A role for niches in hematopoietic cell development

Stem cells reside in a physical niche, a particular microenvironment. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, maintenance and regeneration. Hematopoietic stem cells (HSC) emerge at distinct allocation territories during ontogenesis, notably the aorto-gonadal region, the fetal liver. Adult HSC expand and differentiate exclusively in the bone marrow (BM). They can be mobilized into the blood stream. This implies that stem cells are not autonomous units of development; rather, tissue specific niches control their destiny. Interaction of HSCs with their stem cell niches is critical for adult hematopoiesis in the BM. A niche is composed of stromal cells, which either through direct cell-to-cell contact or via release of soluble factors maintain the typical features of stem cells, mainly stem cell quiescence, maintenance or expansion. HSCs are keeping the balance of the quiescence and the self-renewal in the stem cell niche, and are maintaining long-term hematopoiesis. Therefore, an understanding of cellular and chemical architecture of the stem cell niche is vital in understanding stem cell behavior. This review summarizes the recent developments in our understanding of the stem cell niche with particular focus on the HSC niche.     

A role for niches in hematopoietic cell development

Stem cells reside in a physical niche, a particular microenvironment. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, maintenance and regeneration.Hematopoietic stem cells (HSC) emerge at distinct allocation territories during ontogenesis, notably the aorto-gonadal region, the fetal liver. Adult HSC expand and differentiate exclusively in the bone marrow (BM). They can be mobilized into the blood stream. This implies that stem cells are not autonomous units of development; rather, tissue specific niches control their destiny. Interaction of HSCs with their stem cell niches is critical for adult hematopoiesis in the BM. A niche is composed of stromal cells, which either through direct cell-to-cell contact or via release of soluble factors maintain the typical features of stem cells, mainly stem cell quiescence, maintenance or expansion. HSCs are keeping the balance of the quiescence and the self-renewal in the stem cell niche, and are maintaining long-term hematopoiesis.Therefore, an understanding of cellular and chemical architecture of the stem cell niche is vital in understanding stem cell behavior. This review summarizes the recent developments in our understanding of the stem cell niche with particular focus on the HSC niche.     

Differential activity of bone marrow hematopoietic stem cell subpopulations for EPC development and ischemic neovascularization

Although endothelial progenitor cells (EPCs) differentiate from minor populations of stem cells in bone marrow (BM), the differential role of hematopoietic stem cell (HSC) subpopulations in EPC development is largely unclear. Morphological characterization of EPC colonies has revealed that c-kit+/Sca-1+/lineage (Lin)-(KSL) cells mainly develop small EPC-colony forming units (CFUs) not large EPC-CFUs. In contrast, c-kit+/Sca-1−/Lin− (KL) cells develop large EPC-CFUs not small EPC-CFUs. Neither c-kit-/Sca-1+/Lin− (SL) cells nor c-kit−/Sca-1−/Lin− (L) cells develop EPC-CFUs to an appreciable extent. Hindlimb ischemia enhances formation of large EPC-CFUs from all HSC subpopulations, suggesting an important role for ischemia in functional EPC development. Real time RT-PCR analysis shows that KSL, KL and SL cells but not L cells express various factors at high levels, maintaining a BM-EPC pool. In hindlimb ischemia, transplanted KSL, KL and SL cells efficiently differentiate into endothelial lineage cells in situ and augment capillary density. The percentage of Ki-67+ cycling cells among transplanted cells in ischemic tissue was also greater for KSL, KL and SL cells than L cells. Moreover, the frequency of VEGF- or SDF-1-expressing cells was higher transplanted KSL, KL or SL cells than L cells. Thus, KSL, KL and SL cells are not different in their angiogenic competence under ischemic conditions. In conclusion, although KSL cells are clearly the most potent contributors to EPC development, KL and SL cells may also contribute to neovascularization via both autocrine and paracrine mechanisms in response to ischemic signals.     

Differential expression of microRNAs in hematopoietic cell lineages

Objectives: MicroRNAs (miRNAs) play key roles in a wide variety of normal and pathological cellular processes. A number of studies identified hematopoietic‐specific miRNAs that are necessary for correct function of blood cells. Out of our microarray data, we chose 13 miRNAs that showed differential expression in peripheral blood cells (miR‐15b, miR‐16, miR‐24, miR‐30c, miR‐106b, miR‐142‐3p, miR‐142‐5p, miR‐150, miR‐155, miR‐181, miR‐223, miR‐342, and miR‐451) and examined their expression in separated hematopoietic cell lineages. Methods: Using quantitative real‐time polymerase chain reaction, we measured relative expression of the miRNAs in fractions of reticulocytes, platelets, granulocytes, monocytes, B‐ and T‐lymphocytes as well as in several hematopoietic cell lines. Results: We observed that miR‐16 and miR‐142‐3p were highly expressed in all native cell lineages, miR‐451 reached the maximal expression in reticulocytes, miR‐223 in platelets, granulocytes and monocytes, and miR‐150 in B‐ and T‐lymphocytes. Hierarchical clustering analysis grouped the lineage samples according to their origin based on the expression of these miRNAs. To validate discrimination power of the miRNAs, we quantified expression of the 13 miRNAs in several immortalized cell lines. Although the cell lines showed miRNA expression patterns considerably different from those of native cell lineages, clustering analysis distinguished between myeloid, lymphoid and non‐hematopoietic cells. Conclusions: In conclusion, the study reports the expression levels of 13 miRNAs in particular blood cell lineages as well as immortalized cell lines. We demonstrate that the expression profiles of these miRNAs may be used for discrimination of the hematopoietic cell lineages.     

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.     

Polypeptides controlling hematopoietic cell development and activation

Summary Recombinant DNA technology has been central in answering some of the most relevant questions in the research of regulation of the functional status of hematopoietic progenitor cells and their progeny. This leading article will focuse on recent results that have emerged from studies utilizing recombinant molecules that control hematopoietic blood cell development and activation. The following features will be detailed: The molecular and biological characteristics and biochemistry of hematopoietic growth factors, synergizing factors and releasing factors, their role in the regulation of hematopoiesis and activation of normal and leukemic cells, their cellular sources, and regulation of production.     

Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.     

Growth and development after hematopoietic cell transplant in children

Hematopoietic cell transplantation (HCT) following high-dose chemotherapy or chemoradiotherapy for children with malignant or nonmalignant hematologic disorders has resulted in an increasing number of long-term disease-free survivors. The preparative regimens include high doses of alkylating agents, such as CY with or without BU, and may include TBI. These agents impact the neuroendocrine system in growing children and their subsequent growth and development. Children receiving high-dose CY or BUCY have normal thyroid function, but those who receive TBI-containing regimens may develop thyroid function abnormalities. Growth is not impacted by chemotherapy-only preparative regimens, but TBI is likely to result in growth hormone deficiency and decreased growth rates that need to be treated with synthetic growth hormone therapy. Children who receive high-dose CY-only have normal development through puberty, whereas those who receive BUCY have a high incidence of delayed pubertal development. Following fractionated TBI preparative regimens, approximately half of the patients have normal pubertal development. These data demonstrate that the growth and development problems after HCT are dependent upon the preparative regimen received. All children should be followed for years after HCT for detection of growth and development abnormalities that are treatable with appropriate hormone therapy.     

Polypeptides controlling hematopoietic blood cell development and activation

Summary Colony-stimulating factors (CSFs) have entered the clinical arena. Several investigators have explored, in first clinical phase I studies, different routes of administration to define the optimum biological dose, maximum tolerated dose, toxicity, and pharmacokinetics of these reagents. It has been demonstrated that recombinant human (rh) granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF) can be safely administered over a broad dose range to increase number of circulating granulocytes in man. More recently, GM-CSF and G-CSF have been involved in phase Ib/II studies to assess the granulopoietic responses of patients with granulocytopenia due to various underlying disease states including myelodysplastic syndrome, aplastic anemia, cyclic neutropenia, Kostmann's syndrome, and the acquired immuno-deficiency syndrome. Both factors were also investigated with respect to their potential to prevent chemotherapy induced granulocytopenia or to accelerate recovery from that condition. The short-term effects of rh GM-CSF after autologous bone marrow transplantation for various solid tumors and lymphoid malignancies were assessed as well. In this article we will focus on recent results that have emerged from in vivo studies utilizing CSFs.     

Kinase requirements in human cells: IV. Differential kinase requirements in cervical and renal human tumor cell lines

Functional differences among human cells have been difficult to identify by standard biochemical methods. Loss-of-function shRNA screens provide an unbiased method to compare protein requirements across cell lines. In previous work, we have studied kinase requirements in two settings, either among a panel of cells from numerous tissues or between two cell lines that differ only by the expression of a chosen oncoprotein or tumor suppressor protein. Here we examine the patterns of kinase requirements between two unrelated cells, the cervical carcinoma cell line HeLa and the renal carcinoma cell line 786-O. By using time courses of cell proliferation after shRNA transduction and by introducing different levels of the shRNAs, we were able to carefully compare the kinase requirements. These comparisons identified 10 kinases that were required in HeLa but not 786-O, and 5 kinases that were required in 786-O but not HeLa. The patterns of growth inhibition due to particular sets of shRNAs in a tumor cell line were shown to be similar in some but not all cell lines derived from the same tissue-specific cancer type. Differential kinase requirements promise to be useful in distinguishing important cell-to-cell functional variations and may lead to the identification of fingerprints for different physiological cell states.     

Effect of peripheral blood progenitor cell dose on hematopoietic recovery: identification of minimal progenitor cell requirements for rapid engraftment

Repeated high-dose chemotherapy (HDC) with stem cell support is advocated for curative treatment of epithelial ovarian cancer patients, requiring large quantities of progenitor cell harvest. Although the switchover to peripheral blood stem cell transplantation has generally made possible the harvest of large quantities of progenitor cells, the minimum threshold is still pertinent for planning the safe conduct of HDC. However, as the minimum threshold for safe peripheral blood stem cell transplantation (PBSCT) is not yet established, this study was designed to clarify the minimum amount of progenitor cells required for prompt recovery of hematopoietic. Retrospective analysis was performed on 52 HDCs administered in 37 ovarian cancer patients. After autologous bone marrow aspiration (10 patients) or peripheral blood stem cell harvest (27 patients), colony-forming unit granulocyte macrophage (CFU-GM) were enumerated prior to cryopreservation. Numbers of CFU-GM were again calculated before reinfusion and the patients were divided into eight groups: 0.13-<0.4, 0.4-<0.7, 0.7-<1.0, 1.0-<3.5, 3.5-<5.0, 5.0-<10.0, 10.0-<20.0 and >20.0 (x 10(5)/kg). The minimum CFU-GM threshold (x 10(5)/kg) was found to be 1.0-<3.5 for platelets and 3.5-<5.0 for white blood cells. Higher infusion doses did not lead to significant benefits in hematopoietic reconstruction. These results indicate that preservation of a minimum of 7-10 x 10(5)/kg CFU-GM is recommended for the safe conduct of tandem HDCs.     

Modeling human hematopoietic cell development from pluripotent stem cells

Understanding the steps and cues that allow hematopoietic cells to be generated during development holds great clinical as well as biological interest. Analysis of these events in mice has provided many important insights into the processes involved, but features that might be unique to humans remain challenging to elucidate because they cannot be studied directly in vivo. Human embryonic stem or induced pluripotent stem cells offer attractive in vitro alternatives to analyze the process. Here we review recent efforts to develop defined and quantitative systems to address outstanding developmental questions against a background of what we know about the development of hematopoietic cells in the fetus and derived from mouse embryonic stem cells.     

Current concepts on hematopoietic stem cell transplantation outcome registries; Emphases on resource requirements for new registries

There is tremendous variability in size, scope, and resource requirements for registries depending on the number of patients and participating sites. The outcome registries are organized systems to collect uniform data using an observational study methodology. Patient registries are used to determine specified outcomes for a population for predetermined scientific, clinical, or policy purposes. Historically, outcome registries established in the development of hematopoietic stem cell transplantation (HSCT) have now evolved into myriads of locoregional and international transplant activity and outcome resources. Over time, these registries have contributed immensely in determining trends, patterns, and treatment outcomes in HSCT. There is wider variation in the goals, mission, objectives, and outcomes of the ongoing registries depending on the organizational structure. There is a growing trend toward overarching relationship of these registries to serve as complementary and interoperable resources for high potential collaborative research. In addition to capacity building, standardized, accredited, and optimally operational registries can provide unmatched and unparalleled research data that cannot be obtained otherwise. Moving forward, HSCT data collection, collation, and interpretation should be an integral part of the treatment rather than an option. Quality assurance and continuous quality improvement of the data are pivotal for credibility, measurable/quantifiable outcomes, clinically significant impact, and setting new benchmarks.     

Differential requirements for the activation domain and FOG-interaction surface of GATA-1 in megakaryocyte gene expression and development

GATA1 is mutated in patients with 2 different disorders. First, individuals with a GATA1 mutation that blocks the interaction between GATA-1 and its cofactor Friend of GATA-1 (FOG-1) suffer from dyserythropoietic anemia and thrombocytopenia. Second, children with Down syndrome who develop acute megakaryoblastic leukemia harbor mutations in GATA1 that lead to the exclusive expression of a shorter isoform named GATA-1s. To determine the effect of these patient-specific mutations on GATA-1 function, we first compared the gene expression profile between wild-type and GATA-1-deficient megakaryocytes. Next, we introduced either GATA-1s or a FOG-binding mutant (V205G) into GATA-1-deficient megakaryocytes and assessed the effect on differentiation and gene expression. Whereas GATA-1-deficient megakaryocytes failed to undergo terminal differentiation and proliferated excessively in vitro, GATA-1s-expressing cells displayed proplatelet formation and other features of terminal maturation, but continued to proliferate aberrantly. In contrast, megakaryocytes that expressed V205G GATA-1 exhibited reduced proliferation, but failed to undergo maturation. Examination of the expression of megakaryocyte-specific genes in the various rescued cells correlated with the observed phenotypic differences. These studies show that GATA-1 is required for both normal regulation of proliferation and terminal maturation of megakaryocytes, and further, that these functions can be uncoupled by mutations in GATA1.     

Requirement of Shp-2 tyrosine phosphatase in lymphoid and hematopoietic cell development

Shp-1 and Shp-2 are cytoplasmic phosphotyrosine phosphatases with similar structures. Mice deficient in Shp-2 die at midgestation with defects in mesodermal patterning, and a hypomorphic mutation at the Shp-1 locus results in the moth-eaten viable (me(v)) phenotype. Previously, a critical role of Shp-2 in mediating erythroid/myeloid cell development was demonstrated. By using the RAG-2-deficient blastocyst complementation, the role of Shp-2 in lymphopoiesis has been determined. Chimeric mice generated by injecting Shp-2(-/-) embryonic stem cells into Rag-2-deficient blastocysts had no detectable mature T and B cells, serum immunoglobulin M, or even Thy-1(+) and B220(+) precursor lymphocytes. Collectively, these results suggest a positive role of Shp-2 in the development of all blood cell lineages, in contrast to the negative effect of Shp-1 in this process. To determine whether Shp-1 and Shp-2 interact in hematopoiesis, Shp-2(-/-):me(v)/me(v) double-mutant embryos were generated and the hematopoietic cell development in the yolk sacs was examined. More hematopoietic stem/progenitor cells were detected in Shp-2(-/-):me(v)/me(v) embryos than in Shp-2(-/-) littermates. The partial rescue by Shp-1 deficiency of the defective hematopoiesis caused by the Shp-2 mutation suggests that Shp-1 and Shp-2 have antagonistic effects in hematopoiesis, possibly through a bidirectional modulation of the same signaling pathway(s).