Maturation Of Hematopoietic Stem Cells Research Articles

Placenta as a site for hematopoietic stem cell development

The discovery of a major hematopoietic stem cell (HSC) pool in mid-gestation mouse placenta has defined the placenta as yet another important anatomical site that participates in HSC development. Placental HSC activity starts in parallel with the AGM region, before HSCs are found in circulation or have colonized the fetal liver. Moreover, placental hematopoietic activity culminates in a rapid expansion of the definitive HSC pool, which occurs during the time when the fetal liver HSC reservoir begins to grow. Furthermore, hematopoietic cells in mid-gestation mouse placenta are not instructed for differentiation along the myeloerythroid lineage, as in the fetal liver. These findings suggest that the placenta provides a supportive niche where the definitive hematopoietic stem cell pool can be temporarily established during development. Future studies are needed to characterize the developmental events that lead to the establishment of placental HSC pool, and to define the microenvironmental signals that support this process. Furthermore, if the stem cell-promoting properties of the placental niche can be harnessed in vitro to support HSC formation, maturation, and/or expansion in culture, these assets may greatly improve hematopoietic stem cell-based therapies in the future.

Genesis of Hematopoietic Stem Cells In Vitro and In Vivo: New Insights into Developmental Maturation

Hematopoietic stem cells first arise in the mammalian embryo in a primitive state, not capable of reconstituting hematopoiesis in irradiated adult recipients. As development proceeds, these cells eventually mature to acquire definitive, adult characteristics, including adult reconstitution ability. Mouse embryonic stem cells induced to undergo hematopoiesis in vitro readily generate primitive hematopoietic stem cells but rarely generate the definitive type. Recent work has stimulated a new appreciation of the events involved in the developmental maturation of hematopoietic stem cells. Application of this knowledge to in vitro differentiation systems will be critical to the successful development of hematopoietic therapies from embryonic stem cells.

Identification and Hematopoietic Potential of CD45 − Clonal Cells with Very Immature Phenotype (CD45 − CD34 − CD38 − Lin − ) in Patients with Myelodysplastic Syndromes

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38-Lin- cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34-CD38-Lin- cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site is a matter of debate, and CD45+CD34+CD38-Lin- HSCs were recently reported to be clonal. In the study reported here, we detected CD45-CD34-CD38-Lin- cells in the peripheral blood and bone marrow of patients with MDS and isolated them by successive application of density centrifugation, magnetic cell sorting, and fluorescence-activated cell sorting. Fluorescence in situ hybridization showed that CD45-CD34-CD38-Lin- cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-, they lacked CD117 and CD133 expression. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some patients. Freshly isolated CD45-CD34-CD38-Lin- cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When cocultured with stroma cells, CD45-CD34-CD38-Lin- cells showed only weak potential for proliferation and differentiation, yet they differentiated into CD34+ cells and then mature myeloid cells. This newly identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.

Identification and Hematopoietic Potential of CD45-Negative Clonal Cells with Very Immature Phenotype (CD45−CD34−CD38−Lin−) in Patients with Myelodysplastic Syndromes.

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38−Lin− cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34−CD38−Lin− cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site was reported to be committed myeloid progenitors and, more recently, the CD45+CD34+CD38−Lin− HSCs. In this study, we detected CD45−CD34−CD38−Lin− cells in the peripheral blood and bone marrow of MDS patients. Fluorescence in situ hybridization showed that CD45−CD34−CD38−Lin− cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-negativity, they lacked CD117 and CD133 expressions. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some cases. Freshly-isolated CD45−CD34−CD38−Lin− cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When these cells were co-cultured with stroma cells, CD45−CD34−CD38−Lin− cells showed only weak potential for proliferation/differentiation, yet differentiated to CD34+ cells and then mature myeloid cells. This newly-identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.

Phenotypes and phosphatidylinositol glycan-class A gene abnormalities during cell differentiation and maturation from precursor cells to mature granulocytes in patients with paroxysmal nocturnal hemoglobinuria

To define the phosphatidylinositol glycan-class A (PIG-A) gene abnormality in precursor cells and the changes of expression of glycosylphosphatidylinositol-anchored protein and contribution of paroxysmal nocturnal hemoglobinuria (PNH) clones with PIG-A gene abnormalities among various cell lineages during differentiation and maturation, we investigated CD59 expression on bone marrow CD34(+) cells and peripheral granulocytes from 3 patients with PNH and the PIG-A gene abnormalities in the CD59(-), CD59(+/-), and CD59(+) populations by nucleotide sequence analyses. We also performed clonogeneic assays of CD34(+)CD59(+) and CD34(+)CD59(-) cells from 2 of the patients and examined the PIG-A gene abnormalities in the cultured cells. In case 1, the CD34(+) cells and granulocytes consisted of CD59(-) and CD59(+) populations and CD59(-), CD59(+/-), and CD59(+) populations, respectively. Sequence analyses indicated that mutation 1-2 was in the CD59(+/-) granulocyte population (20 of 20) and the CD34(+)CD59(-) population (2 of 38). In cases 2 and 3, the CD34(+) cells and granulocytes consisted of CD59(+) and CD59(-) cells. Sequence analyses in case 3 showed that mutation 3-2 was not in CD34(+)CD59(-) cells and was present in the CD59(-) granulocyte population. However, PIG-A gene analysis of cultured CD34(+)CD59(-) cells showed that they had the mutation. This analysis also revealed that there were some other mutations, which were not found in CD34(+)CD59(-) cells and CD59(-) or CD59(+/-) granulocytes in vivo, and that sometimes they were distributed specifically among different cell lineages. In conclusion, our findings suggest that PNH clones might contribute qualitatively and quantitatively differentially to specific blood cell lineages during differentiation and maturation of hematopoietic stem cells.

Porcine mononuclear cells adhere to human fibronectin independently of very late antigen-5: implications for donor-specific tolerance induction in xenotransplantation.

To combat the shortage of donor organs, transplantation across species barriers has been proposed. Induction of tolerance would overcome the substantial immunologic barriers to xenotransplantation and would avoid the chronic use of immunosuppressive agents. Successful transplantation of hematopoietic cells induces robust specific tolerance to donor antigens in allogeneic and xenogeneic models. The beta1 integrin class of adhesion molecules and their interactions with extracellular matrix components are thought to be integral to the engraftment and maturation of hematopoietic stem cells. We therefore examined the efficacy of porcine very late antigen-5 (VLA-5) and VLA-4 interactions with the human extracellular matrix (ECM) protein, fibronectin. Peripheral blood mononuclear cells (PBMCs) from humans and miniature swine were flourochrome labeled and adhesion to plates coated with whole human fibronectin (whFN) or its 120 KDa fragment containing the VLA-5 binding region was determined. Flow cytometry and immuno- precipitation were used to identify a monoclonal antibody that cross-reacted on porcine VLA-5. Human and pig PBMC adhesion to human fibronectin (hFN) or 120 kDa fragment-coated plates was assessed following incubation with control ab, anti-VLA-4, anti-VLA-5, or soluble fibronectin. Using rabbit complement, cells expressing VLA-5 were purged from PBMC preparations before performing the adhesion assay. Porcine and human PBMC both adhered to hFN in a divalent cation-dependent and activation-dependent manner. Adhesion to hFN of human but not pig PBMC was blocked by anti-VLA-5 monoclonal antibody SAM-1, although this mAb immunoprecipitated a heterodimeric cell surface molecule (155/135 kDa) resembling VLA-5 from pig PBMC. Complement-mediated depletion of VLA-5-expressing cells ablated specific binding of human but not porcine cells to hFN and its 120 kDa fragment. Addition of soluble fibronectin was capable of blocking adhesion of PBMC of both species to hFN. Anti-VLA-4 reduced the binding of PBMC from both species to hFN to a similar extent. Human and pig cells can specifically adhere to hFN and its 120 kDa fragment, suggesting that this critical cell-ECM interaction is preserved across species. While human cells exclusively use VLA-5 for binding to the 120 kDa fragment, porcine cells could not be shown to adhere to whFN or its 120 kDa fragment via VLA-5. However, porcine VLA-4 is capable of mediating adhesion to human FN. We conclude that disparities in the adhesive interactions of beta1 integrins may be a barrier to the use of porcine hematopoietic stem cell transplantation as a means of inducing donor-specific tolerance in the pig to human species combination.

Development, expansion and maturation of hematopoietic stem cells in mouse embryo

During embryonic development, hematopoiesis occurs in different tissues. Primitive hematopoiesis first occurs in the yolk sac and definitive hematopoietic stem cells (HSCs) arise in the aorta/gonad/mesonephros (AGM) region. Fetal liver then functions as the major hematopoietic organ from the mid to late gestation, before hematopoiesis starts in the bone marrow. We have developed a primary culture system of mouse AGM cells which produces both hematopoietic cells and endothelial cells and found that hemangioblasts, the common precursor of hematopotieic and endothelial cells, are enriched in the cell population which express podocalyxin like protein 1, a CD34-related molecule, but lacks CD45 in the AGM region. We also developed a culture system of E14.5 fetal hepatic cells which support hematopoiesis. AGM-derived HSCs proliferated most vigorously in the presence of such fetal hepatic cells, however HSCs derived from later stages of development proliferated less efficiently in the same co-culture system, suggesting that the proliferation potential of HSCs declines along with development. While AGM-derived HSCs poorly engrafted in the bone marrow of irradiated adult mice, co-culture of AGM-derived HSCs with fetal hepatic cells dramatically increased the bone marrow engraftment. These results indicate that fetal hepatic cells alter the characteristics of HSCs. Interestingly, Oncostatin M (OSM) strongly induced differentiation of immature hepatocytes to metabolically active mature hepatocytes, but it simultaneously reduced their ability to support hematopoiesis. As OSM is produced by hematopoietic cells in the fetal liver, it is likely that OSM plays a role for coordinating the development of hematopoietic cells and the liver. In conclusion, our in vitro system provides a means to study the molecular basis of development, expansion and maturation of HSCs.

Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells.

Clonogenic multipotent mouse hematopoietic stem cells (HSCs) and progenitor cells are contained within the c-kit(+) (K) lineage(-/lo) (L) Sca-1(+) (S) population of hematopoietic cells; long-term (LT) and short-term (ST) HSCs are Thy-1.1(lo). c-kit is a member of the receptor tyrosine kinase family, a class of receptors that are important in the proliferation and differentiation of hematopoietic cells. To establish whether the Flk-2/Flt3 receptor tyrosine kinase was expressed on the most primitive LT-HSCs, we sorted highly purified multipotent stem and progenitor cells on the basis of Flk-2 surface expression and used them in competitive reconstitution assays. Low numbers of Flk-2(-) HSCs gave rise to long-term multilineage reconstitution in the majority of recipients, whereas the transfer of Flk-2(+) multipotent cells resulted in mostly short-term multilineage reconstitution. The KLS subset of adult mouse bone marrow was analyzed for Flk-2 and Thy-1.1 expression. Three phenotypically and functionally distinct populations were isolated: Thy(lo) Flk-2(-) (LT-HSCs), Thy(lo) Flk-2(+) (ST-HSCs), and Thy(-) Flk-2(+) multipotent progenitors. The loss of Thy-1.1 and gain of Flk-2 expression marks the loss of self-renewal in HSC maturation. The addition of Flk-2 antibody to the lineage mix allows direct isolation of LT-HSC from adult bone marrow as c-kit(+) lin(-) Sca-1(+) Flk-2(-) from many strains of mice. Fetal liver HSCs are contained within Flk-2(-) and Flk-2(+) KTLS cells.

PORCINE CELLS ADHERE TO HUMAN FIBRONECTIN INDEPENDENTLY OF VLA-5: IMPLICATIONS FOR PORCINE TO HUMAN XENOTRANSPLANTATION.

510 Purpose: Effective adhesive interactions between species will be critical to the successful use of bone marrow transplantation for the induction of xenotransplantation tolerance. The β1 integrin class of adhesion molecules mediates adhesion of lymphocytes to extracellular matrix (ECM) proteins, and has been implicated in the engraftment and maturation of hematopoietic stem cells. We have compared the role of the integrin very late antigen 5 (VLA-5) in human versus pig cell adhesion to the human ECM protein fibronectin (FN) and in in vitro hematopoiesis. Methods: PBMCs from human and miniature swine were isolated and flourochrome labeled. Labeled cells were placed on tissue culture plates coated with human FN, and adhesion to plates was determined via flouremeter recordings following serial washings. An anti-human VLA-5 antibody, SAM-1, determined via immunprecipitation to be cross-reactive on pig and human VLA-5, was added to the assay to attempt blockade of cell adhesion to FN. Using rabbit complement, cells bearing the VLA-5 epitope recognized by SAM-1 were then purged from the peripheral blood cell preparations before performing the adhesion assay. To further investigate the functional effect of VLA-5 blockade within the extracellular matrix milieu, long term bone marrow cultures (LTBMC) were established in the presence of anti-VLA-5 versus control antibody in both species. Results: Porcine and human PBMC both adhered to human FN. Adhesion of human cells to FN was blocked by anti-VLA-5 monoclonal antibody SAM-1. Adhesion of pig cells to FN was not blocked by anti-VLA-5 mAb SAM-1. Complement-mediated purging of VLA-5- expressing cells ablated specific binding of human cells to fibronectin, but porcine cells lacking VLA-5 retained the ability to adhere to human FN. Antibodies directed against VLA-5 impaired in vitro human hematopoiesis but had no inhibitory effect on porcine LTBMC. Conclusion: Porcine and human cells can use different molecules to adhere to fibronectin. Adhesion to FN is VLA-5-dependent in human but not in pig. Human hematopoiesis appears to be dependent on this interaction, but porcine hematopoiesis may not be. Optimization of porcine interactions with human FN may be critical for successful pig to human xenogeneic chimerism and tolerance induction.

Stem cell responses in myelosuppressed mice following sequential treatment with recombinant human interleukin 1 (rHuIL‐1), recombinant murine interleukin 3 (rMuIL‐3) and recombinant human macrophage colony‐stimulating factor (rHuM‐CSF)

In vivo, recombinant human interleukin 1 alpha (rHuIL-1 alpha) + recombinant human macrophage colony-stimulating factor (rHuM-CSF) (IL-1 + M-CSF) effectively serves as a rescue agent for myelosuppression by enhancing the recovery of hematopoietic stem cell (HSC) subpopulations following treatment with 5-fluorouracil (5-FU). Because in vitro studies have suggested that hematopoietic recovery in 5-FU-treated bone marrow (FUBM) may proceed from a 5-FU resistant, (IL-1 + IL-3 + M-CSF-responsive) high proliferative potential HSC subpopulation of colony forming cells (HPP-CFC), studies were carried out to determine whether the addition of recombinant murine interleukin 3 (rMuIL-3) (IL-3) to either IL-1 or IL-1 + M-CSF would further enhance the recovery of HSC subpopulations in myelosuppressed C57Bl/6 mice. With the exception of the HPP-CFC, IL-3 dampened, rather than enhanced, the accelerated recovery of 8 d and 12 d colony forming units-spleen (8 d and 12 d CFU-S) and the committed macrophage progenitor (CFU-M) associated with in vivo treatment with IL-1 alone. Similarly, IL-3 interfered with the enhanced recovery of those HSC subpopulations in FUBM influenced by the synergistic interaction of IL-1 + M-CSF. This interference, however, was observed only when the rMuIL-3 was administered on day 2 or 3 of a four-day treatment with IL-1 + M-CSF. There was, however, no evidence that IL-3 exerted a negative influence on the restoration of granulocytes in the myelosuppressed animals. Moreover, sequencing studies provided data suggesting that the dampening effects of IL-3 on the synergistic interaction of IL-1 + M-CSF resulted from both an enhanced differentiation of the more primitive HSC subpopulations and a significant, but preferential, mobilization of the more mature 8 d CFU-S and CFU-M to extramedullary organs and that the mobilization of these more mature HSC subpopulations was temporally linked to their generation from the recovering HPP-CFC and 12 d CFU-S subpopulations.