Hematopoiesis

The use of donated red blood cells in transfusion is a well-established cellular therapy. However, problems including insufficient supply, transfusion-transmitted infections and the need for immunological matching hamper even in the best services. These issues may be eliminated by using pluripotent stem cells to generate universal donor group O, Rhesus D-negative red blood cells. Human embryonic stem cells can be maintained and expanded indefinitely and can, therefore, produce the very large cell numbers required for this application. Red blood cell production is also an attractive goal for pluripotent stem cell-derived therapeutics because it is a well-characterized single cell suspension, lacking nucleated cells and with a low expression of HLA molecules. Much progress has been made; however, a number of challenges remain including scale-up, clinical effectiveness and product safety.

Endothelial cells (ECs) are an essential component of the hematopoietic microenvironment, which maintains and regulates hematopoietic stem cells (HSCs). Although ECs can support the regeneration of otherwise lethally-irradiated HSCs, the mechanisms are not well understood. To further understand this phenomenon, we studied HSC regeneration from irradiated bone marrow using co-culture with human aortic ECs (HAECs). Co-culture with HAECs induced a 24-fold expansion of long-term HSCs (CD150 + , lineage lo , Sca-1 + , c-Kit + ; CD150 + LSK cells) in vitro. These cells gave rise to functional hematopoietic stem and progenitor cells (HSPCs) with colony-forming activity, multilineage reconstitution and serial transplantation potential. Furthermore, HAECs significantly reduced DNA damage in irradiated LSK cells within 24 h. Remarkably, we were able to delay the exposure of irradiated bone marrow to the regenerative, HAEC-derived signals for up to 48 h and still rescue functional HSCs. G-CSF is the gold standard for promoting hematopoietic regeneration in vivo. However, when compared to HAECs, in vitro G-CSF treatment promoted lineage differentiation and regenerated 5-fold fewer CD150 + LSK cells. Together, our results show that HAECs are powerful, direct mitigators of HSC injury and DNA damage. Identification of the HAEC-derived factors that rescue HSCs may lead to improved therapies for hematopoietic regeneration after radiation injury.

Over 4 years, 43 cases of severe aplastic anaemia (SAA) were seen. Etiologically 58.1% had idiopathic, 39.5% secondary and 2.3% congenital aplastic, anaemia. Idiopathic SAA was more common in patients between 0-15 years of age, whereas secondary SAA was more frequent in 16-60 years age group. Males were more commonly affected with a male:female ratio of 3.3:1. Amongst 17 cases of secondary SAA, chloramphenicol was responsible for 4, septran for 3, insecticides for 2 and anti-scabitic drugs, anti-diabetic drugs and "kushta from hakeem" in one patient each. Hepatitis associated SAA was suspected in 3 cases.

Growth factor independence 1 (Gfi1) is a transcriptional repressor essential for the function and development of many different hematopoietic lineages. The Gfi1 protein expression is regulated by the ubiquitin-proteasome system. In granulocytes, Gfi1 is rapidly degraded by the proteasome, while it is more stable in monocytes. How the ubiquitination and degradation of Gfi1 is regulated is unclear. Here, we show that the ubiquitin ligase Triad1 interacts with the DNA-binding domain of Gfi1. Unexpectedly, we found that Triad1 inhibited Gfi1 ubiquitination, resulting in a prolonged half-life. Down-regulation of endogenous Triad1 by siRNAs resulted in increased Gfi1 ubiquitination. In U937 cells, Triad1 caused an increase in endogenous Gfi1 protein levels and slowed cell proliferation in a similar manner when Gfi1 itself was expressed. A Triad1 mutant that lacks the Gfi1-binding domain did not affect Gfi1 levels and proliferation. Because neither proteasome-ubiquitin nor Triad1 ubiquitin...

In a chemical screening, we tested the antiangiogenic effects of fumagillin derivatives and identified fumagillin as an inhibitor of definitive hematopoiesis in zebrafish embryos. Fumagillin is known to target methionine aminopeptidase II (MetAP2), an enzyme whose function in hematopoiesis is unknown. We investigated the role of MetAP2 in hematopoiesis by using zebrafish embryo and human umbilical cord blood models. Zebrafish metap2 was expressed ubiquitously during early embryogenesis and later in the somitic region, the caudal hematopoietic tissue, and pronephric duct. metap2 was inhibited by morpholino and fumagillin treatment, resulting in increased mpo expression at 18 hours postfertilization and reduced c-myb expression along the ventral wall of dorsal aorta at 36 hours postfertilization. It also disrupted intersegmental vessels in Tg(fli1:gfp) embryos without affecting development of major axial vasculatures. Inhibition of MetAP2 in CB CD34+ cells by fumagillin had no effect ...

blood flow in the proximal femur was measured in 45 healthy male and female adults by means of a Xe-133 washout method. On the basis of a two-compartment exponential model, blood flow was calculated assuming that the compartments were hematopoietic (red) marrow and nonhematopoietic tissues of bone. Between the ages of 20 and 55 the bone perfusion was 8.3 +/- 1.4 (1 s.d.) ml/100 g/min, decreasing thereafter in older patients. During the same period the red-marrow blood flow (RMBF) decreased linearly: RMBF (ml/100 g/min) = -0.14 x age + 24.5. The nonhematopoietic bone perfusion changed like the bone perfusion. The fractional masses of red marrow and nonhematopoietic tissues of bone in the greater trochanteric region of the femur were determined morphometrically from the bone biopsies of seven adult cadavers. They agreed with the values estimated by the Xe-133 washout method. The blood-flow values will be used as reference values for the Xe-133 bone circulation method.

blood flow in the proximal femur was measured in 45 healthy male and female adults by means of a Xe-133 washout method. On the basis of a two-compartment exponential model, blood flow was calculated assuming that the compartments were hematopoietic (red) marrow and nonhematopoietic tissues of bone. Between the ages of 20 and 55 the bone perfusion was 8.3 +/- 1.4 (1 s.d.) ml/100 g/min, decreasing thereafter in older patients. During the same period the red-marrow blood flow (RMBF) decreased linearly: RMBF (ml/100 g/min) = -0.14 x age + 24.5. The nonhematopoietic bone perfusion changed like the bone perfusion. The fractional masses of red marrow and nonhematopoietic tissues of bone in the greater trochanteric region of the femur were determined morphometrically from the bone biopsies of seven adult cadavers. They agreed with the values estimated by the Xe-133 washout method. The blood-flow values will be used as reference values for the Xe-133 bone circulation method.

The variation of HoxB4 expression levels might be a key regulatory mechanism in the differentiation of human embryonic stem cell (hESC)-derived hematopoietic stem cells (HSCs). In this study, hESCs ectopically expressing high and low levels of HoxB4 were obtained using lentiviral gene transfer. Quantification throughout differentiation revealed a steady increase in transcription levels from our constructs. The effects of the two expression levels of HoxB4 were compared regarding the differentiation potential into HSCs. High levels of HoxB4 expression correlated to an improved yield of cells expressing CD34, CD38, the stem cell leukemia gene, and vascular epithelium-cadherin. However, no improvement in myeloid cell maturation was observed, as determined by colony formation assays. In contrast, hESCs with low HoxB4 levels did not show any elevated hematopoietic development. In addition, we found that the total population of HoxB4-expressing cells, on both levels, decreased in developi...

Long-term cultured pre-B cells are able to differentiate into immunoglobulin (Ig)M-positive B cells (IgM+ cells) when transplanted into severe combined immunodeficient (SCID) mice. Based on previous studies, here we report the development of a reconstitution assay in nonobese diabetic/SCID (NOD/SCID) mice using pre-B cells, which allows us to study the role of calpains (calcium-activated endopeptidases) during B cell development as well as in B cell clonal deletion. Using this model, we show that calpastatin (the natural inhibitor of calpains) inhibits B cell receptor–induced apoptosis in IgM+ cells derived from transplanted mice. We thus hypothesize an important function for calpain in sculpting the B cell repertoire.

Mice homozygous for the b allele of the MHC gene, H-2D, have a high incidence of recovery from Friend virus infections, while mice heterozygous for the b allele at H-2D have a very low incidence of recovery. Previous experiments indicated that the low recovery rates associated with heterozygosity at H-2D might be related to a gene dosage effect requiring the expression of two H-2Db alleles for high recovery. We investigated the effects of reduced H-2Db expression on recovery from Friend disease by using H-2b homozygous mice carrying a single beta 2-microglobulin gene disruption. These mice had reductions in cell surface H-2Db expression comparable to those of H-2Da/b heterozygotes. Numerous cell types with various levels of H-2Db expression were examined, and in each case, the expression levels in the beta 2-microglobulin mutants closely reflected those observed in the H-2Da/b heterozygotes. We found, however, that reduced expression did not affect recovery from Friend disease, indi...

Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the majority of hematopoietic stem cell activity, as defined by long-term competitive repopulation of irradiated animals and by a secondary transplant assay for spleen colony-forming units (CFU- S), could be localized in Ly-6b haplotype mice to a fraction of bone marrow cells that expresses the Ly-6A/E (Sca-1) molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains expressing the Thy-1.1 allele indicated that the vast majority of activity was included in the Thy-1low population. In contrast, hematopoietic stem cell activity found in the bone marrow of Thy-1.2 genotype mouse strains was recovered in both the Thy-1neg and the Thy- 1low populations. However, similar to Thy-1.1 strains, most activity was localized to the Ly-6A/...

This perspective summarizes several important advances in hematopoietic stem cell (HSC) biology in the past few years and places these advances in the context of future directions in stem cell research. The potential utility of stem cells for gene therapy, tissue engineering, and the treatment of neurological and other forms of disease is simply too significant to ignore, and yet our knowledge and ability to deliver these forms of therapy in a safe and efficacious manner will require additional advances in the understanding of the basic biology of stem cells.

The stem cell pool can be fractionated by using the mitochondrial dye, rhodamine-123, into Rholow hematopoietic stem cells and Rhohigh progenitors. Rholow stem cells permanently engraft all lineages, whereas Rhohighprogenitors transiently produce erythrocytes, without substantial platelet or granulocyte production. We hypothesized that the inability of the Rhohigh cells to produce platelets in vivo was due to the fact that these cells preferentially engraft in the spleen and lack marrow engraftment. Initially, we demonstrated that Rhohigh progenitors produced more megakaryocytes in vitro than Rholow stem cells did. To study the activity of the Rholow and Rhohighsubsets in vivo, we used mice allelic at the hemoglobin and glucose phosphate isomerase loci to track donor-derived erythropoiesis and thrombopoiesis. Rholow stem cells contributed to robust and long-term erythroid and platelet engraftment, whereas Rhohigh progenitors contributed only to transient erythroid engraftment and pr...

Transcriptional mechanisms governing hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation are not fully understood. Sequence-specific ssDNA-binding protein 2 (SSBP2) is a candidate acute myelogenous leukemia (AML) suppressor gene located at chromosome 5q14. SSBP2 binds the transcriptional adaptor protein Lim domain-binding protein 1 (LDB1) and enhances LDB1 stability to regulate gene expression. Notably, Ldb1 is essential for HSC specification during early development and maintenance in adults. We previously reported shortened lifespan and greater susceptibility to B cell lymphomas and carcinomas in Ssbp2(-/-) mice. However, whether Ssbp2 plays a regulatory role in normal HSC function and leukemogenesis is unknown. In this study, we provide several lines of evidence to demonstrate a requirement for Ssbp2 in the function and transcriptional program of hematopoietic stem and progenitor cells (HSPCs) in vivo. We found that hematopoietic tissues were hypoplastic i...

This article describes the isolation of a novel cell population (B220loc-kit+CD19−) in the fetal liver that represents 70% of T-cell precursors in this organ. Interestingly, these precursors showed a bipotent T-cell and natural killer cell (NK)– restricted reconstitution potential but completely lacked B and erythromyeloid differentiation capacity both in vivo and in vitro. Moreover, not only mature T-cell receptor (TCR)αβ+ peripheral T cells but also TCRγδ+ and TCRαβ+CD8αα+ intestinal epithelial cells of extrathymic origin were generated in reconstituted mice. The presence of this population in the fetal liver of athymic embryos indicates its prethymic origin. The comparison of the phenotype and differentiation potential of B220loc-kit+CD19−fetal liver cells with those of thymic T/NK progenitors indicates that this is the most immature common T/NK cell progenitor so far identified. These fetal liver progenitors may represent the immediate developmental step before thymic immigration.

Lin28A is a highly conserved RNA-binding protein that concurs to control the balance between stemness and differentiation in several tissue lineages. Here, we report the role of miR-128a/Lin28A axis in blocking cell differentiation in acute myeloid leukemia (AML), a genetically heterogeneous disease characterized by abnormally controlled proliferation of myeloid progenitor cells accompanied by partial or total inability to undergo terminal differentiation. First, we found Lin28A underexpressed in blast cells from AML patients and AML cell lines as compared with CD34+ normal precursors. In vitro transfection of Lin28A in NPM1-mutated OCI-AML3 cell line significantly triggered cell-cycle arrest and myeloid differentiation, with increased expression of macrophage associate genes (EGR2, ZFP36 and ANXA1). Furthermore, miR-128a, a negative regulator of Lin28A, was found overexpressed in AML cells compared with normal precursors, especially in acute promyelocytic leukemia (APL) and in &#39...

The International Society of Experimental Hematology holds its annual meeting every northern summer. This year the meeting comprised of eight plenary sessions with distinguished invited speakers on in vivo imaging and tracking of hematopoietic stem cells (HSCs), HSC niches, epigenetic regulations of stem cells and regulation of stem cell fate, leukemogenesis, and mesenchymal stem cells. The small size of the meeting (300 attendees) permitted excellent discussion and face-to-face contacts between students, junior scientists and experts. Owing to the large number of keynote speakers, this report focuses on the most novel, unpublished data presented during the meeting.

There is still controversy concerning the nature of the stem cells from bone marrow that colonize the thymus during embryogenesis and continually throughout life. To identify the bone marrow stem cells that home to and populate the thymus, we screened murine bone marrow cells for the presence of a population of surface phenotype similar to the earliest known intrathymic precursor. We have identified a population characterized by expression of an intermediate level of heat stable antigen, a very low level of Thy-1, and high levels of CD44 and class I major histocompatibility complex antigens. It is negative for B- cell, granulocyte, macrophage, and erythrocyte markers (B220, Gr-1, Mac- 1, and TER 119). All these markers are common to the intrathymic precursors and bone marrow stem cells. However, this new bone marrow population is Sca-2+, similar to the intrathymic precursor, which makes a clear distinction from the Sca-2- bone marrow hematopoietic stem cells previously characterized...

Renal ossicles are ossified structures developed after the implantation of a bone marrow (BM) plug beneath the kidney capsule. The authors have investigated the origin of the hematopoietic cells in murine renal ossicles by conducting sex-mismatched implants into Ly-5 congenic mice. BM plugs from transgenic mice provided additional genotypic tracers. Flow cytometry analyses on nonadherent cells from long-term cultures established with ossicles excised at 17 to 40 weeks postimplantation evidenced the presence of 5% to 70% of donor-derived myeloid cells. The genetic analysis of the day 12 colony-forming unit (CFU-S12) population in ossicles excised at 10 to 40 weeks postimplantation revealed that 16% to 93% of the colonies were of donor origin. Moreover, we describe for the first time the presence of long-term repopulating cells of donor origin in ossicles excised at 10 to 19 weeks postimplantation.

We report our experience of peripheral blood and bone marrow changes in patients with HIV disease. Abnormalities were most commonly seen in patients with advanced disease. In AIDS group IV patients (CDC classification) anaemia (92%) neutropenia (85%) monocytopenia (75%) and thrombocytopenia (61%) have their highest incidence, the reason being a combination of factors such as infection, myelosuppressive drugs and HIV infection itself. Bone marrow examinations were performed most commonly for microbiological culture (25%) and the investigation of anaemia (16%). Morphological changes in the bone marrow were non‐specific and not pathognomic; however erythroid hypoplasia was found to be a distinctive feature associated with MAI infection. The procedure provided a high yield for microbiological culture, particularly in MAI infection.

Transient albuminuria induced by pathogen-associated molecular patterns (PAMPs) in mice via engagement of Toll-like receptors (TLRs) is widely studied as a partial model for some forms of human nephrotic syndrome (NS). In addition to TLRs, CD80 has been shown to be essential for PAMP-mediated albuminuria. However, the mechanistic relationships between TLRs, CD80 and albuminuria remain unclear. Here, we show that albuminuria and CD80-uria induced in mice by many TLR ligands are dependent on expression of TLRs and their downstream signalling intermediate MyD88 exclusively in hematopoietic cells and, conversely, on CD80 expression exclusively in non-hematopoietic cells. TNFα is critical for TLR-mediated albuminuria and CD80-uria, and induces CD80 expression in cultured renal podocytes. IL-10 from hematopoietic cells ameliorates TNFα production, albuminuria and CD80-uria but does not prevent TNFα-mediated induction of podocyte CD80 expression. Chitohexaose, a small molecule originally o...

Similar to other lymphoid organs, the bone marrow (BM) is a potential reservoir for HIV-1. Although hematologic abnormalities are common in AIDS patients, the mechanisms by which HIV-1 contributes to these abnormalities are poorly understood. Hemapoietic suppression can be mediated by HIV-1 and some of its associated proteins. We have previously reported that AIDS peripheral blood sera inhibit normal CFU-granulocyte macrophage (CM). In this study, we have found that sera obtained from AIDS BM patients are more inhibitory to CFU-CM than those from peripheral blood ( p < 0.05). To determine a candidate suppressor factor, we studied the role of p24 and found that it exerts varying degrees of suppression to CFU-CM, but minimal inhibition on erythroid colonies. We have found that the suppressive levels of p24 in in vitro assays are similar to the average circulating levels in AIDS patients. We have also observed that most of the inhibition caused by p24 is mediated by soluble factor(s) produced by the BM stroma. Cross-linking studies with membrane proteins from normal BM stroma and CD34+ cells indicated a putative 26-kDa p24 receptor, indicating that p24 binds to a receptor present on normal BM cells. These results implicate another HIV-1 protein, p24, in hemapoietic suppression and add another plausible mechanism by which HIV-1 contributes to BM failure in AIDS. Furthermore, these findings suggest a potential risk for similar in vivo suppression in the early phase of HIV-1 infection when viremia is elevated, and also in long-term survivors who have low, although persistent, HIV-1 replication.

The neuropeptide Substance P (SP) is widely distributed in the peripheral nervous system. Its biologic effects have been extensively studied in the immune system. However, even though the bone marrow (BM) is innervated with SP-immunoreactive fibers and some of its cells not only express SP receptors (T and B cells, endothelial cells, and macrophages) but also produce SP (macrophages, eosinophils, and endothelial cells), the effects of SP on hematopoiesis are scanty. Furthermore, SP induces the production of hematopoietic growth factors (HGFs) (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) from human monocytes. In this study, we have found a potent in vitro stimulatory effect of SP (10(-8) to 10(-12) mol/L) on hematopoiesis for both erythroid and granulocytic progenitors in short-term methyl- cellulose BM cultures. SP alone, in the absence of exogenous HGFs, is able to sustain hematopoiesis in vitro. This stimulatory effect of SP is: (1) mostly mediated by the adherent...