Roles of Sca-1 in hematopoietic stem/progenitor cell function

OBJECTIVE: This study was focused on studying the role of Sca-1 (Ly-6 A/E) in hematopoietic stem/progenitor cell self-renewal, activation, and lineage fate. MATERIALS AND METHODS: Sca-1(-/-) bone marrow cells were transplanted into wild-type recipient mice and assessed for self-renewal activity and lineage choice. In addition, Sca-1(-/-) mice were injected with 5-FU and Lin(-) cells were analyzed. Sca-1 was also overexpressed in mouse and human stem/progenitor cells to assess the effect of Sca-1 overexpression on stem/progenitor differentiation and proliferation. RESULTS: Self-renewal of Sca-1(-/-) HSC appeared to be normal, but lineage skewing was observed in B cells, NK cells, and granulocytes/macrophages derived from Sca-1(-/-) HSC. There was also a decrease in c-kit expression on activated Sca-1(-/-) progenitor cells. Overexpression of mouse Sca-1 decreased the in vitro myeloid activity of both mouse and human progenitors. CONCLUSION: These data indicate that Sca-1 plays a role in hematopoietic progenitor/stem cell lineage fate and c-kit expression. In addition, mouse Sca-1 overexpression affects human as well as mouse stem/progenitor cell activity, suggesting the possibility of a functional human Sca-1 homologue.     

Inhibitory effects of tumor necrosis factor on hematopoiesis seen in vitro are translated to increased numbers of both committed and multipotent progenitors in TNF-deficient mice

OBJECTIVES: The effects of TNF deficiency on myelopoiesis were evaluated in long-term (LTBMC) and short-term bone marrow cultures (STBMC) and compared to hematopoietic activity in vivo in TNF-deficient mice. METHODS: LTBMC and STBMC were established from bone marrow of TNF-deficient mice in the presence or absence of soluble TNF. Total cell production was measured over time, as well as the number of colony-forming units in culture (CFU-C). Morphology of nonadherent (NA) cells in LTBMC was assessed after 10 weeks. Bone marrow cells (BMC) and peripheral blood (PB) cells were used to determine lineage distribution within the hematopoietic system. BMC were sorted to obtain Lin(-)c-kit(+)Sca-1- and Lin(-)c-kit(+)Sca-1+ cells, which were plated in semisolid media to determine CFU-C numbers or injected into irradiated recipients to determine colony formation in the spleen (CFU-S). RESULTS: TNF-deficient LTBMC and STBMC show increased proliferative capacity, which can be inhibited by exogenous TNF to wild-type levels. Morphological analysis of NA cells from TNF-deficient LTBMC revealed increased numbers of cells at early stages of granulocytic differentiation (myeloblasts/promyelocytes) paralleled by a sharp decrease in the number of terminally differentiated polymorphonuclear neutrophils. Slightly elevated numbers of leukocytes, mainly neutrophils, were detected in PB of TNF-deficient mice. In bone marrow of TNF-deficient mice a significant increase in the number of both CFU-GM within Lin(-)c-kit(+)Sca-1- population and CFU-S within Lin(-)c-kit(+)Sca-1+ population was observed. CONCLUSIONS: TNF has inhibitory effects on granulocyte-macrophage progenitors in vitro and on committed and primitive hematopoietic progenitors in vivo. However, in adult organism TNF deficiency is mostly compensated and controlled.     

Hematopoietic origin of glomerular mesangial cells

It was recently reported that crude bone marrow cells have the ability to differentiate into glomerular mesangial cells. However, the exact nature of the engrafting cells in the bone marrow was not known. We tested the hypothesis that hematopoietic stem cells are capable of reconstituting the mesangial cells by transplanting a clonal population of cells derived from a single stem cell. We cultured Lin(-), Sca-1(+), c-kit(+), CD34(-) bone marrow cells from transgenic enhanced green fluorescent protein (EGFP) mice (C57BL/6-Ly-5.2 background) individually for 1 week in the presence of interleukin-11 and steel factor. We then transplanted viable clones individually into lethally irradiated C57BL/6-Ly-5.1 mice. Kidneys from 5 recipient mice showing high levels (60%-90%) of multilineage hematopoietic reconstitution were examined 2 to 6 months later, using differential interference contrast and epifluorescence microscopy. EGFP(+) cells with a morphology characteristic of mesangial cells were evident within the glomeruli. Transplantation of 100 noncultured Lin(-), Sca-1(+), c-kit(+), CD34(-) bone marrow cells also generated mesangial cells. Cultured EGFP(+) glomerular cells from recipient mice contracted in response to angiotensin II. EGFP(+) mesangial cells seen in male-to-male transplants revealed only one Y-chromosome. These data demonstrate that a single hematopoietic stem cell is capable of differentiating into glomerular mesangial cells and that the process does not involve cell fusion.     

Expression change of Flk-2/Flt-3 on murine hematopoietic stem cells in an activating state

OBJECTIVE: The receptor tyrosine kinase Flk-2/Flt-3 (Flt-3) represents an important molecule involved in early hematopoiesis. Murine hematopoietic stem cells (HSCs) have been shown to be negative for the expression of Flt-3. We now present clear evidence for the expression change of Flt-3(-) HSCs in an activating state, and the reversibility of Flt-3 expression by HSCs in vivo. MATERIALS AND METHODS: Bone marrow cells isolated from Ly5.1 mice were sorted on the basis of Flt-3 expression and transplanted into lethally irradiated Ly5.2 recipients. After 24 weeks, peripheral blood was analyzed for donor contribution by flow cytometry. RESULTS: Although long-term engraftment was predominantly detected in Flt-3(-) populations as previously described, a 6-day cultivation of Lin(-/low)c-kit(+)Sca-1(+) Flt-3(-) bone marrow cells with stem cell factor and interleukin-11 resulted in the generation of Flt-3(+) HSCs with long-term engraftment capabilities. However, the Flt-3 ligand had no significant effect on self-renewal of the Flt-3(+) HSCs. Next, to examine reversible expression of this receptor molecule, Flt-3(+) cells converted in vitro from Ly5.1 Lin(-/low)c-kit(+)Sca-1(+) Flt-3(-) bone marrow cells were isolated and transplanted into Ly5.2 primary recipients. After 24 weeks, Ly5.1 Lin(-/low) bone marrow cells were again separated into Flt-3(-) and Flt-3(+) cells and retransplanted into Ly5.2 secondary recipients. The majority of donor HSCs with long-term engraftment capabilities were detected in the Flt-3(-) populations, indicating the reversion of Flt-3(+) to Flt-3(-) HSCs. CONCLUSIONS: These observations suggest that Flt-3 is a useful cell-surface marker of HSC activation and that this phenotypic change is reversible.     

Expansion of hematopoietic stem cell phenotype and activity in Trp53-null mice

OBJECTIVES: To study the effects of transformation-related protein 53 (Trp53) and other genes on hematopoiesis and hematopoietic stem cells (HSCs). METHODS: Frequencies of murine bone marrow cells (BMCs) with the Lin(-)Sca-1(+)c-kit(+)CD34- phenotype were analyzed by flow cytometry, and were increased in mice with germ-line deletion of the Trp53 (Trp53(-/-)) gene but not in 25 other deletions of genes involved in cell cycling, development, cancer, or hematopoiesis. Therefore, Trp53(-/-) and wild-type Trp53(+/+) mice were compared using the following assays: complete blood counts, day-9 colony-forming unit spleen (CFU-S), and competitive repopulation. In the latter assay, donor repopulating ability was analyzed at one, three, and five months, while recipient survival and recipient blood and bone marrow cell composition were analyzed at five months, after transplantation. RESULTS: In comparison to wild-type controls, Trp53(-/-) mice had normal blood and bone marrow cell counts, increased CD11b(+), and decreased CD45R(+) cell proportions in blood and bone marrow, twice as many Lin(-)Sca-1(+)c-kit(+)CD34(-) BMCs, and 37% more day-9 CFU-S. In the competitive repopulation assay, Trp53(-/-) BMCs engrafted lethally irradiated recipients two to four times better than Trp53(+/+) BMCs. The Trp53(-/-) engraftment advantage increased with time in the recipients. Recipients of Trp53(-/-) donors had two to three times more Lin(-)Sca-1(+)c-kit(+)CD34(-) BMCs than recipients of Trp53(+/+) donors at five months after transplantation. However, only 44% of recipients of Trp53(-/-) donors survived five months after trans-plantation, compared with 92% of recipients of Trp53(+/+) donors. CONCLUSION: The Trp53-null allele expands bone marrow Lin(-)Sca-1(+)c-kit(+)CD34(-) cells and the overall activity of HSCs; however, it increases recipient mortality.     

Hematopoietic potential of murine skeletal muscle-derived CD45(-)Sca-1(+)c-kit(-) cells

OBJECTIVE: Somatic stem cells, which are poorly defined in postnatal mammalian tissues, are attractive candidates for examination of stem cell plasticity. Our goal was to determine the identity of neonatal muscle-derived cells that contain hematopoietic potential and to explore the status of CD45 expression on these cells. MATERIALS AND METHODS: Skeletal muscle from thighs of 4- to 7-day-old mice was harvested, enzymatically digested, and flow cytometrically sorted to yield CD45(-)Sca-1(+)c-kit(-) cells. These cells were examined in hematopoietic colony-forming assays and competitive repopulation assays, and were expanded ex vivo. Additionally, CD45, c-kit, PU.1, and beta globin major expression was tracked over time in cultured cells to assess the possibility of manipulating stem cell differentiation in vitro. RESULTS: Freshly isolated CD45(-)Sca-1(+)c-kit(-) cells were devoid of hematopoietic lineage markers and contained no colony-forming activity but displayed superior long-term competitive repopulating ability when compared to freshly isolated muscle-derived CD45(+)Sca-1(+)c-kit(+) cells. CD45(-)Sca-1(+)c-kit(-) cells expanded ex vivo in 5 ng/mL murine stem cell factor, mFlt-3L, and megakaryocyte growth and development factor (MGDF) for 9 days increased their in vivo hematopoietic repopulating potential 5.3-fold relative to fresh cells. Although fresh cells did not transcribe mRNA of several hematopoietic genes, a small fraction of cells cultured for 9 days acquired cell surface c-kit, and only these cells expressed c-kit and PU.1 mRNA and maintained competitive repopulating ability, suggesting at least myeloid and perhaps lymphoid developmental potential. CONCLUSION: Neonatal murine muscle-derived cells expressing the phenotype CD45(-)Sca-1(+) c-kit(-) are putative adult somatic stem cells with in vitro and in vivo hematopoietic differentiation potential.     

Deficiency of Src family kinases compromises the repopulating ability of hematopoietic stem cells

OBJECTIVE: Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. RESULTS: Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1(+)Lin(-) cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK(-/-) cells, although, this increase did not affect the homing potential of more primitive Lin(-)Sca-1(+) SFK(-/-) cells. The stem cell-repopulating defect observed in mice transplanted with SFK(-/-) bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK(-/-) bone marrow cells. CONCLUSIONS: Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells.     

Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells, and overexpression of Hoxa-9 markedly expands hematopoietic stem cells, suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However, sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia, indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays, Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability, a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures, Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation, with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo.     

Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to early-acting cytokines

Quantitative trait analysis may shed light on mechanisms regulating hematopoiesis in vivo. Strain-dependent variation existed among C57BL/6 (B6), DBA/2, and BXD recombinant inbred mice in the responsiveness of primitive progenitor cells to the early-acting cytokines kit ligand, flt3 ligand, and thrombopoietin. A significant quantitative trait locus was found on chromosome 2 that could not be confirmed in congenic mice, however, probably because of epistasis. Because it has been shown that alleles of unknown X-linked genes confer a selective advantage to hematopoietic stem cells in vivo in humans and in cats, we also analyzed reciprocal male D2B6F1 and B6D2F1 mice, revealing an X-linked locus regulating the responsiveness of progenitor and stem cells to early-acting factors. Among DBA/2, B6, and BXD recombinant inbred mice, correlating genetic variation was found in the absolute number and frequency of Lin(-)Sca1(++)kit(+) cells, which are highly enriched in hematopoietic progenitor and stem cells, and in the number of Lin(-)Sca1(++)kit(-) cells, a population whose biologic significance is unknown, suggesting that both populations are functionally related. Suggestive quantitative trait loci (QTLs) for the number of Lin(-)Sca1(++) cells on chromosomes 2, 4, and 7 were confirmed in successive rounds of mapping. The locus on chromosome 2 was confirmed in congenic mice. We thus demonstrated genetic variation in the response to cytokines critical for hematopoiesis in vivo and in the pool size of cells belonging to a phenotype used to isolate essentially pure primitive progenitor and stem cells, and we identified loci that may be relevant to the regulation of hematopoiesis in steady state.     

Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR(+) cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin(-/lo)Sca-1(+) and c-kit(+)Sca-1(+) subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR(+)c-kit(+)Sca-1(+) bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR(+) cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.     

Enhanced green fluorescent protein targeted to the Sca-1 (Ly-6A) locus in transgenic mice results in efficient marking of hematopoietic stem cells in vivo

OBJECTIVE: Hematopoietic stem cells are important clinically, both as targets of disease and as reagents for cellular therapy. Studies in hematopoietic stem cell biology have been hampered by difficulties in purifying and manipulating these cells. To facilitate these studies, we sought to develop a system for targeting genes of interest to the hematopoietic stem cell compartment in transgenic mice. MATERIALS AND METHODS: We used Sca-1, a glycosyl phosphatidylinositol-anchored protein expressed on the surface of all hematopoietic stem cells in commonly used inbred mouse strains. We created a mutant Sca-1 allele in which the enhanced green fluorescent protein (EGFP) cDNA is integrated into the Sca-1 locus by homologous recombination in embryonic stem cells. RESULTS: EGFP protein is detectable in all hematopoietic tissues of mice heterozygous for the mutant Sca-1 allele. Growth and development of these mice are normal. No adverse effects of long-term, high-level EGFP expression were noted. Sca-1 positive cells coexpress EGFP in all tissues and lineages examined, as predicted by the targeting strategy. Sca-1 and EGFP expression are coordinately up-regulated in splenocytes from mutant mice. The Lin(-)EGFP(+) bone marrow population contains all progenitor activity in Sca-1(+)(/EGFP) mice. The Lin(-)EGFP(+) bone marrow cells are equivalent to Lin(-)Sca-1(+) cells in long-term repopulation and serial transplantation assays. CONCLUSION: The hematopoietic stem cell compartment appears to be targeted in Sca-1(+)(/EGFP) mutant mice. This system should be useful for studying the normal biology of hematopoietic stem cells and for targeting other genes to this cellular compartment.     

Bone marrow production of lung cells: the impact of G-CSF, cardiotoxin, graded doses of irradiation, and subpopulation phenotype

OBJECTIVE: Previous studies have demonstrated the production of various types of lung cells from marrow cells under diverse experimental conditions. Our aim was to identify some of the variables that influence conversion in the lung. METHODS: In separate experiments, mice received various doses of total-body irradiation followed by transplantation with whole bone marrow or various subpopulations of marrow cells (Lin(-/+), c-kit(-/+), Sca-1(-/+)) from GFP(+) (C57BL/6-TgN[ACTbEGFP]1Osb) mice. Some were given intramuscular cardiotoxin and/or mobilized with granulocyte colony-stimulating factor (G-CSF). RESULTS: The production of pulmonary epithelial cells from engrafted bone marrow was established utilizing green fluorescent protein (GFP) antibody labeling to rule out autofluorescence and deconvolution microscopy to establish the colocaliztion of GFP and cytokeratin and the absence of CD45 in lung samples after transplantation. More donor-derived lung cells (GFP(+)/CD45(-)) were seen with increasing doses of radiation (5.43% of all lung cells, 1200 cGy). In the 900-cGy group, 61.43% of GFP(+)/CD45(-) cells were also cytokeratin(+). Mobilization further increased GFP(+)/CD45(-) cells to 7.88% in radiation-injured mice. Up to 1.67% of lung cells were GFP(+)/CD45(-) in radiation-injured mice transplanted with Lin(-), c-kit(+), or Sca-1(+) marrow cells. Lin(+), c-kit(-), and Sca-1(-) subpopulations did not significantly engraft the lung. CONCLUSIONS: We have established that marrow cells are capable of producing pulmonary epithelial cells and identified radiation dose and G-CSF mobilization as variables influencing the production of lung cells from marrow cells. Furthermore, the putative lung cell-producing marrow cell has the phenotype of a hematopoietic stem cell.     

Contrasting effects of P-selectin and E-selectin on the differentiation of murine hematopoietic progenitor cells

OBJECTIVE: The two endothelial selectins, P- and E-selectin, are critically important for adhesion and homing of hematopoietic progenitor cells (HPC) into the bone marrow. Little is known, however, about the roles of these two selectins in hematopoiesis. Here, we demonstrate that the most primitive HPC capable of long-term in vivo repopulation express P-selectin glycoprotein ligand-1/CD162 (PSGL-1), a receptor common to both P- and E-selectin. In addition, we demonstrate that P-selectin delays the differentiation of HPC whereas E-selectin enhances their differentiation along the monocyte/granulocyte pathway, describing different roles for these selectins in the regulation of hematopoiesis. MATERIALS AND METHODS: Murine bone marrow HPC were isolated according to their expression of c-kit and PSGL-1, transplanted into lethally irradiated congenic recipients, and chimerism analyzed 6 months posttransplant. Bone marrow lineage-negative (Lin(-)) Sca-1(+)c-kit(+) cells were then cultured on immobilized P- or E-selectin for 4 weeks in the presence of cytokines. Hematopoietic potential was assessed using in vitro phenotyping and colony-forming assays and in vivo spleen colony-forming unit (CFU-S) and long-term competitive repopulation assays. RESULTS: Long-term competitive repopulating HSCs were Lin(-)c-kit(bright) and expressed intermediate levels of PSGL-1. Both P- and E-selectin slowed the proliferation of Lin(-)Sca-1(+)c-kit(+) cells during the first two weeks of liquid culture. After two weeks, however, cells cultured on immobilized P-selectin showed increased proliferation with increased production of both colony-forming cells (CFC) and CFU-S(12) compared to the other cultures. In contrast, E-selectin enhanced the differentiation of Lin(-)Sca-1(+)c-kit(+) cells into cells that expressed the granulocyte maturation marker, Gr-1, accompanied by loss of CFC potential from these cultured cells. Finally, the long-term repopulation potential of these cells was not maintained following culture on either selectin. CONCLUSION: These results suggest that the two endothelial selectins, E-selectin and P-selectin, have very different effects on HPC. E-selectin accelerates the differentiation of maturing HPC towards granulocyte and monocyte lineages while maintaining the production of more immature CFU-S(12) in ex vivo liquid suspension culture. In marked contrast, P-selectin delays the differentiation of Lin(-)Sca-1(+)c-kit(+) cells, allowing enhanced ex vivo expansion of CFC and CFU-S(12) but not HSCs.     

c-Kit expression and stem cell factor-induced hematopoietic cell proliferation are up-regulated in aged B6D2F1 mice

Expression of c-Kit (CD117) and stem cell factor/c-Kit-mediated cell proliferation were tested in vitro in young and old B6D2F1 mice to study the role of c-Kit signaling in hematopoietic stem cell (HSC) senescence. Increasing age is associated with a significant increase in bone marrow (BM) cells without affecting mature blood cells. The number of c-Kit-expressing BM cells increased significantly in old mice when compared to young controls, to 201% in total BM cells, 261% in Lin(-) cells, 517% in Lin(-)CD34(+)Sca1(+) progenitor cells, and 1272% in Lin(-)CD34(-)Sca1(+) HSCs. Sorted Lin(-)Sca1(+)CD117(+) BM cells from an old mouse expanded 5-fold when cultured in vitro for 72 hours with stem cell factor at 25 ng/ml, which was significantly higher than a 2.5-fold expansion of the same cells from a young donor. HSCs and progenitor cells from B6D2F1 mice maintain extremely high proliferative potentials and do not reach proliferative arrest at old age during a normal life span.     

SOCS up-regulation mobilizes autologous stem cells through CXCR4 blockade

The chemokine CXCL12 influences self-renewal and differentiation of hematopoietic stem cell precursors in bone marrow by directing them toward specific stromalcell components. CXCL12 up-regulates members of the SOCS family through JAK/STAT activation, a mechanism that attenuates chemokine responses. SOCS expression may thus modulate retention of hematopoietic precursors (Sca-1(+) c-Kit(+)Lin(-) cells) in bone marrow. We show that in bovine growth hormone transgenic mice and in growth hormone-treated mice, SOCS up-regulation correlated with a large number of Sca-1(+) c-Kit(+)Lin(-) cells in blood. Retroviral transduction of SOCSs blocked in vitro migration of Sca-1(+)c-Kit(+)Lin(-) cells, as well as their capacity to reconstitute lethally irradiated mice. Furthermore, in lethally irradiated mice reconstituted with bone marrow infected by a tetracycline-regulated, SOCS-expressing lentiviral vector, doxycycline treatment promoted rapid, extensive precursor mobilization to the periphery. The results indicate that by blocking CXCR4-mediated functions, SOCSs modulate hematopoietic precursor cell retention in bone marrow, and suggest the therapeutic interest of SOCS manipulation in several pathologic situations.     

Mouse Pum1 and Pum2 genes,members of the Pumilio family of RNA-binding proteins,show differential expression in fetal and adult hematopoietic stem cells and progenitors

Self-renewal is the common functional property of all types of stem cells and is thought to be regulated by unknown conserved intrinsic and extrinsic molecular mechanisms. Recently, an evolutionarily conserved Pumilio family of RNA-binding proteins that regulate asymmetric cell division was found to be essential for stem cell maintenance and self-renewal in Drosophila and Caenorhabditis elegans. Based on conserved function in invertebrates and lower vertebrates it was recently proposed that an ancestral function of Pumilio proteins is to support proliferation and self-renewal of stem cells. This raises an interesting possibility that Pumilio could be part of evolutionarily conserved intrinsic molecular mechanism that regulates self-renewal of mammalian stem cells. Here we describe cloning and comparative sequence analysis of Pum1 and Pum2 genes, mouse members of the Pumilio family, and for the first time demonstrate expression of Pumilio genes in mammalian hematopoietic stem cells (HSC). Pum1 and Pum2 share 51 and 55% overall similarity with the fly Pum, whereas their RNA-binding domains show a very high degree of evolutionary conservation (86-88% homology). Both genes are expressed in a variety of tissues suggesting that they have widespread function. During blood cell development Pum1 and Pum2 exhibit differential expression in cell populations enriched for HSC and progenitors. Both genes are highly transcribed in populations of adult HSC (Rho-123(low)Sca-1(+)c-kit(+)Lin(-) cells). In a more heterogeneous population of HSC (Lin(-)Sca-1(+)) and in progenitors (Lin(-)Sca-1(-) cells) Pum1 is not transcribed, whereas Pum2 expression is significantly down-regulated. Ongoing in vitro and in vivo functional analysis of mouse Pumilio genes will help to elucidate the biological role of mammalian Pumilio genes and determine whether they play any role in maintenance of mammalian stem cells, such as HSC.     

Induction of donor-type chimerism and transplantation tolerance across major histocompatibility barriers in sublethally irradiated mice by Sca-1(+)Lin(-) bone marrow progenitor cells: synergism with non-alloreactive (host x donor)F(1) T cells.

Induction of transplantation tolerance by means of bone marrow (BM) transplantation could become a reality if it was possible to achieve engraftment of hematopoietic stem cells under nonlethal preparatory cytoreduction of the recipient. To that end, BM facilitating cells, veto cells, or other tolerance-inducing cells, have been extensively studied. In the present study, we show that BM cells within the Sca-1(+)Lin(-) cell fraction, previously shown to be enriched for early hematopoietic progenitors, are capable of reducing specifically antidonor CTL-p frequency in vitro and in vivo, and of inducing split chimerism in sublethally 7-Gy-irradiated recipient mice across major histocompatibility complex barriers. The immune tolerance induced by the Sca-1(+)Lin(-) cells was also associated with specific tolerance toward donor-type skin grafts. The minimal number of cells required to overcome the host immunity remaining after 7 Gy total body irradiation is very large and, therefore, it may be very difficult to harvest sufficient cells for patients. This challenge was further addressed in our study by demonstrating that non-alloreactive (host x donor)F(1) T cells, previously shown to enhance T-cell-depleted BM allografts in lethally irradiated mice, synergize with Sca-1(+)Lin(-) cells in their capacity to overcome the major transplantation barrier presented by the sublethal mouse model.     

Reversible expression of CD34 by murine hematopoietic stem cells.

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34(-). We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34(-) and CD34(+) cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage(-) c-kit(+) Sca-1(+) CD34(-) bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34(-) and CD34(+) cell populations and tested for their engraftment capabilities, only CD34(+) cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34(+) stem cells to CD34(-) state. We transplanted Ly-5.1 CD34(+) post-5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34(-) cell fraction, indicating the reversion of CD34(+) to CD34(-) stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.     

Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y

OBJECTIVE: The aim of this study was to determine the function of primitive hematopoietic stem cells (PHSC) at phases G(0) and G(1) of the cell cycle. MATERIALS AND METHODS: A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G(0) and G(1) subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function. RESULTS: We confirmed that the Rh(lo)Lin(-)Kit(+)Sca-1(+) PHSC were relatively quiescent when compared with the more mature Rh(hi)Lin(-)Kit(+)Sca-1(+) HSC and Rh(hi)Lin(-)Kit(+)Sca-1(-) progenitors. In addition, cells with Rh(lo)Lin(-)Kit(+)Sca-1(+), Rh(lo)Ho(lo)Lin(-)Sca-1(+), or Rh(lo)Ho(sp)Lin(-)Sca-1(+) phenotypes identified the same cell population. We further subfractionated the Rh(lo)Ho(lo/sp)Lin(-)Sca-1(+) PHSC using PY into PY(lo) and PY(hi) subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the PY(lo)Rh(lo)Ho(lo/sp) PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G(1) phase (PY(hi)). We found a dose-dependent PY-mediated cytotoxicity that at moderate concentration affected most of the murine hematopoietic compartment but spared the early HSC compartment. CONCLUSION: Our data confirm that the HSC compartment is hierarchically ordered on the basis of quiescence and further extend this concept to PY-mediated cytotoxicity. PY supravital dye can be used to reveal functional heterogeneity within the Rh(lo)Ho(lo/sp) PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population.