Human Human Hematopoietic Stem/progenitor Cells Research Articles

Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo

Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.

Prediction of hub genes and key pathways associated with the radiation response of human hematopoietic stem/progenitor cells using integrated bioinformatics methods

Hematopoietic stem cells (HSCs) are indispensable for the maintenance of the entire blood program through cytokine response. However, HSCs have high radiosensitivity, which is often a problem during radiation therapy and nuclear accidents. Although our previous study has reported that the combination cytokine treatment (interleukin-3, stem cell factor, and thrombopoietin) improves the survival of human hematopoietic stem/progenitor cells (HSPCs) after radiation, the mechanism by which cytokines contribute to the survival of HSPCs is largely unclear. To address this issue, the present study characterized the effect of cytokines on the radiation-induced gene expression profile of human CD34+ HSPCs and explored the hub genes that play key pathways associated with the radiation response using a cDNA microarray, a protein–protein interaction-MCODE module analysis and Cytohubba plugin tool in Cytoscape. This study identified 2,733 differentially expressed genes (DEGs) and five hub genes (TOP2A, EZH2, HSPA8, GART, HDAC1) in response to radiation in only the presence of cytokines. Furthermore, functional enrichment analysis found that hub genes and top DEGs based on fold change were enriched in the chromosome organization and organelle organization. The present findings may help predict the radiation response and improve our understanding of this response of human HSPCs.

Evaluation of the Effects of Power-Frequency Magnetic Field Exposure on B-Cell Differentiation From Human Hematopoietic Stem/Progenitor Cells.

The causal relationship between exposure to power-frequency magnetic fields (MFs) and childhood leukemia has long been controversial. The most common type of childhood leukemia is acute B-lymphoblastic leukemia caused by abnormal proliferation of B cells in the early differentiation process. Here, we focused on B-cell early differentiation and aimed to evaluate the effects of exposing cells to power-frequency MF. First, we optimized an in vitro differentiation protocol of human hematopoietic stem/progenitor cells (HSPCs) to B-cell lineages. Following validation of the responsiveness of the protocol to additional stimulations and the uniformity of the experimental conditions, human HSPCs were continuously exposed to 300 mT of 50 Hz MF for 35 days of the differentiation process. These experiments were performed in a blinded manner. The percentages of myeloid or lymphoid cells and their degree of differentiation from pro-B to immature-B cells in the MF-exposed group showed no significant changes compared with those in the control group. Furthermore, the expression levels of recombination-activating gene (RAG)1 and RAG2 in the B cells were also similar to those in the control group. These results indicate that exposure to 50 Hz MF at 300 mT does not affect the human B-cell early differentiation from HSPCs. © 2023 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

Repressive β-Catenin-Ikaros Complexes Are Essential to Prevent Clonal Evolution of Human Lymphopoiesis

Introduction β-catenin signaling represents a central driver of proliferation and survival in multiple epithelial, neuronal, and mesenchymal lineages, but is dispensable for hematopoietic development. Illegitimate activation of Wnt/β-catenin signaling induce development arrest at progenitor stages and underlies development of myeloid dysplastic syndrome (MDS). Moreover, β-catenin is required for emergence and self-renewal of leukemia stem cells (LSC), which are responsible for initiation and relapse of acute myeloid leukemia (AML). Experimental approach and results Our global interactome and gene editing experiments revealed that β-catenin engages lymphoid-specific Ikaros zinc finger (IKZF) factors to repress MYC and restrict lymphoid cell proliferation. Therefore, we reasoned that genetic deletion of β-catenin in human hematopoietic stem/progenitor cells (HSPC) could selectively benefit lymphoid development, which could be useful under lymphopenic conditions, immunosenescence and myeloid skewing in elderly. To this end, we introduced Crispr/Cas9 mediated deletion of β-catenin in CD34+ HSPCs from human cord blood and injected them into immune-deficient recipients for assessing multi-lineage reconstitution of human hematopoiesis. Compared to the controls, mice that received β-catenin-deleted progenitor cells showed increased human chimerism in the peripheral blood at 10 and 15 weeks after transplantation. Comparing absolute cell numbers, β-catenin-deficient progenitors gave rise to markedly increased human B-cell and T-cell production in the bone marrow and spleen, while myeloid populations were largely unchanged. Mice receiving β-catenin deficient cells contained significantly more human thymocytes compared to the control mice. These findings suggest that genetic deletion of β-catenin does not adversely impact human hematopoiesis and instead relieves lymphoid-intrinsic repression of B- and T-lymphopoiesis by β-catenin:IKZF complexes. Therefore, we hypothesized that deletion of β-catenin in pre-leukemic cells from MDS patients would favor generation of normal lymphocytes and prevent transformation into AML. To test this hypothesis, we deleted β-catenin in CD34+ HSPCs derived from bone marrow of MDS patients and injected them into immune-deficient recipients. 16 weeks after transplantation, we detected human B cells only in the blood of the recipients that received β-catenin deficient HSPCs but the mice that were injected with control cells only had myeloid cells in their blood. Interestingly, β-catenin deletion increased lymphoid cell generation by 5-and 2-fold in the bone marrow and spleen, respectively. Pseudo-bulk analysis of single-cell RNA sequencing of MDS-derived bone marrow cells revealed that upon β-catenin deletion, genes that define leukemic stem cell signature in AML (LSC17) such as CD34, SOCS2, EMP1 etc. were repressed. Strikingly, β-catenin deleted bone marrow progenitors from MDS patients could migrate to the thymus and give rise to de-novo thymopoeisis, while none of the mice that received control cells could generate human thymocytes. Although β-catenin deletion improved human lymphopoiesis from pre-leukemic HSPCs, we observed evolution into T-ALL and CD8+ TCR γδ T-cell lymphoma in two cases studied. In the first case human thymocytes generated from HSPCs with 5q- deletion, were arrested at CD4CD8 double positive stage (CD7+ CD3-), resembling a subgroup of T-cell acute lymphoblastic leukemia (T-ALL) with 5q- deletions. In the second case, deleting β-catenin in HSPCs carrying EZH2G660V mutation led to clonal expansion of CD8+ γδ-T cells in spleen of the mice. Conclusion In summary, deletion of β-catenin in human HSPCs improved lymphoid output in humanized mice. However, in the absence of β-catenin-IKZF mediated tumor-suppression, HSPCs carrying 5q- deletion and EZH2G660V mutation evolved into T-ALL and γδ-T cell lymphoma, respectively. These results suggest, although targeting Wnt/β-catenin pathway may be a promising strategy to treat MDS and AML and improve lymphopoiesis, interference with β-catenin-IKZF mediated restraint of lymphoid expansion may unleash pathological lymphocytes and lead to development of lymphoid malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NOTCH-mediated exvivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

SummaryActivation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

A Novel Source of Endogenous DNA Damage That Requires Repair By the Fanconi Anemia Pathway

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome. Impaired DNA interstrand crosslink (ICL) repair is the underlying mechanism for BMF in FA. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage are most likely to play an important role in the pathogenesis of FA. Metabolic by-products, such as reactive aldehydes, have been implicated in the acceleration of BMF or leukemia in both humans and mice lacking the ICL repair pathway. However, the potential contribution of other detoxifying metabolic enzymes to genome maintenance has not been systematically investigated. Identification of all sources of endogenous DNA damage will allow us to develop novel strategies to prevent DNA damage from arising, and possibly prevent BMF and leukemia in FA as well as other BMF syndromes. To determine whether detoxifying enzymes other than ALDH2 or ADH5 play a role in the protection of HSPC, we performed a metabolism-focused CRISPR/Cas9 synthetic lethality screen (3000 metabolism genes, 10 sgRNA per gene), using wild-type and FANCD2-/- Jurkat cells. From the screen, we identified ALDH9A1 as the most significantly depleted gene in FANCD2-/- Jurkat cells compared with wild-type. Eight out of ten sgALDH9A1 were significantly depleted in FANCD2-/- Jurkat cells, indicating robust effect of the ALDH9A1 knockout. ADH5, a known synthetic lethal gene with FA, was also depleted in FANCD2-/- Jurkat cells, but to a lesser degree. In vitro fluorescence-based competition assay confirmed synthetic lethal interaction between the two genes, in two independent FANCD2-/- Jurkat clones. To determine whether ALDH9A1 deficiency also caused cell death in FA-deficient human hematopoietic stem progenitor cells (HSPCs), we performed an in vitro validation assay using human umbilical cord blood (UCB). UCB CD34+ cells were edited by ribonucleoprotein delivery of Cas9 and sgRNA, in which either of sgCTRL, sgFANCD2 or sgALDH9A1, or both sgFANCD2 and sgALDH9A1 were used. Edited cells were grown on methylcellulose for 10 to 14 days, after which individual colonies were scored and harvested for sequencing. While CD34+ cells that were targeted by both sgFANCD2 and sgALDH9A1 (double KO) achieved lower editing efficiency for each gene compared with cells targeted by single guides, they produced the fewest hematopoietic colonies and the lowest frequency of GEMM (Granulocyte, Erythrocyte, Macrophage and Megakaryocyte) colonies. We observed fewer colonies targeted for both genes (biallelic double KO; observed to expected ratio 0.33) as compared to either single gene KO. These results suggest that loss of ALDH9A1 is deleterious in FANCD2-deficient HSPC. Lastly, we generated a Fanca-/-Aldh9a1-/- mouse model to examine the in vivo hematopoietic phenotype due to increased endogenous aldehydes. These mice were born at the Mendelian ratio without significant anomalies except rare cases of eye abnormalities. At three months of life, Fanca-/-Aldh9a1-/- mice had lower platelet counts than wild-type, Fanca-/- or Aldh9a1-/- control mice, but total white blood counts and hemoglobin levels were similar between groups. A follow-up result of this mouse model will be presented at the meeting. In conclusion, we identified that cells with ALDH9A1 deficiency require the FA pathway for survival. ALDH9A1 may protect human and mouse HSPC that are deficient in the FA pathway from DNA damage and cell death. Disclosures No relevant conflicts of interest to declare.

Hypoxia and Notch Signaling Synergistically Expand Long-Term Repopulating Human Hematopoietic Stem/Progenitor Cells

Ex vivo culture of human hematopoietic stem/progenitor cells (HSPCs) under either low O2 tension or Notch signal activation driven by Delta1 ligand (Delta1) have been independently shown to primarily expand short-term HSPCs. In contrast, we have previously demonstrated a significant (5-fold) expansion of long-term repopulating HSPCs upon combination of these two strategies, suggesting synergy between Notch and hypoxia signaling pathways. Understanding the molecular mechanisms underlying this synergy in HSPCs could provide new insights to further enhance expansion by combined activation of these pathways. Given the stoichiometric relationship between the number of Notch receptors (Notch 1-4) and Notch signaling intensity, we assessed whether hypoxia regulated Notch receptor expression. G-CSF mobilized human CD34+ cells from 3 healthy subjects were cultured for 0, 4, 24, or 48hrs in the presence of cytokines (SCF, FLT3L, TPO), under hypoxic (1.5-2.0% O2) or normoxic (21% O2) conditions. Using flow cytometry, Notch 3 expression was not detected in CD34+ cells and no differences in the expression level of any of the other Notch receptor isoforms (Notch 1, 2 and 4) were observed regardless of O2 tension. Another unique aspect of Notch signaling is that the receptor must undergo enzymatic cleavage to initiate signal transduction. These cleavage events culminate in the nuclear translocation of the intracellular domain of notch (ICDN) where it binds to Notch promoter regions, leading to the expression of Notch regulated genes. It has been suggested that a shift in energy metabolism from aerobic to glycolytic may lead to an enhancement of these cleavage events. To directly measure the differences in Notch cleavage events, CD34+ cells from 2 healthy donors were cultured for 24 hours under hypoxic or normoxic conditions, followed by a 1-hour exposure to Delta1. Cells were then collected, processed and stained with antibodies specific to the cleaved ICDN of Notch 1, 2, and 4. Using ImageStream analysis, preliminary data suggest that hypoxia accelerates the rate of Notch 2 receptor intracellular signaling by as much as 2-fold at this early time point. Another potential mechanism by which hypoxia may modulate Notch signaling is via protein-protein interaction of the ICDN and HIF1α, a master transcriptional regulator of cellular response to hypoxia. To assess this possible interaction, CD34+ cells were cultured in chamber slides as described above. After exposure to Delta1, cells were fixed, permeabilized, and labeled with antibodies specific for cleaved ICDN and HIF1α. Ten cells per condition (i.e. normoxia or hypoxia, +/- Delta1) were imaged with a Zeiss 780 inverted confocal microscope. Using Imaris and Interactive Data Language (IDL) software, all high resolution images were analyzed to determine the subcellular localization of each protein. As expected, exposure to Delta1 increased the levels of cleaved ICDN, and hypoxia increased the levels of HIF1α detected. Interestingly, the combination of hypoxia and Delta1 exposure resulted in a significant increase in the ICDN-HIF1α Pearson's coefficient of colocalization (PCC = 0.51) compared to control groups without Delta1 (PCC [normoxia] = 0.17, p<0.0001; PCC [hypoxia] = 0.26, p<0.05). Finally, to assess whether the observed ICDN-HIF1α interaction led to a recruitment of HIF1α to Notch promoter regions and thereby upregulate Notch-driven genes, ChIP-qPCR was performed on cells cultured under the same conditions described above. Samples were fixed, lysed, and immunoprecipitated through an anti-HIF1α column. Selected protein/DNA conjugates were then eluted and qPCR was performed on the captured DNA fragments. In cultures exposed to hypoxia and Delta1, ChiP-qPCR results indicated a significant enhancement of HIF1α /promoter interaction at the Hey-2 gene, a major downstream target of HIF1α. No DNA from this region was detected in the normoxia+Delta1 control sample (0.02% of input vs. below the level of background, p<0.05). Taken together, these data suggest that the synergy between hypoxia and Notch in human HSPCs may be driven by an enhancement of Notch signaling events, a protein-protein interaction between ICDN and HIF1α, and a recruitment of HIF1α to the promoter region of Hey-2, a gene traditionally thought to be regulated by Notch alone. Experiments are ongoing to further understand these mechanisms and their relationship to HSPC expansion. DisclosuresNo relevant conflicts of interest to declare.

Microgravity Impairs DNA Damage Repair in Human Hematopoietic Stem/Progenitor Cells and Inhibits Their Differentiation into Dendritic Cells.

Astronauts on missions beyond low-Earth orbit are exposed to a hostile environment in which they are continually bombarded with unique high-energy species of radiation, while in conditions of microgravity (μG), which can alter radiation response and immunity. In the present studies, we examined the impact exposing human hematopoietic stem/progenitor cells (HSC) to μG had upon their capacity to repair DNA damage and their ability to generate immune cells critical for mounting an effective antitumor response. To this end, we first treated a human HSC-like cell line with an acute dose of the radiomimetic drug bleomycin, cultured them in normal gravity (1G) or simulated μG, and quantitated double-strand breaks through γ-H2AX foci. Calculating the median fluorescence intensity ratio at 1-to-4 h post-bleomycin revealed a 26% decrease in 1G, but a 20% increase in μG, suggesting that μG compromised HSC DNA damage repair and thus has the potential to enhance the genotoxic effects of space radiation. We next examined whether μG negatively affected the development of dendritic cells (DC), critical regulators of both the innate and acquired arms of the immune system. Primary human HSC were cytokine induced in 1G or μG and analyzed for generation of plasmacytoid (CD123+) and myeloid (CD11c+) DC. HSC cultured in 1G gave rise to significantly higher numbers of both myeloid and plasmacytoid DC than those cultured in μG, suggesting μG impairs production of these critical antigen-presenting cells. Our studies thus indicate that conditions of μG present during spaceflight perturb multiple pathways that could potentially enhance astronaut risk from exposure to space radiation.

Hypoxia Potentiates Notch-Induced Expansion of Human Hematopoietic Stem/Progenitor Cells

Activation of Notch signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with Notch ligand Delta1 has enabled a clinically relevant ex vivo expansion of short-term HSPCs. In vitro studies have also revealed a role of low O2 tension in HSPC regulation. A molecular link has been demonstrated in several stem/progenitor cell populations between Notch and hypoxia pathways but their interaction has not been investigated in human HSPCs. G-CSF mobilized human CD34+ cells from 4 healthy subjects were cultured in the presence of cytokines (SCF, FLT3L and TPO) in hypoxia (1.5-2% O2) or normoxia (21% O2) in vessels coated with fibronectin alone or combined with increasing concentrations of the immobilized ligand Delta1 (2.5, 5, 10 and 20 µg/mL). After 21 days in culture, cells were counted and characterized using CFU assays, flow cytometry for lineage (Glycophorin A+, CD13+, CD20+, CD3+ and CD41+ cells) and HSC (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholow) phenotypes, and transplantation in immunodeficient (NSG) mice. In normoxia, the total number of cells increased 118-fold compared to baseline in the absence of Delta1 with limited residual CD34+ cells (1.5 ± 0.7%), extensive differentiation toward the myeloid lineage (96.3 ± 0.3% CD13+ cells) and minimal engraftment potential in NSG mice (0.2 ± 0.2% human CD45+ cells). With increasing concentrations of Delta1 in normoxia, consistent with the hypothesis that Delta1 delays differentiation, the total number of cells increased less (41-, 25-, 11- and 7-fold relative to baseline, respectively) CD34+ cells expanded more (4-, 4-, 3- and 2-fold relative to baseline, respectively), and CFU numbers increased more (8-, 7-, 4- and 3-fold relative to baseline, respectively) than without Delta1. However, phenotypically defined HSCs were undetectable or markedly decreased at the lowest Delta1 concentrations used (2.5 and 5 µg/mL) and their numbers were maintained or only minimally increased at the highest Delta-1 concentrations tested (10 and 20 µg/mL) relative to uncultured CD34+ cells. Accordingly, only cells cultured with 10 and 20 µg/mL Delta1 resulted in levels of engraftment in NSG mice (5.5 ± 5.4% and 5.4 ± 0.9% human CD45+ cells, respectively) comparable to uncultured cells (7.0 ± 0.1% human CD45+ cells). In hypoxia, total cell counts increased less than in normoxia both without (8-fold relative to baseline) and with increasing concentrations of Delta1 (11-, 11-, 9-, 9-fold relative to baseline, respectively) due to diminished myeloid differentiation. Total CD34+ cells decreased 1.7-fold in hypoxia in the absence of Delta1, but expanded modestly in the presence of Delta1 (3-, 3-, 2- and 2-fold, respectively). CFU numbers followed a similar trend. However, in hypoxic cultures with 2.5, 5 and 10 µg/mL Delta1, phenotypically defined HSCs increased 2.5-, 6.6- and 1.3-fold, respectively, compared to uncultured cells. Importantly, hypoxia combined with 2.5, 5 and 10 µg/mL Delta1 concentrations resulted in increased human cell engraftment in NSG mice (21.2 ± 4.4%, 29.3 ± 11% and 11.8 ± 5.4% human CD45+ cells, respectively) compared to uncultured cells (7.0 ± 0.1% human CD45+ cells). When 20 µg/mL Delta1 was used in hypoxia, engraftment potential in NSG mice was decreased (1.1 ± 0.6% human CD45+ cells). We next performed limiting dilution analysis to measure the frequencies of long-term repopulating HSCs (LT-HSCs) within the CD34+ cell compartment at baseline and after 21 days in hypoxic or normoxic cultures supplemented with the optimized concentrations of Delta1 (10 µg/mL in normoxia and 5 µg/mL in hypoxia). LT-HSCs in uncultured CD34+ cells were measured at the expected frequency (1 in 7,706; 95% CI of 3,446 to 17,232). When analyzed at 3 months post-transplantation, a limited (1.5-fold) increase in LT-HSC frequency (1 in 5,090; 95% CI 2.456 to 10,550) was obtained from Delta1 normoxic cultures compared to uncultured cells. In contrast, the frequency of LT-HSCs (1 in 1,586; 95% CI 680 to 3,701) was 4.9-fold higher in hypoxic Delta1 cultures compared to uncultured cells, and 4.2-fold higher than in normoxic Delta1 cultures. Similarly, absolute numbers of LT-HSCs per 100,000 Day 0 equivalent CD34+ cells increased from 13 (baseline) to 216 (normoxia) and 694 (hypoxia). Our data indicate that hypoxia potentiates Notch-induced expansion of human HSPCs and may be of benefit in stem cell transplantation and gene therapy applications. DisclosuresCheruku:Novartis: Research Funding. Larochelle:Novartis: Research Funding.

IMiDs Cause Thrombocytopenia By the Inhibition of Proplatelet Formation, through Blocking Estradiol Autocrine Loop in Human Megakaryocytes

Introduction:Immunomodulatory drugs (IMiDs), lenalidomide (LEN) and pomalidomide (POM), are key agents for hematologic malignancies including multiple myeloma and 5q- syndrome. Thrombocytopenia is one of main side effects of IMiDs, which sometimes interrupts treatment. We here investigated the mechanism of IMiDs-mediated thrombocytopenia.Materials and methods:Colony-forming and differentiation activities of normal human hematopoietic stem/progenitor cells (HSPCs) were evaluated in the presence or absence of 10 μM LEN and POM. The alteration of gene expression profile in megakaryocytes (MKs) exposed to LEN was assessed by DNA microarray. To evaluate the stability of the protein responsible for the IMiDs-mediated inhibition of proplatelet formation (PPF), Western blot and co-immunoprecipitation analyses were performed.Results:We first tested whether IMiDs affect colony-forming potential and lineage readout of human HSPCs. Unexpectedly, the addition of LEN and POM to the culture of HSPCs resulted in a dramatic increase in number of megakaryocytic colonies (CFU-MK and CFU-MegE), whereas the number of erythroid colonies (BFU-E) was decreased. HSPCs preferentially differentiated into CD41+CD105- MK lineage cells in the presence of IMiDs, suggesting that IMiDs enhanced commitment into the MK lineage in human hematopoiesis at the bipotent megakaryocytic/erythroid stage. In clinical setting, however, IMiDs do not cause thrombocytosis, but often lead to thrombocytopenia. We therefore examined whether IMiDs inhibit MK maturation at a later stage of their development. Treatment with IMiDs did not result in morphological changes of MKs in terms of cell size and nuclear ploidy. However, in the presence of LEN (5.7 fold, p < 0.01) and POM (4.1 fold, p < 0.01), MKs did not display PPF, a mechanically indispensable step for platelet production. Transcriptome analysis of MKs suggested that estradiol (E2) signaling was attenuated by LEN (NES = -2.56, P < 0.01). Consistent with a previous finding that the E2 autocrine signaling triggers PPF, we found that the addition of 10nM E2 in the medium effectively recovered PPF of MKs in the presence of IMiDs. Thus, it is suggested that IMiDs-mediated blockade of endogenous E2 signaling impairs PPF. IMiDs modulate proteolysis by binding cereblon (CRBN), the substrate adaptor for ubiquitin ligase complex. Western blot analysis revealed that IMiDs reduced the protein level of aromatase (AROM), a key enzyme for a final step of E2 biosynthesis, without affecting mRNA level of AROM in MKs. We also found that AROM was co-immunoprecipitated with IMiDs-bound CRBN. These results collectively suggest that AROM is the novel substrate of CRBN, and that in the presence of IMiDs, AROM is degradated by binding to IMIDs-bound CRBN, which hampers E2 synthesis to impair PPF in MKs.Conclusions:IMiDs do not inhibit, but rather promote the megakaryocytic lineage commitment of human HSPCs at an early stage of hematopoiesis. IMiDs, however, inhibited PPF through the blockade of E2 autocrine signaling as a consequence of degradation of AROM in MKs, which may be the main cause for thrombocytopenia in IMiDs-treated patients. Thus, development of new IMiDs having low affinity to AROM may be a key to resolve unwanted thrombocytopenia in IMiDs treatment. DisclosuresAkashi:Asahi Kasei Pharma Corporation: Research Funding; Shionogi & Co., Ltd: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Celgene: Research Funding; Bristol Meyers Squibb: Research Funding; Astellas Pharma: Research Funding; Sunitomo Dainippon Pharma: Consultancy.

Human Hematopoietic Stem/Progenitor Cells (HSPCs) and Mesenchymal Stromal Cells (MSCs) Express Several Functional Pituitary and Gonadal Sex Hormone Receptors - Identification of Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) As New Growth Factors for HSPCs and MSCs

Background. Evidence has accumulated that murine hematopoietic stem/progenitor cells (HSPCs) share several markers with the germline, a connection supported by our recent reports that pituitary and gonadal sex hormones (SexHs) regulate development of murine HSPCs (Stem Cells & Develop. 2015, 24, 927-937). In contrast to mice, relatively little is known about the role of SexHs in human hematopoiesis, except that androgens have been employed successfully to treat certain cases of bone marrow (BM) failure. In particular, no studies have been performed to study the roles of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in this process. It is known that the blood level of FSH increases with age and is high in older patients, which correlates with increased risk of hematopoietic malignancies.Aim of the study. To better address the effects of SexHs, and particularly pituitary SexHs, on human hematopoiesis, we tested human HSPCs purified from umbilical cord blood (UCB) and BM for expression of receptors for pituitary SexHs, including FSH, LH, and prolactin (PRL), as well as the receptors for gonadal SexHs, including progesterone, estrogens, and androgen. We then tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. In parallel, we tested the effect of SexHs on human mesenchymal stromal cells (MSCs). Finally, based on our observation that at least some of the UCB-derived, CD45- very small embryonic-like stem cells become specified into CD45+ HSPCs (Leukemia 2011;25,1278-1285), we also evaluated the expression of pituitary and gonadal SexH receptors on these cells.Results. We report for the first time that, like their murine counterparts, human HSPCs, small CD45- very small embryonic-like stem cells endowed with hematopoietic specification potential, and MSCs expressed functional pituitary and gonadal SexH receptors at the mRNA and protein level and responded by MAPKp42/44 and AKT phosphorylation to SexH stimulation. Most importantly, human HSPCs proliferated in vitro in response to SexH stimulation, as did MSCs. Finally, FSH and LH also chemoattracted MSCs in a Transwell migration assay and stimulated these cells to secrete several factors that enhanced in vitro angiogenesis.Conclusions. We report for the first time that FSH, LH, estrogens, and progesterone stimulate growth of human HSPCs and MSCs. In addition, we confirmed that androgens and prolactin stimulate proliferation of human HSPCs. These results are important for understanding the interplay of SexHs in the development and aging of BM, as plasma levels of some of these hormones change with age. Finally, these results further support a developmental link between hematopoiesis and the germline as previously proposed by us and others. DisclosuresNo relevant conflicts of interest to declare.

CD133+ CD34+ HSPCs Are Not Significantly Increased in Fetal Liver Compared to Adult or Umbilical Cord HSPCs

The use of CD133 has recently been described to further characterize human hematopoietic stem/progenitor cells (HSPCs). This led to a revised model of hematopoiesis, in which hematopoietic stem cells and multipotent progenitors (HSCs/MPPs: CD34+ CD133+ CD45RA-) divide asymmetrically giving rise to lympho-myeloid progenitors (LMPPs: CD34+ CD133+ CD45RA+) and erythro-myeloid progenitors (EMPs: CD34+ CD133low CD45RA-). The revised model also suggests, that adult/postnatal multipotent HSCs/MPPs can easily be identified as CD133+ CD34+ cells containing erythroid potentials. Interestingly, previous studies also demonstrated that a substantial fraction of lineage-restricted LMPPs lacking erythro-megakaryocytic lineage-potentials was able to engraft NOD/SCID, therefore questioning the mouse model as a readout for multipotent HSPCs.Since adult/postnatal and fetal human HSPCs differ in their gene expression profiles, differentiation capabilities and cell surface marker expression, we were interested in studying whether the current revised model of hematopoiesis in adult/postnatal stem cells would also apply to hematopoiesis during embryogenesis. Thus, we comprehensively analyzed the CD133 expression, lineage-relationships and content of multipotent HSPCs in the human fetal liver (FL).Consistent with findings in adult tissues, CD133 expression allowed for a facile discrimination of distinct HSC/MPP, LMPP and EMP subpopulations in the human FL. We next determined whether the phenotypic findings would also apply to biologic assays. Freshly isolated FL HSPCs were enriched for phenotypical EMPs (30 ± 5%) and HSCs/MPPs (38 ± 3%); the latter population demonstrated extensive proliferation in vitro and also priming towards erythro-myeloid (EMP) differentiation as shown for adult HSPCs. Previous studies have identified the FL with the highest content of HSCs, containing approximately 100/200-fold more HSCs than umbilical cord blood (UCB) and BM respectively. However in contrast to previous reports, the frequency of multipotent CD133+ HSC/MPPs with erythroid differentiation potential in the FL was much lower than expected (0.87 ± 0.16%), with similar frequencies as in UCB and approximately 2-fold higher than in BM. Upon transplantation in neonatal NOD/SCID mice, rapid and stable (29-89%) multi-lineage engraftment in the peripheral blood (PB) was detected after 8-18 weeks showing human B cells, T cells and monocytes. Surprisingly, analysis of engraftment of more primitive human HSPCs in the murine BM revealed that multi-lineage engraftment of human cells was predominantly driven by short-term engrafting LMPPs with very low levels of multipotent human HSPCs in the BM (0.12% of CD34+ cells).In summary, our data suggests that CD133 expression on FL-HSPCs can be used to identify corresponding functional subpopulations as previously described for adult human stem cell sources and UCB. Surprisingly however, the frequency of multipotent CD133+ HSC/MPPs was substantially lower than expected and, similar to findings with UCB, engraftment of FL-derived CD34+ HSPCs in mice was mostly driven by committed progenitor cells (LMPPs). These data thus challenge the general assumption that the human FL contains greater amounts of multipotent HSCs/MPPs than adult stem cell sources and the use of the SRC assay as a readout for HSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Concurrent Measures of Fusion and Transduction Efficiency of Primary CD34+ Cells with Human Immunodeficiency Virus 1-Based Lentiviral Vectors Reveal Different Effects of Transduction Enhancers

Lentiviral vectors (LVs) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking for precisely evaluating parameters that control the efficiency of transduction in relation to the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer-based human immunodeficiency virus-1 fusion assay to measure the entry of nonreplicative recombinant LVs in various cell types, including primary human hematopoietic stem progenitor cells (HSPCs), and to quantify the level of transduction of the same initially infected cells. The assay utilizes recombinant LVs containing β-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) and encoding a truncated form of the low-affinity nerve growth factor receptor (ΔNGFR). After infection of target cells with BLAM-LVs, the vector entry rapidly leads to BLAM-Vpr release into the cytoplasm, which is measured by cleavage of a fluorescent substrate using flow cytometry. Parallel cultures of the same infected cells show transduction efficiency resulting from ΔNGFR expression. This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing, for instance, the postentry restrictions of LVs known to occur in cells of hematopoietic origin, especially human HSPCs. Furthermore, this BLAM-LV assay allowed us to evaluate the effect of cytokine prestimulation of HSPCs on the entry step of LVs. The assay also shows that transduction enhancers such as Vectofusin-1 or Retronectin can partially relieve the postentry block, but their effects differ in how they promote LV entry. In conclusion, one such assay should be useful to study hematopoietic postentry restrictions directed against LVs and therefore should allow improvements in various LV-based gene therapy protocols.

Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34(+) HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8(+) and CD4(+) T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4-5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8(+) and CD4(+) T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34(+) HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.

Deletion of the LTR Enhancer/Promoter Has No Impact on the Integration Profile of MLV Vectors in Human Hematopoietic Progenitors

Moloney murine leukemia virus (MLV)-derived gamma-retroviral vectors integrate preferentially near transcriptional regulatory regions in the human genome, and are associated with a significant risk of insertional gene deregulation. Self-inactivating (SIN) vectors carry a deletion of the U3 enhancer and promoter in the long terminal repeat (LTR), and show reduced genotoxicity in pre-clinical assays. We report a high-definition analysis of the integration preferences of a SIN MLV vector compared to a wild-type-LTR MLV vector in the genome of CD34+ human hematopoietic stem/progenitor cells (HSPCs). We sequenced 13,011 unique SIN-MLV integration sites and compared them to 32,574 previously generated MLV sites in human HSPCs. The SIN-MLV vector recapitulates the integration pattern observed for MLV, with the characteristic clustering of integrations around enhancer and promoter regions associated to H3K4me3 and H3K4me1 histone modifications, specialized chromatin configurations (presence of the H2A.Z histone variant) and binding of RNA Pol II. SIN-MLV and MLV integration clusters and hot spots overlap in most cases and are generated at a comparable frequency, indicating that the reduced genotoxicity of SIN-MLV vectors in hematopoietic cells is not due to a modified integration profile.

Expansion on Stromal Cells Preserves the Undifferentiated State of Human Hematopoietic Stem Cells Despite Compromised Reconstitution Ability

Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38−CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38−CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.

Human hematopoietic stem/progenitor cells overexpressing Smad4 exhibit impaired reconstitution potential in vivo

AbstractHematopoietic stem cells (HSCs) constitute a rare population of tissue-specific cells that can self-renew and differentiate into all lineages of the blood cell system. These properties are critical for tissue regeneration and clinical applications of HSCs. Cord blood is an easily accessible source of HSCs. However, the number of HSCs from one unit is too low to effectively transplant most adult patients, and expansion of HSCs in vitro has met with limited success because of incomplete knowledge regarding mechanisms regulating self-renewal. Members of the TGF-β superfamily have been shown to regulate HSCs through the Smad signaling pathway; however, its role in human HSCs has remained relatively uncharted in vivo. Therefore, we asked whether enforced expression of the common-Smad, Smad4, could reveal a role for TGF-β in human hematopoietic stem/progenitor cells (HSPCs) from cord blood. Using a lentiviral overexpression approach, we demonstrate that Smad4 overexpression sensitizes HSPCs to TGF-β, resulting in growth arrest and apoptosis in vitro. This phenotype translates in vivo into reduced HSPC reconstitution capacity yet intact lineage distribution. This suggests that the Smad pathway regulates self-renewal independently of differentiation. These findings demonstrate that the Smad signaling circuitry negatively regulates the regeneration capacity of human HSPCs in vivo.

The bone marrow-expressed antimicrobial cationic peptide LL-37 enhances the responsiveness of hematopoietic stem progenitor cells to an SDF-1 gradient and accelerates their engraftment after transplantation

We report that the bone marrow stroma-released LL-37, a member of the cathelicidin family of antimicrobial peptides, primes/increases responsiveness of murine and human hematopoietic stem/progenitor cells (HSPCs) to an α-chemokine stromal-derived factor-1 (SDF-1) gradient. Accordingly, LL-37 is upregulated in irradiated BM cells and enhances the chemotactic responsiveness of hematopoietic progenitors from all lineages to a low physiological SDF-1 gradient as well as increases their i) adhesiveness, ii) SDF-1-mediated actin polymerization, and iii) MAPKp42/44 phosphorylation. Mice transplanted with bone marrow (BM) cells ex vivo primed by LL-37 showed accelerated recovery of platelet and neutrophil counts by ~3–5 days compared to mice transplanted with unprimed control cells. These priming effects were not mediated by LL-37 binding to its receptor and depended instead on incorporation of the CXCR4 receptor into membrane lipid rafts. We propose that LL-37, which has primarily antimicrobial functions and is harmless to mammalian cells, could be clinically applied to accelerate engraftment as ex vivo priming agent for transplanted human HSPCs. This novel approach would be particularly important in cord blood transplantations, where the number of HSCs available is usually limited.

Heavy Ion Beam Irradiation Regulates the mRNA Expression in Megakaryocytopoiesis from Human Hematopoietic Stem/Progenitor Cells

Heavy ion beams are a high-LET radiation that has greater biological effect than electron beams or X-rays. However, little is known about the effect of heavy ion beams on the proliferation and differentiation of human hematopoietic stem/progenitor cells (HSPCs). The present study examined the effect of heavy ion beams on gene expression in human HSPCs, especially during early stage of megakaryocytopoiesis. Human CD34+ cells were exposed to monoenergetic carbon-ion beams (290 MeV/nucleon, LET = 50 KeV/m) that were generated by an accelerator (Heavy Ion Medical Accelerator in Chiba). The expression of various genes related to early hematopoiesis, megakaryocytopoiesis/erythropoiesis, cytokine receptors and oxidative stress were analyzed by real-time RT-PCR. Friend leukemia virus integration 1, an early hematopoiesis-related gene, showed significantly higher mRNA expression than the control at 6 hr after irradiation. In contrast, no significant differences were observed in almost all of the other early hematopoiesis-related genes, cytokine receptor-coded genes and megakaryocytopoiesis/erythropoiesis-differentiation pathway-related genes, respectively. An analysis of the response of the genes to oxidative stress revealed the expression of heme oxygenase 1 to show a 1.5-fold and 11.9-fold increase from the day 0 control at 24 hr after 0.5 Gy and 2 Gy irradiation, respectively. Similarly, the NAD(P)H dehydrogenase-quinone 1 expression also showed a 22.0-fold and a 21.8-fold increase at 6 hr in comparison to the initial control. These results showed that the heavy ion beams affect megakaryocytopoiesis/ erythropoiesis differentiation of human HSPCs on the gene expression level.

Kruppel-Like Factor 4 Upregulates p21 and Downregulates Proliferation of Human and Mouse HSPCs, but Is Not Essential for Mouse HSPC Repopulation.

Several Kruppel-like factor family members, including KLF1, KLF2, KLF3, and KLF6 have pivotal roles in hematopoiesis. Experiments in zebrafish have suggested that KLF4 may play a similar role. Here we found that enforced expression of KLF4 in hematopoietic cells induced cell cycle arrest without triggering apoptosis. Based on the high levels of expression of KLF4 in mouse and human hematopoietic stem-progenitor cells (HSPCs), we hypothesized and demonstrated that KLF4 regulates proliferation of these cells through regulation of p21cip1/waf1 (p21). Nevertheless, KLF4−/− mouse fetal liver cells had normal numbers of all mature lineages and provided radioprotection, similar to wild type (wt) controls. Furthermore, in long-term competitive repopulation assays, KLF4−/− mouse HSPCs demonstrated hematopoietic potency equivalent to wt. We found that KLF2 is expressed at higher levels than KLF4 in mouse HSPCs and is a more potent activator of p21, suggesting that KLF2 (and/or other KLF family members) may play a compensatory role in KLF4−/− HSPCs. Thus, although is not essential for their normal development and function, KLF4 expression is sufficient to induce p21-mediated cell cycle arrest in hematopoietic cells.