Recent studies in normal mice have suggested that transplanted bone marrow cells can transdifferentiate into pancreatic beta-cells at relatively high efficiency. Herein, adopting the same and alternative approaches to deliver and fate map-transplanted bone marrow cells in the pancreas of normal as well as diabetic mice, we further investigated the potential of bone marrow transplantation as an alternative approach for beta-cell replacement. In contrast to previous studies, transplanted bone marrow cells expressing green fluorescence protein (GFP) under the control of the mouse insulin promoter failed to express GFP in the pancreas of normal as well as diabetic mice. Although bone marrow cells expressing GFP under the ubiquitously expressed beta-actin promoter efficiently engrafted the pancreas of normal and hyperglycemic mice, virtually all expressed CD45 and Mac-1/Gr-1, demonstrating that they adopt a hematopoietic rather than beta-cell fate, a finding further substantiated by the complete absence of GFP(+) cells expressing insulin and the beta-cell transcription factors pancreatic duodenal homeobox factor-1 and homeodomain protein. Thus, transplanted bone marrow cells demonstrated little, if any, capacity to adopt a beta-cell fate.

Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood , 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

Cytokine signaling through the tyrosine kinase receptor flt3 and the hematopoietin Interleukin-7-receptor a chain (IL7Ra) is critically involved in regulation of B lymphopoiesis. Specifically, we have recently demonstrated that mice double deficient in flt3 and IL7Ra signaling fail to support B cell commitment and development during embryogenesis as well as in adult hematopietic tissues (Sitnicka et al, J. Exp. Med ; 198:1495, 2003). Previous studies have through generation of IL7Ra knockout mouse on a Bcl-2 transgenic background suggested that IL-7 induced triggering of anti-apoptotic pathways is critically involved in T cell but not B cell development (Akashi et al, Cell ; 89:1033, 1997, Kondo et al, Immunity ; 7:155, 1997). Thus, we here investigated whether anti-apoptotic signaling might rather be involved in flt3-dependent B cell development, through generation of a H2k driven human Bcl-2 transgenic/Flt3 ligand (FL)−/− (Bcl-2/FL−/−) mice. Strikingly, numbers of Common Lymphoid Progenitors (CLP; lin−c-kitloSca-1loIL7Ra+), pro-B (B220+CD19+AA4.1+CD43+), pre-B (B220+CD43−IgM−) and mature B cells (B220+CD43−IgM+) in the BM of 7–8 weeks old Bcl-2/FL−/− mice were all comparable to those observed in wild type mice. More importantly, the pronounced reductions in CLP, pro-B and pre-B progenitors seen in FL−/− mice on a wild type background were compensated by approximately 50% when FL-deficiency was rather induced on a bcl-2 transgenic background (Bcl-2/FL−/−). Thus, in contrast to IL7Ra, the critical role of flt3 in B cell commitment and development might involve triggering of anti-apoptotic pathways.

We recently demonstrated that signaling through the cytokine tyrosine kinase receptor flt3 and interleukin-7 receptor a (IL-7Ra) is indispensable for fetal and adult B cell commitment and development (Sitnicka et al., J. Exp. Med. 198: 1495, 2003). These receptors are also implicated to be important in regulation of T cell development, but their potential interdependence remains unexplored. We recently showed that flt3 ligand (FL)-deficient mice have reduced levels of early thymic progenitors as well as the common lymphoid progenitor (CLP) (Sitnicka et al., Immunity, 17:463, 2002). In the present study we investigated T cell development in mice deficient in FL and IL-7Ra expression. Strikingly, when compared to FL−/− and IL-7Ra−/− mice, FL−/−xIL-7Ra−/− (double deficient) mice (8-10 week old) lack visible lymph nodes and Peyer's Patches. Thymic cellularity was dramatically reduced to only 0.3% of FL−/− and wild type (WT) controls and to only 4% of IL-7Ra−/− mice. In agreement with previous studies, IL-7Ra−/− thymocytes revealed a partial block at the progression from the DN2 (CD4−CD8−CD44+CD25+) to DN3 (CD4−CD8−CD44−CD25+) stage, while in FL−/−xIL-7Ra−/− mice DN1 (CD4−CD8−CD44+CD25−), DN2 and DN3 thymic progenitors were undetectable. Thus, severe reductions in early thymocyte development in FL−/−xIL-7Ra−/− mice support a similar role for cross talk between these two signaling pathways in T cell development as recently demonstrated for B cell genesis.

Several recent studies have suggested that bone marrow (BM) cells can contribute to non-hematopoietic cell lineages through cell fusion rather than transdiffentiation. As this phenomenon has been observed in multiple organs, including the brain and heart, without prior infliction of organ-specific insults, it has been proposed that BM cells might contribute to replacement of non-hematopoietic cell lineages during steady state, and that BM transplantation might be developed as a therapeutic modality in diseases of these organs. However, as all observations of BM-derived cell fusion in vivo have been made in lethally irradiated mice reconstituted with genetically marked BM cells, we addressed to what degree cell fusion occurs normally and/or in response to whole body irradiation. To be able to distinguish between these possibilities we used c-kit deficient (w 41 /w 41 ) mice, which unlike wild type mice do not require irradiation-induced myeloablation to facilitate reconstitution of transplanted BM cells. Noteworthy, no BM-derived cell fusion events were observed in the brain (purkinje neurons) or heart (cardiomyocytes) when unconditioned w 41 /w 41 mice were reconstituted with beta actin GFP transgenic BM cells. In striking contrast, following whole body irradiation (875 rad), BM-derived cell fusion was observed in recipient cardiomyocytes and purkinje neurons of all BM transplanted mice. Thus, spontaneous adult BM-derived cell fusion does not occur in steady state but is potently facilitated by irradiation-induced injuries to the organs in which cell fusion occurs.

Extensive studies of mice deficient in one or several cytokine receptors have failed to support an indispensable role of cytokines in development of multiple blood cell lineages. Whereas B1 B cells and Igs are sustained at normal levels throughout life of mice deficient in IL-7, IL-7Rα, common cytokine receptor gamma chain, or flt3 ligand (FL), we report here that adult mice double deficient in IL-7Rα and FL completely lack visible LNs, conventional IgM+ B cells, IgA+ plasma cells, and B1 cells, and consequently produce no Igs. All stages of committed B cell progenitors are undetectable in FL−/− × IL-7Rα−/− BM that also lacks expression of the B cell commitment factor Pax5 and its direct target genes. Furthermore, in contrast to IL-7Rα−/− mice, FL−/− × IL-7Rα−/− mice also lack mature B cells and detectable committed B cell progenitors during fetal development. Thus, signaling through the cytokine tyrosine kinase receptor flt3 and IL-7Rα are indispensable for fetal and adult B cell development.

Flt3 has emerged as a potential regulator of hematopoietic stem cells (HSC). Sixty percent of cells in the mouse marrow Lin−Sca1+c-kit+ HSC pool expressed flt3. Although single cell cloning showed comparable high proliferative, myeloid, B, and T cell potentials of Lin−Sca1+c-kit+flt3+ and Lin−Sca1+c-kit+flt3− cells, only Lin−Sca1+c-kit+flt3− cells supported sustained multilineage reconstitution. In striking contrast, Lin−Sca1+c-kit+flt3+ cells rapidly and efficiently reconstituted B and T lymphopoiesis, whereas myeloid reconstitution was exclusively short term. Unlike c-kit, activation of flt3 failed to support survival of HSC, whereas only flt3 mediated survival of Lin−Sca1+c-kit+flt3+ reconstituting cells. Phenotypic and functional analysis support that Lin−Sca1+c-kit+flt3+ cells are progenitors for the common lymphoid progenitor. Thus, upregulation of flt3 expression on Lin−Sca1+c-kit+ HSC cells is accompanied by loss of self-renewal capacity but sustained lymphoid-restricted reconstitution potential.

This article presents our studies on the adenoviral transduction efficiency, level of transgene expression, cell cycle status, and multilineage reconstitution ability of human CD34+ hematopoietic cells transduced under proliferating and survival growth conditions. Bone marrow and umbilical cord blood CD34+ cells were cultured in serum-free medium under survival conditions with thrombopoietin (Tpo) alone, or under proliferating conditions with Tpo, c-Kit ligand (KL), and Flt3 ligand (FL). Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) gene under the control of the PGK-1 promoter were used to transduce CD34+ cells. Approximately 10% of CD34+ cells were EGFP+ under both culture conditions. In contrast, up to 50% of CD34+CD38- cells were EGFP+, whereas a maximum of 8% of CD34+ CD38high cells were EGFP+ (p < 0.001). Both colony-forming unit cells (CFU-C) and 5-week long-term culture-initiating cells (LTC-ICs) were efficiently transduced. Under survival conditions, a substantial fraction of transduced CD34+ cells remained quiescent. The nondividing CD34+EGFP+ cells contained LTC-ICs capable of reconstituting long-term culture for as long as 10 weeks. CD34+EGFP+ cells also retained the ability to engraft and multilineage-reconstitute NOD/SCID mice. These observations demonstrate that primitive human hematopoietic progenitor cells can be efficiently transduced by adenoviral vectors.

The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G1 arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit–granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34+ cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of -RB to liquid cultures of CD34+ cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34+ cells mediated by -RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34+ cells incubated with oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

HEMATOPOIESIS IS A life-long process responsible for replenishing both hematopoietic progenitor cells and mature blood cells from a pool of pluripotent, long-term reconstituting stem cells.[1][1] The daily turnover in a normal adult of approximately 1012 blood cells is tightly regulated, involving,

It has been demonstrated recently that in vivo administration of murine IL-12 to mice enhances the activity of cytotoxic NK cells and lymphocyte-activated killer cells, and that it has antitumor and antimetastatic activity. However, one side effect observed in response to systemic IL-12 treatment is anemia. In the present study, we examined for the first time the ability of IL-12 to affect directly the growth of murine erythroid progenitor cells in vitro. Whereas IL-12 alone or in combination with Erythropoietin (Epo) showed no stimulatory effect on erythroid progenitors, IL-12 potently enhanced the number of erythroid burst-forming unit (BFU-E) colonies formed in response to Epo+IL-4 by 63% and Epo+stem cell factor by 80%. The stimulatory effect of IL-12 occurred in a concentration-dependent fashion, with maximum enhancing effect observed at 50 ng/ml. Furthermore, single cell experiments suggested that the stimulatory effect of IL-12 on erythroid colony formation was directly mediated. Thus, IL-12 can directly enhance murine erythropoiesis in vitro, suggesting that IL-12-induced anemia is mediated through an indirect mechanism.

The present studies have investigated, for the first time, the synergistic effects of interleukin-4 (IL-4) and IL-11 on the growth of single murine bone marrow progenitor cells. These studies suggest that IL-4 and IL-11 are synergistic hematopoietic growth factors, enhancing colony formation of bone marrow progenitors from normal mice in the presence of colony-stimulating factors or stem cell factor, whereas neither IL-4 nor IL-11, alone or in combination, resulted in colony formation. However, in the presence of a neutralizing anti-TGF-beta antibody, IL-11 plus IL-4 induced clonal growth of primitive Lin-Sca1+ progenitors. Furthermore, here we report several observations extending the knowledge about IL-4 and IL-11 as synergistic factors. In addition to the established ability of IL-11 to enhance IL-3- and GM-CSF-induced colony formation, IL-11 also enhanced the number of G-CSF- and CSF-1-stimulated colonies of mature (Lin-) and primitive (Lin-Sca-1+) hematopoietic progenitors cultured at the single-cell level. In contrast, IL-4 bifunctionally regulated the growth of Lin- progenitors, whereas the growth of single Lin-Sca=1+ progenitors was unaffected or enhanced in the presence of IL-4. Finally, IL-4 and IL-11, in combination, potently synergized to enhance the high-proliferative-potential colony-forming cell colony formation of Lin-Sca-1+ progenitors in response to all four CSFs and to SCF.

Administration of recombinant human interleukin 7 (rh)IL-7 to mice has been reported by our group to increase the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakarocyte macrophage) from the bone marrow to peripheral organs (blood, spleen[s], and liver). We now report that IL-7 also stimulates a sixfold increase in the number of more primitive CFU-S day 8 (CFU-S8) and day 12 (CFU-S12) in the peripheral blood leukocytes (PBL) of mice treated with rhIL-7 for 7 d. Moreover, > 90% of lethally irradiated recipient mice that received PBL from rhIL-7-treated donor mice have survived for > 6 mo whereas none of the recipient mice that received an equal number of PBL from diluent-treated donors survived. Flow cytometry analysis at 3 and 6 mo after transplantation revealed complete trilineage (T, B, and myelomonocytic cell) repopulation of bone marrow, thymus, and spleen by blood-borne stem/progenitor cells obtained from rhIL-7-treated donor mice. Thus, IL-7 may prove valuable for mobilizing pluripotent stem cells with long-term repopulating activity from the bone marrow to the peripheral blood for the purpose of gene modification and/or autologous or allogeneic stem cell transplantation.

TNF-alpha is a pleiotropic cytokine with stimulatory as well as inhibitory effects on hematopoiesis. We have previously demonstrated that TNF-alpha directly inhibits CSF-induced proliferation of primitive murine lineage-negative bone marrow progenitors (Lin-) and stem cell antigen-1 hematopoietic progenitors through the 75-kDa TNF receptor (TNF-R2), whereas TNF-alpha-induced inhibition of more committed Lin- progenitors is mediated through the 55-kDa TNF-R (TNF-R1), indicating a differential role of the two TNF-Rs in hematopoiesis. Numerous studies have demonstrated the ability of stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, to synergize with other hematopoietic growth factors, but little is known about cytokines capable of inhibiting hematopoiesis induced by SCF. While TNF-alpha has been demonstrated to enhance SCF-induced proliferation of myeloid leukemia blasts, the present report demonstrates that TNF-alpha, by signaling through TNF-R2, inhibits SCF-induced proliferation of normal murine Lin- and stem cell antigen-1 hematopoietic progenitors. SCF-stimulated proliferation of the hematopoietic cell line FDC-P1 was also potently inhibited by TNF-alpha and was accompanied by down-regulation of c-kit cell surface expression as well as c-kit mRNA levels. Finally, treatment of the FDC-P1 cell line with TNF-alpha resulted in increased levels of the tumor suppressor p53 mRNA, suggesting another mechanism by which hematopoietic effects of TNF-alpha may be mediated.

Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF- beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4- , and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF- beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF- beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.