Expansion Of HSCs Research Articles

Ectopic Expression of Mutated HOXB4 Proteins with Increased Intracellular Stability Affects Both Long and Short Term Hematopoietic Reconstitution.

The capacity of recombinant Hoxb4 protein to induce ex vivo expansion of HSCs identifies this protein as a potential HSC expanding factor. However, its short extra- and intra-cellular half-life (3–4 hours and 40–60 minutes, respectively) are hampering clinical applications of Hoxb4. The analyses of Hoxb4 molecular structure lead us to generate amino acid substitutions: Leu7→Ala, Tyr23→Ala and Tyr28→Ala in the Hoxb4 protein in order to decrease its degradation. Indeed, these modifications increased the intracellular stability of Hoxb4 protein ~3-fold compared to wild type Hoxb4 (Hoxb4(WT)). The ability of mutated Hoxb4 protein to favor expansion of hematopoietic progenitors was first examined in cultures initiated with 10% Hoxb4(WT)-GFP, 10% mutated Hoxb4-YFP expressing cells and 80% non-transduced cells. After an 18-day culture, the proportion of Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) cells increased to 50–60% in comparison to 30% for Hoxb4(WT) (p < 0.05), and no difference between the proliferation of Hoxb4(Tyr28→Ala) and Hoxb4(WT) cells could be identified. Western blot analyses showed that these Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) cells expressed ~ 4-fold higher and Hoxb4(Tyr28→Ala) cells ~ 8-fold lower levels of Hoxb4 protein than Hoxb4(WT) cells. The long-term reconstituting ability of these constructs was then evaluated in vivo using competitive repopulation assays. At 8 weeks after transplantation, Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) contributed to 11.5±2 and 13.1±1.8% of peripheral blood leukocytes (PBL) compared to 26.2± 4.3% determined for Hoxb4(WT), while after 16 weeks the progeny of Hoxb4(WT) cells generated the majority (≥65%) of the transplant-derived PBL in all recipients. Likewise, 16 weeks post transplantation Hoxb4 positive cells represented ≥80% of bone marrow, while cells expressing mutated Hoxb4 were present at ~10–12%level. Flow cytometry analysis of bone marrow, spleen and thymus revealed that mutated Hoxb4, like Hoxb4(WT) was expressed by all hematopoietic lineages, and that repopulation differences observed between mutated and WT Hoxb4 expressing cells were almost entirely attributable to myeloid lineage cells. However, short-term, non-competitive repopulation experiments showed that in the first 4 weeks post transplantation, mutated Hoxb4 expressing progenitors had a significantly greater contribution to the PBL recovery in comparison to Hoxb4(WT) (range 50–70% vs 16–30%, respectively; p < 0.05) for all three mutant proteins. Interestingly, this difference became less pronounced and non-significant after week 8 post transplantation. Together, these studies strongly suggest that different intracellular levels of Hoxb4 protein are affecting different types of hematopoietic progenitors. Early ex vivo expansion of clonogenic progenitors was achieved with mutated Hoxb4 proteins without impairing HSC long-term reconstituting ability. Thus, mutated Hoxb4 could represent a useful tool to accelerate engraftment after HSC transplantation.

The molecular mechanisms that control thrombopoiesis

Our understanding of thrombopoiesis--the formation of blood platelets--has improved greatly in the last decade, with the cloning and characterization of thrombopoietin, the primary regulator of this process. Thrombopoietin affects nearly all aspects of platelet production, from self-renewal and expansion of HSCs, through stimulation of the proliferation of megakaryocyte progenitor cells, to support of the maturation of these cells into platelet-producing cells. The molecular and cellular mechanisms through which thrombopoietin affects platelet production provide new insights into the interplay between intrinsic and extrinsic influences on hematopoiesis and highlight new opportunities to translate basic biology into clinical advances.

The Placenta Is a Niche for Hematopoietic Stem Cells

The hematopoietic system develops during embryogenesis at temporally and anatomically restricted sites. The anatomical origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct HSC development. Here, we show that the mouse placenta functions as a hematopoietic organ that harbors a large pool of pluripotent HSCs during midgestation. The onset of HSC activity in the placenta parallels that of the AGM (aorta-gonad-mesonephros) region starting at E10.5-E11.0. However, the placental HSC pool expands until E12.5-E13.5 and contains >15-fold more HSCs than the AGM. The expansion of the CD34(+)c-kit(+) HSC pool in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver. Importantly, the placental HSC pool is not explained by rare circulating HSCs, which appear later. These data support an important, but unappreciated, role for the placenta in establishing the mammalian definitive hematopoietic system.

Placenta Is a Niche for Hematopoietic Stem Cells.

The hematopoietic system in the embryo develops in anatomically distinct sites, facilitating rapid generation of erythroid cells and formation of a pool of pluripotent HSCs. The origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct the genesis, maturation, expansion and differentiation of HSCs. In avians, de novo hematopoiesis occurs not only in the yolk sac and the AGM but also in another mesodermal appendage, the allantois. In mammals, the allantois forms the umbilical cord and fetal placenta upon fusion with the chorion. The placenta has not been recognized as a hematopoietic organ, although Melchers reported fetal B-cell potential in murine placenta 25 years ago (Nature 1979, 277:219). Recently, Alvarez-Silva et al. showed that the placenta is a rich source for multipotential hematopoietic progenitors prior to the fetal liver (Development 2003, 130:5437).We have performed spatial and temporal analysis of HSCs during mouse development and for the first time assessed HSC activity in the placenta. Hematopoietic organs from E10.5-18.5 embryos (CD45.1/CD45.2) were treated with collagenase and transplanted in limiting dilutions (3–1/1000 embryo equivalents, ee) into irradiated CD45.2+ adult hosts with CD45.1+ support BM cells. Reconstitution was analyzed by FACS and HSCs were quantified as repopulating units (RUs/ee = ([reconstituted recipients] /[total recipients]) /[transplanted dose]).Our data show that the placenta functions as a hematopoietic organ that during midgestation harbors a large pool of pluripotent HSCs. The onset of HSC activity in the placenta parallels that of the AGM starting at E10.5–11.0. However, the placenta HSC pool expands until E12.5–13.5 (>50 RUs) contrasting lack of HSC expansion in the AGM. The expansion of CD34+c-kit+ HSCs in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver and is not accompanied with myeloerythroid differentiation. A far greater expansion of placenta HSCs compared to that of clonogenic progenitors (17-fold vs. 2-fold at E11.5–12.5) suggests that the placenta provides a favorable niche for HSCs. Indeed, placenta HSCs possess functional properties of authentic adult-type HSCs by providing high level multilineage reconstitution for >5 months and exhibiting self-renewal capacity upon serial transplantation. Importantly, placenta HSCs are distinct from circulating HSCs that appear in low numbers after E11.5. HSC activity in the placenta declines towards the end of gestation while HSCs in the fetal liver and blood continue to increase, possibly reflecting mobilization of placenta HSCs to the fetal liver and other developing hematopoietic organs.The early onset of HSC activity in the placenta suggests that the allantois and its derivatives may participate in de novo genesis and maturation of HSCs together with the AGM and possibly the yolk sac. As the main blood volume from the dorsal aorta reaches the fetal liver via umbilical vessels and the placenta, placenta may also provide a niche where nascent HSCs, or pre-HSCs, from the AGM colonize for maturation and expansion prior to seeding fetal liver. While further studies are needed to define the precise origin of placenta HSCs and the function of placenta microenvironment as an HSC supportive niche, the unique kinetics and magnitude of HSC activity suggest an important, previously unappreciated role for the placenta in establishing the definitive hematopoietic system.

Ex Vivo Expanded HOXB4-Transduced Bone Marrow Cells Enhance Allogeneic Repopulation in a Sublethal Irradiation Model of BMT.

The successful development of molecular strategies to improve the competitive capacity of donor hematopoietic cells would have major implications for bone marrow transplantation (BMT). The forced expression of HOXB4 has been previously shown to increase the competitive capacity of donor marrow in congenic competitive repopulation assays. However, a major clinical objective is to improve allogeneic engraftment after BMT utilizing reduced intensity conditioning regimens. We hypothesized that expansion of HSCs resulting from forced expression of HOXB4 would enhance stable allogenic engraftment after sublethal myeloablative conditioning.BM mononuclear cells (BMMNC) isolated from B6(H2Kb) donor mice treated with 5-FU (150mg/Kg body weight) were prestimulated with cytokines (IL3, IL6 and SCF) and transduced with an MSCV based retroviral vector expressing either HOXB4-ires-GFP or only GFP. Following incubation with viral supernatants, BMMNC were expanded in the absence of virus, examined for transduction efficiency, and phenotyped by flow cytometry prior to administration of 2.5 x 106 cells to Balb/c (H2Kd) sublethally irradiated (450cGy) mice. Data is expressed as mean ± SEM. Transduction efficiency with both vectors was 45–50% as measured by GFP expression. Phenotypic analysis of transduced cultures showed no significant difference in surface phenotype between the HOXB4 or GFP transduced BM cells. The majority of the cells were CD45R/B220 positive (70%) and/or CD11b positive (55%). As early as 4 weeks post-transplantation, GFP and H2Kb positive donor derived cells were present in the peripheral blood of recipients. At 4 weeks, HOXB4 transduced donor cells engrafted at a higher rate 55% (5 out of 9 animals; donor cells in PB = 81.92 ± 10.25%; n = 5) compared to 33% engraftment (3 out of 9 animals) with GFP transduced cells (donor cells in PB = 39.11 ± 20.66; n = 3). Long term donor cell chimerism (16 weeks) could be measured in 3 of 9 HOXB4 transduced recipients while GFP engrafted recipients failed to survive beyond 6 weeks. Multilineage analysis of donor derived phenotypes in long term HOXB4 engrafted mice in PB, BM and spleen are shown in the table. Analysis of thymus in one of the HOXB4 engrafted recipients showed CD4 +/GFP+ H2Kb (76.49%) or CD8+/GFP+ H2Kb (76.95%) cells, indicating the development of T-cell lineages from donor derived HOXB4 transduced HSCs.Our results indicate engraftment and long term donor chimerism throughout the hematopoietic compartment in a murine allogenic model. However, forced HOXB4 expression in HSCs also resulted in increased myeloid lineage expression in chimeric animals, consistent with earlier observations in congenic transplants (Antonchuk et al, 2002). Though the mechanisms of HOXB4 induced HSC self renewal and multilineage hematopoiesis are still not clear, its capability to contribute to enhanced engraftment across major histocompatibility complex barriers supports its potential utility for allogeneic BMT.Lineage Engraftment (%) of Donor Cells (GFP+/H2Kb+) in Hematopoietic TissuesTissueCD3CD11bCD19CD45PB4.14 ± 0.3983.77 ± 10.1411.25 ± 11.7389.47 ± 8.17BM1.56 ± 1.165.81 ± 9.85.76 ± 2.980.54 ± 6.2Spleen12.15 ± 10.145.92 ±25.8124.15 ± 21.585.87 ± 5.3