ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.

Yolk sac erythromyeloid progenitors expressing gain of function PTPN11 have functional features of JMML but are not sufficient to cause disease in mice.

BACKGROUND: Accumulating evidence suggests the origin of juvenile myelomonocytic leukemia (JMML) is closely associated with fetal development. Nevertheless, the contribution of embryonic progenitors to JMML pathogenesis remains unexplored. We hypothesized that expression of JMML-initiating PTPN11 mutations in HSC-independent yolk sac erythromyeloid progenitors (YS EMPs) would result in a mouse model of pediatric myeloproliferative neoplasm (MPN). RESULTS: E9.5 YS EMPs from VavCre+;PTPN11D61Y embryos demonstrated growth hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and hyperactive RAS-ERK signaling. Mutant EMPs engrafted the spleens of neonatal recipients, but did not cause disease. To assess MPN development during unperturbed hematopoiesis we generated CSF1R-MCM+;PTPN11E76K ;ROSAYFP mice in which oncogene expression was restricted to EMPs. Yellow fluorescent protein-positive progeny of mutant EMPs persisted in tissues one year after birth and demonstrated hyperactive RAS-ERK signaling. Nevertheless, these mice had normal survival and did not demonstrate features of MPN. CONCLUSIONS: YS EMPs expressing mutant PTPN11 demonstrate functional and molecular features of JMML but do not cause disease following transplantation nor following unperturbed development. Developmental Dynamics 246:1001-1014, 2017. © 2017 Wiley Periodicals, Inc.

Transcriptional profiling of erythroid progenitors from G-CSF mobilized and nonmobilized peripheral blood.

PURPOSE: The purpose of this study was to examine the gene expression profile of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB)-derived progenitors, used in transplantation. METHODS: We correlated gene expression patterns of highly enriched steady-state peripheral blood (PB)- and mPB-derived CD71+ cells by microarray and ingenuity pathway analyses, to identify the transcriptional program during in vitro erythroid differentiation. RESULTS: The gene expression was more than doubled in mPB-derived (4180 genes) compared to PB-derived erythroid progenitors (1667 genes) while PB-and mPB-derived erythroid progenitors shared 1534 common genes. Comparative analysis of transcript levels showed differential expression of 54 genes between cultured erythroid progenitors of PB and mPB origin, where we identified common 13 downregulated and 30 upregulated genes. The most significant genes in mPB-derived erythroid progenitors were P4HB, DDIA3, ARPC2 and ATP5G3. Regarding G-CSF stimulation the G-CSF receptor CSF2RB (1.1-fold) was linked via STAT3 to erythroid-specific ALAS2 (2.9-fold) and GATA2 (1.3-fold) factors, all upregulated in mPB-derived erythroid progenitors, coupled to common upregulated NUDC gene involved in the proliferation of erythroid cells. CONCLUSION: This report provides an extensive transcriptional profile of cultured erythroid progenitors and leads to a better understanding of diversity among the progenitor sources.

Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(-) fraction.

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

Establishment of culturing system for ex-vivo expansion of angiogenic immature erythroid cells, and its application for treatment of patients with chronic severe lower limb ischemia.

Angiogenesis therapy by bone marrow-mononuclear cell implantation (BMI) has been utilized. We found that erythroid cells played an essential role in angiogenesis by BMI. We then tried to establish a novel cell therapy by implantation of ex vivo expanded immature erythroblasts cultured from hematopoietic stem/precursor cells. Immature to mature erythroblasts were purified from human bone marrow, and mRNA expression were analyzed. Strongly expressed VEGF and PLGF in immature erythroid cells decreased according to erythroid maturation. To expand very immature erythroid cells, we established a two-step culturing system, i.e., bone marrow cells were cultured in the presence of Flt-3L, SCF and TPO for 7 days, and the cells were further cultured in the presence of SCF, IGF-I and EPO for an additional 7 days. The in vivo angiogenic effects of implantation of the ex vivo expanded cells were stronger than that of BMI in mouse limb ischemia model. Three patients with severe chronic lower limb ischemia accompanied by Burger's disease or collagen arteritis were enrolled in a pilot clinical trial of the novel cell therapy by transplantation of ex-vivo expanded immature erythroid cells. In the clinical trial, most clinical symptoms such as rest pain and skin ulcers improved in 4 weeks, and did not recur in the one-year follow-up. No adverse events were observed in any of the patients. Moreover this novel cell therapy required only a small amount of bone marrow collection. Further enrollment of patients with chronic severe lower limb ischemia is necessary to confirm the efficacy and safety of this novel cell therapy, and to estimate the necessary amount of bone marrow aspirate.

CD71+ Erythroid Cells in Human Neonates Exhibit Immunosuppressive Properties and Compromise Immune Response Against Systemic Infection in Neonatal Mice.

Newborns are highly susceptible to infectious diseases. The underlying mechanism of neonatal infection susceptibility has generally been related to their under-developed immune system. Nevertheless, this notion has recently been challenged by the discovery of the physiological abundance of immunosuppressive erythroid precursors CD71+erythroid cells (CECs) in newborn mice and human cord blood. Here, as proof of concept, we show that these cells are also abundant in the peripheral blood of human newborns. Although their frequency appears to be more variable compared to their counterparts in mice, they rapidly decline by 4 weeks of age. However, their proportion remains significantly higher in infants up to six months of age compared to older infants. We found CD45 expressing CECs, as erythroid progenitors, were the prominent source of reactive oxygen species (ROS) production in both humans and mice. Interestingly, a higher proportion of CD45+CECs was observed in the spleen versus bone marrow of neonatal mice, which was associated with a higher ROS production by splenic CECs compared to their siblings in the bone marrow. CECs from human newborns suppressed cytokine production by CD14 monocytes and T cells, which was partially abrogated by apocynin in vitro. Moreover, the depletion of CECs in neonatal mice increased the number of activated effector immune cells in their spleen and liver, which rendered them more resistant to Listeria monocytogenes infection. This was evident by a significant reduction in the bacteria load in the spleen, liver and brain of treated-mice compared to the control group, which enhanced their survival rate. Our finding highlights the immunoregulatory processes mediated by CECs in newborns. Thus, such tightly regulated immune system in newborns/infants may explain one potential mechanism for the asymptomatic or mild COVID-19 infection in this population.

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin.

A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD50) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications.The therapeutic use of single-chain antibodies (VHHs) is limited by their short half-life in the circulation. Here the authors engineer mouse and human red blood cells to express VHHs against botulinum neurotoxin A (BoNT/A) on their surface and show that an infusion of these cells into mice confers long lasting protection against a high dose of BoNT/A.

Stimulation of erythroid engraftment by recombinant human erythropoietin in ABO-compatible, HLA-identical, allogeneic bone marrow transplant patients.

Recombinant human erythropoietin (rhEpo) was given i.v. at a dose of 50 U/kg/tid to eight patients undergoing an HLA-matched, ABO-compatible bone marrow transplantation (BMT), from day +1 up to day +30. Compared to the data recorded in 13 similar BMT patients who had not received the hormone, the administration of rhEpo resulted in a faster erythroid engraftment: in fact, the time required to reach a stable hematocrit value greater than or equal to 35% decreased from 123.0 to 58.0 days after BMT. Moreover, the number of blood reticulocytes on day +21 was about fourfold greater in the rhEpo group than in the controls, while the number of the most immature, high RNA content reticulocytes (HFR), as determined by a flow cytometric technique, was more than sixfold greater; finally, the recovery time of both total and HFR reticulocytes was significantly reduced by rhEpo. The stimulation of erythroid progenitors also resulted in a reduction in red blood cell (RBC) transfusion requirements: the number of RBC units delivered in the first 30 days following BMT decreased from 8.1 in the controls to 4.0, while the total number of RBC units before transfusion independence was about threefold lower than in the control. Finally, the time of transfusion dependence was significantly shortened by rhEpo. No clinically significant adverse effect directly attributable to rhEpo was recorded. These data suggest that the administration of rhEpo may be beneficial in hastening erythroid engraftment, and possibly in reducing RBC transfusion requirements following BMT.

Transient and permanent engraftment potential of murine hematopoietic stem cell subsets: differential effects of host conditioning with gamma radiation and cytotoxic drugs.

Transplant of sorted donor (B6-Gpi-1a) hematopoietic stem cell subsets and host (B6-Gpi-1b) treatment with total body irradiation (TBI) or cytotoxic drugs were compared for induction of short- and long-term engraftment in a murine chimera model of congenic bone marrow transplantation (BMT). Parallel studies on donor and host marrow were performed in vitro in long-term bone marrow cultures to determine early and late cobblestone area forming cell (CAFC) frequencies in the grafts or in the transplant recipients 1 day after conditioning. Bone marrow cells (BMC) sorted for high wheat germ agglutinin affinity (WGA ) were enriched about 30-fold for early developing CAFC (colony-forming unit-spleen [CFU-S]) but not for primitive late CAFC (pre-CFU-S). This fraction showed only temporary engraftment when transplanted in irradiated recipients. In contrast, the low affinity (WGA+) fraction were preferentially enriched (200- to 300-fold) for late developing CAFC and were very effective for providing stable long-term engraftment following transplantation. Substituting radiation for chemotherapy in the host conditioning treatment also had diverse effects on the development of bone marrow engraftment. Pretreatment with 5-fluorouracil (5-FU, 200 mg/kg) allowed a discrete wave of donor engraftment that peaked at 2 to 4 weeks and then subsided to leave mostly host cells at 10 weeks and beyond. Busulfan preparation gave over 50% engraftment at 1 month after BMT but this continued to increase to reach stable donor chimerism of 80 to 90% beyond 10 weeks. The level of long-term engraftment given by 50 mg/kg busulfan appeared similar to that induced by doses of 6 to 8 Gy TBI. The changing patterns of erythroid chimerism for each preparative agent showed a remarkable correlation with depletion of defined hematopoietic stem cell subsets as quantified using the CAFC assay at 1 day after recipient treatment. These findings collectively show that the level of depletion of host CFU-S (CAFC-10) determines the extent of early and transient repopulation, whereas the degree of pre-CFU-S (CAFC-35) depletion determines the percentage of stable chimerism. Preliminary data on bone marrow CAFC content at 9 months after busulfan and BMT revealed a long-term reduction of the stem cell pool by about 50% but with relatively minor effects on supporting bone marrow stromas. The differential cytotoxic effects on stem cell subsets in relation to subsequent donor marrow engraftment offer a systematic and mechanistic approach toward identifying more effective chemotherapeutic compounds for use in clinical BMT conditioning regimens.

Dynamics of erythropoietic recovery following bone marrow transplantation: role of marrow proliferative capacity and erythropoietin production in autologous versus allogeneic transplants.

The mechanisms of erythrocyte recovery after BMT are not known. We investigated the respective role of marrow function and erythropoietin production in 31 ABMT and 47 allogeneic BMT by analysing peripheral counts, serum erythropoietin levels, and serum transferrin receptor (TfR) levels which have been shown to be a quantitative measurement of erythropoiesis. Median times to complete neutrophil (25 vs 48 days, p < 0.0001) and platelet (45 vs 263 days, p < 0.001) recovery were faster after allogeneic BMT than ABMT, but complete erythrocyte recovery was slower (218 vs 101 days, p < 0.001). After ABMT, erythrocyte recovery paralleled that of neutrophils and platelets, and erythropoietin levels remained appropriate for the degree of anemia. After allogeneic BMT, erythrocytes developed independently of the other cell lines and defective erythropoietin production delayed recovery of adequate erythropoietic activity. This correlated with an alteration of renal function only in those patients remaining erythropoietin deficient beyond day 180. However, supranormal erythropoietin levels in interstitial pneumonia suggests that erythropoietin response to hypoxia is not abrogated. CMV infection could also affect erythropoiesis through erythropoietin production after ABMT as well as allogeneic BMT. It is concluded that after ABMT the development of erythropoiesis is determined by the overall marrow proliferative activity and erythropoietin plays only a facilitating role. After allogeneic BMT, erythropoiesis depends on erythropoietin levels which remain inadequate for prolonged periods of time. The results suggest that the administration of recombinant human erythropoietin could reduce transfusion requirements after BMT.

Ex vivo expansion of megakaryocyte precursor cells in autologous stem cell transplantation for relapsed malignant lymphoma.

To evaluate the impact of ex vivo expanded megakaryocyte (MK) progenitors on high-dose chemotherapy-induced thrombocytopenia, we conducted a phase II study in 10 patients with relapsed lymphoma. Two fractions of peripheral blood progenitor cells (PBPC) were cryopreserved, one with enough cells for at least 2 x 10(6) CD34+ cells/kg and a second obtained after CD34+ selection. Ten days before autologous stem cell transplantation, the CD34+ fraction was cultured with MGDF+SCF for 10 days. After BEAM (BCNU, cyclophosphamide, cytarabine, and melphalan) chemotherapy, patients were reinfused with standard PBPC and ex vivo expanded cells. No toxicity was observed after reinfusion. The mean fold expansion was 9.27 for nucleated cells, 2 for CD34+ cells, 676 for CD41+ cells, and 627 for CD61+ cells. The median date of platelet transfusion independence was day 8 (range: 7-12). All patients received at least one platelet transfusion. In conclusion, ex vivo expansion of MK progenitors was feasible and safe, but this procedure did not prevent BEAM-induced thrombocytopenia. Future studies will determine if expansion of higher numbers of CD34+ cells towards the MK-differentiation pathway will translate into a functional effect in terms of shortening of BEAM-induced thrombocytopenia.

In utero stem cell therapy.

In utero stem cell transplantation offers the potential for treating a number of genetic disorders. The combination of fetal immunotolerance and fetal marrow space makes the fetus an excellent transplant recipient. Experiments on the mouse, sheep and rhesus monkey have indicated that in utero transplantation is feasible. Human trials are currently beginning.

Prospects for human gene therapy.

Retroviral vectors containing marker genes and the sequence for human proteins have been used to transduce cultured lymphocytes, which have then been reinfused into patients. Circulating hematopoietic progenitor cells from human fetal cord blood obtained at the time of term and premature deliveries as early as 19 weeks of gestation have been shown to express such transduced genes in vitro. Cord blood cells from fetal sheep sampled and transduced ex vivo and transfused back in utero expressed marker genes up to two years after birth. Although the efficiency of gene transfer into cells and their long-term expression need to be improved, the potential exists for treating some genetic diseases after prenatal diagnosis either in utero or shortly after birth.

[The infection of mice with the influenza virus suppresses bone marrow precursor differentiation in the erythroid and granulocyte directions]. / Infitsirovanie myshei virusom grippa ugnetaet differentsirovku kostnomozgovykh predshestvennikov v éritroidnom i granulotsitarnom napravleniiakh.

Mice (CBA X C57BL/6)F1 were inoculated intranasally with nonlethal doses of influenza A/PR/8/34 virus (500 EID50). At different intervals postinoculation, bone marrow cells were collected from them and inoculated intravenously to lethally irradiated syngeneic recipients with the purpose of determination of the histological types of colony-forming units of spleen (CFUs). On day 3 after inoculation, inhibition of CFUs differentiation in erythroid and granulocytic directions was observed, and in 7 days only granulocytic shoot of hemopoiesis was inhibited. Lymph node cells of the donors in 3 days postinoculation inhibited the colony-forming activity of bone marrow cells of intact mice with suppression of differentiation of erythrocytes and granulocytes. The possible mechanisms of influenza virus-mediated effect on the processes of bone marrow hemopoiesis are discussed.

Donor lymphocyte infusions for relapse after allogeneic transplantation: when, if and for whom?

Donor lymphocyte infusion (DLI) using unstimulated leukapheresis is one of the most effective treatment strategies for patients with hematological malignancies; its graft-versus-leukemia effects make it especially effective in chronic myeloid leukemia patients who relapsed after allogeneic stem cell transplantation (allo-HSCT). However, DLI application is limited by the development of graft-versus-host disease and aplasia, and thus cannot be routinely applied for prophylaxis. Therefore, important questions remain to be answered, such as when, and whom to DLI? Recent advances enable DLI using allografts of granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells; allodepleted donor T cells; and infusions of donor-derived, ex vivo-expanded, CD8(+) cytotoxic T lymphocyte, which can decrease relapse and improve transplant outcomes. Preemptive immunotherapy of relapse was also introduced based on the determination of mixed chimerism and minimal residual disease. In this review, we summarize the latest developments in recent strategies that will affect future DLI efficacy - focusing on the disadvantages and advantages of each protocol for the treatment, preemptive therapy, and prophylaxis of relapse.

Red blood cell production from immortalized progenitor cell line.

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If immortalized erythroid progenitor cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. We have developed a robust method to establish immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of mouse embryonic stem (ES) cells and have established many immortalized erythroid progenitor cell lines so far. Although their precise characteristics varied among cell lines, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. Considering the number of human ES cell lines that have been established so far and the number of induced pluripotent stem cell lines that will be established in future, the intensive testing of a number of these lines for establishing immortalized erythroid progenitor cell lines may allow the establishment of such cell lines similar to the mouse erythroid progenitor cell lines.