischemia-reperfusion in mice

MyD88 is an adaptor protein critical for innate immune response against microbial infection and in certain noninfectious tissue injury. The present study examined the role of MyD88 in myocardial inflammation and injury after ischemia-reperfusion (I/R). I/R was produced by coronary artery ligation for 30 min followed by reperfusion. The ratios of area at risk to left ventricle (LV) were similar between wild-type (WT) and MyD88-deficient (MyD88-/-) mice. However, 24 h after I/R, the ratios of myocardial infarction to area at risk were 58% less in MyD88(-/-) than in WT mice (14 +/- 2% vs. 33 +/- 6%, P = 0.01). Serial echocardiographic studies demonstrated that there was no difference in baseline LV contractile function between the two groups. Twenty-four hours after I/R, LV ejection fraction (EF) and fractional shortening (FS) in WT mice were reduced by 44% and 62% (EF, 51 +/- 2%, and FS, 22 +/- 1%, P < 0.001), respectively, and remained depressed on the seventh day after I/R. In comparison, EF and FS in MyD88(-/-) mice were 67 +/- 3% and 33 +/- 2%, respectively, after I/R (P < 0.001 vs. WT). Similarly, LV function, as demonstrated by invasive hemodynamic measurements, was better preserved in MyD88(-/-) compared with WT mice after I/R. Furthermore, when compared with WT mice, MyD88(-/-) mice subjected to I/R had a marked decrease in myocardial inflammation as demonstrated by attenuated neutrophil recruitment and decreased expression of the proinflammatory mediators keratinocyte chemoattractant, monocyte chemoattractant protein-1, and ICAM-1. Taken together, these data suggest that MyD88 modulates myocardial inflammatory injury and contributes to myocardial infarction and LV dysfunction during I/R.     

Hemopoietic precursor cell defects in nonanemic but stem cell-deficient W44/W44 mice

Hematopoietic stem cell deficiencies cause a severe macrocytic anemia in W/Wv mice. W44/W44 mice, on the other hand, are not anemic, but, since they accept marrow implants without prior total body irradiation, they have inherited a stem cell lesion. In an attempt to identify the aberrant stem cell(s), we have determined the concentration in W44/W44 marrow of hematopoietic precursors known to be deficient in W/Wv marrow. The in vitro erythroid burst-forming units (BFU-E), the in vivo spleen colony-forming units (CFU-S), and the cells that repopulate the erythroid compartment of stem cell-deficient mice were examined. The progenitors of 7-day bursts are dramatically reduced in W/Wv marrow but are present in normal concentrations in W44/W44 marrow. W44/W44 marrow CFU-S, unlike W/Wv, generate visible spleen colonies 10 days after injection into lethally irradiated recipients. The colonies are, however, smaller and at least 2 times less numerous than those produced from equivalent numbers of +/+ marrow. An additional defect was the inability of W44/W44 stem cells to compete with genetically marked +/+ cells during erythroid repopulation. An estimate of the number of W44/W44 stem cells needed to compete with +/+ cells was provided by enriching W44/W44 progenitors fivefold. Twice as many enriched W44/W44 marrow cells as unfractionated +/+ cells were required to replace competitor cells. This suggests that there are up to 10 times fewer stem cells somewhere in the W44/W44 erythrogenerative pathway. The data support the conclusion that an erythroid progenitor less mature than the BFU-E is one of the cells most severely affected by expression of the mutant gene.     

The mast cell-committed progenitor. II. W/Wv mice do not make mast cell-committed progenitors and S1/S1d fibroblasts do not support development of normal mast cell-committed progenitors

We have previously reported that the population of mesenteric lymph node cells from normal BALB/c mice infected 14 days with the rodent nematode Nippostrongylus brasiliensis (Nb-MLN) contains a nongranulated mast cell-committed progenitor (MCCP) which does not require IL-3 for proliferation and differentiation if either a fibroblast monolayer or soluble factors produced by monolayers of 3T3 fibroblasts or embryonic skin are present in the culture. When Nb-MLN were cloned in a methylcellulose culture system using fibroblast conditioned medium as the only source of growth factors, numerous colonies of pure mast cells developed. We wished to determine whether the mast cell deficiency of W/Wv or S1/S1d mice could be explained by the failure of these mice to make either the MCCP or the factor to support proliferation and differentiation of the MCCP. We found that Nb-MLN from W/Wv mice were only able to produce mast cell colonies in response to a source of IL-3 such as conditioned medium from pokeweed mitogen-stimulated spleen cells (CM), and cultures given fibroblast conditioned medium as the only source of growth factors did not produce mast cell colonies. In contrast, Nb-MLN from mast cell deficient S1/S1d mice developed many mast cell colonies in methylcellulose cultures supplemented with either fibroblast conditioned medium or conditioned medium from PWM-stimulated spleen cells. These data suggest that S1/S1d mice but not W/Wv mice produce the mast cell progenitor that responds to fibroblast conditioned medium. To determine if mast cell deficient mice make the fibroblast derived factors that support development of the MCCP, monolayers were prepared from skin connective tissues of S1/S1d and W/Wv mice and Nb-MLN from normal BALB/c mice were cloned in the presence of conditioned medium from these monolayers. Fibroblast conditioned medium from monolayers prepared from W/Wv but not S1/S1d mice supported development of numerous mast cell colonies. Taken together, these data demonstrate that W/Wv mice are incapable of producing normal MCCP whereas S1/S1d fibroblasts fail to produce the appropriate factor to support the MCCP. In accordance with these data, a candidate for the gene product of each of these mutant alleles is discussed.     

Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.     

Resistance to friend virus-induced erythroleukemia in W/W(v) mice is caused by a spleen-specific defect which results in a severe reduction in target cells and a lack of Sf-Stk expression

The characteristic progression and specificity of Friend virus for the erythroid lineage have allowed for the identification of a number of host-encoded loci that are required for disease progression. Several of these loci, including the Friend virus susceptibility gene 2 (Fv2), dominant white spotting gene (W), and Steel gene (Sl), regulate the initial polyclonal expansion of infected erythroid progenitor cells. W and Sl encode the Kit receptor tyrosine kinase and its ligand, stem cell factor, respectively. W mutant mice are severely anemic, and earlier work suggested that this defect in erythroid differentiation is the cause for the resistance to Friend virus-induced erythroleukemia. Here we show that in bone marrow, W/W(v) mice have near normal numbers of target cells and the initial infection of bone marrow occurs normally in vivo. In contrast, spleen cells from W/W(v) mice infected both in vitro and in vivo with Friend virus failed to give rise to erythropoietin-independent colonies at any time following Friend virus infection, suggesting that mutation of the Kit receptor specifically affects target cells in the spleen, rendering the mutant mice resistant to the development of Friend virus-induced erythroleukemia. In addition, we show that the Kit+ pathogenic targets of Friend virus in the spleen are distinct from the pathogenic targets in bone marrow and this population of spleen target cells is markedly decreased in W/W(v) mice and these cells fail to express Sf-Stk. These results also underscore the unique nature of the spleen microenvironment in its role in supporting the progression of acute leukemia in Friend virus-infected mice.     

Heat shock factor 1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of bone marrow stem/progenitor cells

Bone marrow (BM)-derived stem/progenitor cells play an important role in ischemia-induced angiogenesis in cardiovascular diseases. Heat shock factor 1 (HSF1) is known to be induced in response to hypoxia and ischemia. We examined whether HSF1 contributes to ischemia-induced angiogenesis through the mobilization and recruitment of BM-derived stem/progenitor cells using HSF1-knockout (KO) mice. After the induction of ischemia, blood flow and microvessel density in the ischemic hindlimb were significantly lower in the HSF1-KO mice than in the wild-type (WT) mice. The mobilization of BM-derived Sca-1- and c-kit-positive cells in peripheral blood after ischemia was significantly lower in the HSF1-KO mice than in the WT mice. BM stem/progenitor cells from HSF1-KO mice showed a significant decrease in their recruitment to ischemic tissue and in migration, adhesion, and survival when compared with WT mice. Blood flow recovery in the ischemic hindlimb significantly decreased in WT mice receiving BM reconstitution with donor cells from HSF1-KO mice. Conversely, blood flow recovery in the ischemic hindlimb significantly increased in HSF1-KO mice receiving BM reconstitution with donor cells from WT mice. These findings suggest that HSF1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of BM-derived stem/progenitor cells.     

An assessment of mast-cell deficient mice (W/Wv) as a model system to study the role of histamine in implantation and deciduoma formation

The ovaries from mast cell-normal (+/+) and mast cell-deficient (W/Wv) mice were examined with light and electron microscopy. In addition the effect of ovariectomy and subsequent steroid treatment on total uterine histamine content, total mast cell numbers and surface and glandular epithelial cell heights was measured. The ovaries of +/+ mice were normal, displaying various stages of follicular growth and atresia and numerous corpora lutea; the ovaries of W/Wv mice lacked follicles and corpora lutea but contained numerous hyperplastic interstitial cells which contained numerous lipid droplets, vesiculated mitochondria and abundant endoplasmic reticulum suggestive of steroid synthesis. Steroid treatment of ovariectomized +/+ and W/Wv mice caused a significant increase in uterine wet weight and endometrial surface and glandular epithelial cell heights. In +/+ mice, steroid treatment caused a concomitant increase in total mast cells per uterine horn while mast cells were totally absent in W/Wv mice. The increase in uterine histamine in +/+ mice is consistent with the increase in mast cell numbers. Measurable amounts of uterine histamine, which increases slightly after steroid treatment, were demonstrated in W/Wv mice. Since the uteri of +/+ and W/Wv mice respond to steroids in a similar manner with the sole exception being histamine content and mast cell numbers, our results demonstrate the potential of using these animals to investigate the role(s) of uterine mast cells and non-mast cell uterine histamine in the process of implantation and the formation of a decidual cell response.     

Bcl6 is required for the development of mouse CD4+ and CD8α+ dendritic cells

Th2-type inflammation spontaneously shown in Bcl6-knockout (KO) mice is mainly caused by bone marrow (BM)-derived nonlymphoid cells. However, the function of dendritic cells (DCs) in Bcl6-KO mice has not been reported. We show in this article that the numbers of CD4(+) conventional DCs (cDCs) and CD8α(+) cDCs, but not of plasmacytoid DCs, were markedly reduced in the spleen of Bcl6-KO mice. Generation of cDCs from DC progenitors in BM cells was perturbed in the spleen of irradiated wild-type (WT) mice transferred with Bcl6-KO BM cells, indicating an intrinsic effect of Bcl6 in cDC precursors. Although cDC precursors were developed in a Bcl6-KO BM culture with Fms-like tyrosine kinase 3 ligand, the cDC precursors were more apoptotic than WT ones. Also p53, one of the molecular targets of Bcl6, was overexpressed in the precursors. The addition of a p53 inhibitor to Bcl6-KO BM culture protected apoptosis, suggesting that Bcl6 is required by cDC precursors for survival by controlling p53 expression. Furthermore, large numbers of T1/ST2(+) Th2 cells were naturally developed in the spleen of Bcl6-KO mice. Th2 skewing was accelerated in the culture of WT CD4 T cells stimulated with Ags and LPS-activated Bcl6-KO BM-derived DCs, which produced more IL-6 and less IL-12 than did WT DCs; the addition of anti-IL-6 Abs to the culture partially abrogated the Th2 skewing. These results suggest that Bcl6 is required in cDC precursors for survival and in activated DCs for modulating the cytokine profile.     

IFN-gamma production by intrinsic renal cells and bone marrow-derived cells is required for full expression of crescentic glomerulonephritis in mice

The contribution of IFN-gamma from bone marrow (BM) and non-BM-derived cells to glomerular and cutaneous delayed-type hypersensitivity (DTH) was studied in mice. Chimeric IFN-gamma mice (IFN-gamma(+/+) BM chimera), in which IFN-gamma production was restricted to BM-derived cells, were created by transplanting normal C57BL/6 (wild-type (WT)) BM into irradiated IFN-gamma-deficient mice. BM IFN-gamma-deficient chimeric mice (IFN-gamma(-/-) BM chimera) were created by transplanting WT mice with IFN-gamma-deficient BM. WT and sham chimeric mice (WT mice transplanted with WT BM) developed crescentic glomerulonephritis (GN) with features of DTH (including glomerular T cell and macrophage infiltration) in response to an Ag planted in their glomeruli and skin DTH following subdermal Ag challenge. IFN-gamma-deficient mice showed significant protection from crescentic GN and reduced cutaneous DTH. IFN-gamma(+/+) BM chimeric and IFN-gamma(-/-) BM chimeric mice showed similar attenuation of crescentic GN as IFN-gamma-deficient mice, whereas cutaneous DTH was reduced only in IFN-gamma(-/-) BM chimeras. In crescentic GN, IFN-gamma was expressed by tubular cells and occasional glomerular cells and was colocalized with infiltrating CD8(+) T cells, but not with CD4(+) T cells or macrophages. Renal MHC class II expression was reduced in IFN-gamma(+/+) BM chimeric mice and was more severely reduced in IFN-gamma-deficient mice and IFN-gamma(-/-) BM chimeric mice. These studies show that IFN-gamma expression by both BM-derived cells and intrinsic renal cells is required for the development of crescentic GN, but IFN-gamma production by resident cells is not essential for the development of cutaneous DTH.     

Prolonged expulsion of adult Trichinella spiralis and eosinophil infiltration in mast cell-deficient W/Wv mice

Eosinophil infiltrations were observed in the intestine and the muscle of both Trichinella spiralis-infected (WBxC57BL/6)F1-W/Wv mice and their littermates, WBB6F1-+/+, +/W, +/Wv, almost to the same extent. W/Wv mice did not show infiltration of subepithelial mast cells and globule leucocytes in response to T. spiralis infection. Increased numbers of these cells were observed in their littermates. Worms in W/Wv mice were retained for longer periods than those in littermates. Also, no difference was noted in the production of specific serum antibodies between W/Wv mice and their littermates, as determined by passive cutaneous anaphylaxis (PCA) for specific IgE and by indirect haemagglutination (IHA). These results suggest a possible participation of SMC, GL and eosinophils in the expulsion of adult T. spiralis.     

Endothelial Cell-Selective Adhesion Molecule Expression in Hematopoietic Stem/Progenitor Cells Is Essential for Erythropoiesis Recovery after Bone Marrow Injury

Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.     

Fibroblast-dependent growth of mouse mast cells in vitro: duplication of mast cell depletion in mutant mice of W/Wv genotype

In spite of the apparent depletion of mast cells in tissues of mutant mice of W/Wv genotype, cells with many features of mast cells do develop when bone marrow cells of W/Wv mice are cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). In order to resolve this discrepancy and facilitate the analysis of the W mutation, we attempted to establish an in vitro system in which the in vivo defect of W/Wv mice can be reproduced. Cultured mast cells (CMC) were developed from bone marrow cells of either W/Wv or congenic +/+ mice, and then co-cultured with NIH/3T3 mouse fibroblasts in media supplemented only with fetal calf serum (i.e., in the absence of PWM-SCM). Under this condition, CMC from +/+ mice continued to divide and were maintained for more than 4 weeks. The supportive effect of NIH/3T3 cells required close-range interactions with CMC and was not due to synthesis of the known mast cell growth factors, interleukins 3 and 4. By contrast, CMC from W/Wv mice were not maintained, and the number of mast cells remaining after 4 weeks of co-culture was only 1% of the normal +/+ counterparts. Thus, the humoral factor-independent and cell contact-dependent system presented here revealed the intrinsic defects in growth and differentiation of CMC derived from W/Wv mice and might be useful for biochemical and molecular analysis of the gene product(s) encoded at the W locus.     

Transplantation of male germ line stem cells restores fertility in infertile mice

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.     

Mast cells are important in the development of hypersensitivity pneumonitis. A study with mast-cell-deficient mice

We have previously reported that C57B1/6 mice develop lung lesions similar to human hypersensitivity pneumonitis (HP) by repeated transnasal administration of Thermoactinomyces vulgaris antigen. Since the HP-like lesions were induced via respiratory route and by the causative antigen in human HP (farmer's lung), it seems that this murine model is useful for investigating the cell-to-cell interactions in human HP. To clarify the involvement of mast cells (MC) in the development of HP, T. vulgaris (90 micrograms/day) was transnasally administered to MC-deficient WBB6F1-W/Wv mice (W/Wv) and their littermates (+/+) five times a wk for 3 wk. When the lungs were examined by scoring pathological findings and lung indexes, HP-like lesions were significantly less severe in W/Wv than in +/+, whose lesions were equivalent to those of C57B1/6. Bone-marrow-derived cultured MC from +/+ mice (98% purity) were obtained by in vitro culture mixed with WEHI-3B-derived conditioned medium which contained IL-3. When these MC were adoptively transferred to W/Wv mice (10(7) cells/mouse), the HP-like lesions in W/Wv mice were enhanced to be as severe as those in +/+. Importantly, significant numbers of MC were found in the lungs of MC-transferred W/Wv mice. These results suggest that MC play an important role in the development of the murine experimental HP.