Agm1/Pgm3-mediated sugar nucleotide synthesis is essential for hematopoiesis and development.

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.

Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells.

During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta-gonad-mesonephros (AGM) region. Several signalling pathways such as Notch, Wnt, Shh and RA are implicated in this process, yet how these interact to regulate the emergence of HSCs has not previously been described in mammals. Using a combination of ex vivo and in vivo approaches, we report here that stage-specific reciprocal dorso-ventral inductive interactions and lateral input from the urogenital ridges are required to drive HSC development in the aorta. Our study strongly suggests that these inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways. Specifically, Shh produced in the dorsal region of the AGM, stem cell factor in the ventral and lateral regions, and BMP inhibitory signals in the ventral tissue are integral parts of the regulatory system involved in the development of HSCs.

Genetic modification of murine hematopoietic stem cells by retroviruses.

Among the currently available methods for gene transfer, recombinant murine retroviruses remain the best established method for achieving stable integration of a transgene with high efficiency. Pioneering work by a number of groups has demonstrated the feasibility of using this method for gene transfer to primitive, multipotential long-term repopulating hematopoietic stem cells (HSC) (1-4). In the case of the hematopoietic system, it is required that the introduced gene integrates into the genome of HSC in order to be expressed in multiple lineages over an extended period of time. However, HSC are found at low frequency, and are normally in a quiescent or slow cycling state. Both factors represent challenges to successful retroviral gene transfer. The former places a premium on high titer, and the latter dictates methods to trigger HSC cycling during the infection, since stable integration of murine retroviruses requires cell division of the target cell and breakdown of the nuclear membrane (5,6). In general, titers greater than 1 × 10(5) U/mL allow some degree of gene transfer for HSC, but 1 × 10(6) or higher are a reasonable goal for achieving useful efficiencies of at least 20%. For activation of HSC, most protocols invoke a combination of in vivo and in vitro stimulation. The former is most easily and routinely achieved by administration of cytotoxic agents like 5-fluorouracil (5-FU) 4 d prior to bone-marrow harvest. This procedure removes a large proportion of actively cycling, more differentiated cells, thus achieving a.

Formation of embryoid bodies from human pluripotent stem cells using AggreWell™ plates.

Many human embryonic stem (hES) and induced pluripotent stem (hiPS) cell differentiation protocols begin with the formation of three-dimensional aggregates of cells called embryoid bodies (EBs). Traditional EB formation methods result in a heterogeneous population of EB sizes and shapes, which then undergo heterogeneous differentiation efficiencies. AggreWell(TM)400 and AggreWell(TM)800 use the spin-EB method to force the aggregation of a defined number of cells, thereby controlling EB size and generating a population of uniform EBs. Moreover, the dense array of microwells on the bottom surface of AggreWell(TM)400 provide for the rapid and simple production of thousands of EBs at a time.

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.

Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells.

The role of cytokines in regulation of hematopoietic stem cells (HSCs) remains poorly understood. Herein we demonstrate that thrombopoietin (THPO) and its receptor, MPL, are critically involved in postnatal steady-state HSC maintenance, reflected in a 150-fold reduction of HSCs in adult Thpo(-/-) mice. Further, whereas THPO and MPL proved not required for fetal HSC expansion, HSC expansion posttransplantation was highly MPL and THPO dependent. The distinct role of THPO in postnatal HSC maintenance is accompanied by accelerated HSC cell-cycle kinetics in Thpo(-/-) mice and reduced expression of the cyclin-dependent kinase inhibitors p57(Kip2) and p19(INK4D) as well as multiple Hox transcription factors. Although also predicted to be an HSC viability factor, BCL2 failed to rescue the HSC deficiency of Thpo(-/-) mice. Thus, THPO regulates posttransplantation HSC expansion as well as the maintenance of adult quiescent HSCs, of critical importance to avoid postnatal HSC exhaustion.

Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles of thrombopoietin and LNK.

The role of cytokines as regulators of hematopoietic stem cell (HSC) expansion remains elusive. Herein, we identify thrombopoietin (THPO) and the cytokine signaling inhibitor LNK, as opposing physiological regulators of HSC expansion. Lnk(-/-) HSCs continue to expand postnatally, up to 24-fold above normal by 6 mo of age. Within the stem cell compartment, this expansion is highly selective for self-renewing long-term HSCs (LT-HSCs), which show enhanced THPO responsiveness. Lnk(-/-) HSC expansion is dependent on THPO, and 12-wk-old Lnk(-/-)Thpo(-/-) mice have 65-fold fewer LT-HSCs than Lnk(-/-) mice. Expansions of multiple myeloid, but not lymphoid, progenitors in Lnk(-/-) mice also proved THPO-dependent.

HOXB4-induced expansion of adult hematopoietic stem cells ex vivo.

Hox transcription factors have emerged as important regulators of primitive hematopoietic cell proliferation and differentiation. In particular, HOXB4 appears to be a strong positive regulator of hematopoietic stem cell (HSC) self-renewal. Here we demonstrate the potency of HOXB4 to enable high-level ex vivo HSC expansion. Cultures of nontransduced or GFP-transduced murine bone marrow cells experienced large HSC losses over 10-14 days. In sharp contrast, cultures of HOXB4-transduced cells achieved rapid, extensive, and highly polyclonal HSC expansions, resulting in over 1000-fold higher levels relative to controls and a 40-fold net HSC increase. Importantly, these HSCs retained full lympho-myeloid repopulating potential and enhanced in vivo regenerative potential, demonstrating the feasibility of achieving significant ex vivo expansion of HSCs without functional impairment.

Synergistic effects on erythropoiesis, thrombopoiesis, and stem cell competitiveness in mice deficient in thrombopoietin and steel factor receptors.

The degree of redundancy between thrombopoietin (Tpo) and steel factor (SF) cytokine pathways in the regulation of hematopoiesis was investigated by generating mice lacking both c-Mpl and fully functional c-Kit receptors. Double-mutant c-Mpl(-/-)Kit(Wv/Wv) mice exhibited reduced viability, making up only 2% of the offspring from c-Mpl(-/-)Kit(Wv/)(+) intercrosses. The thrombocytopenia and megakaryocytopenia characteristic of c-Mpl(-/-) mice was unchanged in c-Mpl(-/-)Kit(Wv/Wv) mice. However, the number of megakaryocytic colony forming units (CFU-Mks) was significantly reduced, particularly in the spleen. While Kit(Wv/Wv) mice, but not c-Mpl(-/-) mice, are anemic, the anemia was more severe in double-mutant c-Mpl(-/-)Kit(Wv/Wv) mice, indicating redundancy between Tpo and SF in erythropoiesis. At the primitive cell level, c-Mpl(-/-) and Kit(Wv/Wv) mice have similar phenotypes, including reduced progenitors, colony forming units-spleen (CFU-Ss), and repopulating activities. All of these parameters were exacerbated in double-mutant mice. c-Mpl(-/-)Kit(Wv/Wv) mice had 8-fold fewer clonogenic progenitor cells and at least 28-fold fewer CFU-Ss. c-Mpl(-/-) mice also demonstrated a reduced threshold requirement for nonmyeloablative transplant repopulation, a trait previously associated only with Kit(W) mice, and the level of nonmyeloablative engraftment was significantly greater in c-Mpl(-/-) Kit(Wv/Wv) double mutants. Thus, c-Mpl(-/-) Kit(Wv/Wv) mice reveal nonredundant and synergistic effects of Tpo and SF on primitive hematopoietic cells.

Homeostasis and regeneration of the hematopoietic stem cell pool are altered in SHIP-deficient mice.

SH2-containing inositol 5-phosphatase (SHIP) is an important negative regulator of cytokine and immune receptor signaling. SHIP-deficient mice have a number of hematopoietic perturbations, including enhanced cytokine responsiveness. Because cytokines play an important role in the maintenance/expansion of the primitive hematopoietic cell pool, we investigated the possibility that SHIP also regulates the properties of cells in these compartments. Primitive hematopoietic cells were evaluated in SHIP-deficient mice and wild-type littermate controls using the colony-forming unit-spleen (CFU-S) and competitive repopulating unit (CRU) assays for multipotent progenitors and long-term lympho-myeloid repopulating cells, respectively. Absence of SHIP was found to affect homeostasis of CFU-S and CRU compartments. Numbers of primitive cells were increased in extramedullary sites such as the spleen of SHIP-deficient mice, although total body numbers were not significantly changed. In vivo cell cycle status of the CRU compartment was further evaluated using 5-fluorouracil (5-FU). SHIP-deficient CRUs were more sensitive to 5-FU killing, indicating a higher proliferative cell fraction. More strikingly, SHIP was found to regulate the ability of primitive cells to regenerate in vivo, as CRU recovery was approximately 30-fold lower in mice that received transplants of SHIP-deficient cells compared with controls. These results support a major role for SHIP in modulating pathways important in homeostasis and regeneration of hematopoietic stem cells, and emphasize the importance of negative cytokine regulation at the earliest stages of hematopoiesis.

Feasibility of using autologous transplantation to evaluate hematopoietic stem cell-based gene therapy strategies in transgenic mouse models of human disease.

Histoincompatibility between murine donors and recipients of bone marrow (BM) transplants reduces engraftment, and this compromises assessment of hematopoietic stem cells (HSCs) in certain transgenic mice. To study HSCs in the S+S-Antilles mouse model of human sickle cell disease (SCD), we developed an autotransplant protocol. Initial experiments showed no differences between S+S-Antilles mice and normal C57BL/6 (+/+) mice in their radiosensitivity or baseline hematopoietic progenitor numbers. The kinetics of red blood cell (RBC) replacement post-transplant in +/+ recipients of mixtures of transgenic and +/+ BM cells also showed no competitive advantage of the +/+ cells. BM cells were then aspirated from mice 4 days after 5-fluorouracil treatment, transduced with a green fluorescent protein (GFP)-encoding retrovirus, and transplanted into the same recipients that, just before transplant, were irradiated with 800 cGy. We subsequently detected high levels of GFP(+) RBCs (21-79%) and white blood cells (WBCs; 35-88%) in the blood for 11 months and showed that transduced HSCs regenerated in the primary mice also repopulated secondary mice. These findings provide a generally applicable protocol for performing autotransplants in mice and forecast the potential utility of this approach in assessing HSC-based gene therapy protocols in transgenic mouse models of many human diseases.

Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling.

Genetic screens in lower organisms, particularly those that identify modifiers of preexisting genetic defects, have been used successfully to order components of complex signaling pathways. To date, similar suppressor screens have not been used in vertebrates. To define the molecular pathways regulating platelet production, we have executed a large-scale modifier screen with genetically thrombocytopenic Mpl(-/-) mice by using N-ethyl-N-nitrosourea mutagenesis. Here we show that mutations in the c-Myb gene cause a myeloproliferative syndrome and supraphysiological expansion of megakaryocyte and platelet production in the absence of thrombopoietin signaling. This screen demonstrates the utility of large-scale N-ethyl-N-nitrosourea mutagenesis suppressor screens in mice for the simultaneous discovery and in vivo validation of targets for therapeutic discovery in diseases for which mouse models are available.