Impaired B cell terminal differentiation in B cell-specific knockout mice of cell death-defying factor anamorsin.

Anamorsin (AM) is an anti-apoptotic molecule cloned by us as a molecule that confers resistance against apoptosis induced by growth factor deprivation. AM-deficient mice are embryonic lethal, which impedes detailed analyses of the roles of AM in various types of adult cells. To overcome the embryonic lethality, we generated AM conditional knockout (AMflox/flox) mice and cell type-specific genetic modification became possible using the Cre-loxP system. CD19-Cre/AMflox/flox mice with AM deleted specifically in CD19+ B cells exhibited less B220+ B cells in their spleen, peripheral blood, and lymph node compared with control CD19-Cre mice. Using flow cytometry to categorize bone marrow and spleen cells into B cell subsets, we observed significantly less follicular type I cells, which are the most mature follicular B cells, compared with control CD19-Cre mice. These data suggest that AM has an important role in the generation of mature B cells.

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis.

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34+CD38+CD33+ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3+ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.

Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor immune responses in humans.

CD4(+) Treg cells expressing the transcription factor FOXP3 (forkhead box P3) are abundant in tumor tissues and appear to hinder the induction of effective antitumor immunity. A substantial number of T cells, including Treg cells, in tumor tissues and peripheral blood express C-C chemokine receptor 4 (CCR4). Here we show that CCR4 was specifically expressed by a subset of terminally differentiated and most suppressive CD45RA(-)FOXP3(hi)CD4(+) Treg cells [designated effector Treg (eTreg) cells], but not by CD45RA(+)FOXP3(lo)CD4(+) naive Treg cells, in peripheral blood of healthy individuals and cancer patients. In melanoma tissues, CCR4(+) eTreg cells were predominant among tumor-infiltrating FOXP3(+) T cells and much higher in frequency compared with those in peripheral blood. With peripheral blood lymphocytes from healthy individuals and melanoma patients, ex vivo depletion of CCR4(+) T cells and subsequent in vitro stimulation of the depleted cell population with the cancer/testis antigen NY-ESO-1 efficiently induced NY-ESO-1-specific CD4(+) T cells. Nondepletion failed in the induction. The magnitude of the responses was comparable with total removal of FOXP3(+) Treg cells by CD25(+) T-cell depletion. CCR4(+) T-cell depletion also augmented in vitro induction of NY-ESO-1-specific CD8(+) T cells in melanoma patients. Furthermore, in vivo administration of anti-CCR4 mAb markedly reduced the eTreg-cell fraction and augmented NY-ESO-1-specific CD8(+) T-cell responses in an adult T-cell leukemia-lymphoma patient whose leukemic cells expressed NY-ESO-1. Collectively, these findings indicate that anti-CCR4 mAb treatment is instrumental for evoking and augmenting antitumor immunity in cancer patients by selectively depleting eTreg cells.

The endothelial antigen ESAM monitors hematopoietic stem cell status between quiescence and self-renewal.

Whereas most hematopoietic stem cells (HSC) are quiescent in homeostasis, they actively proliferate in response to bone marrow (BM) injury. Signals from the BM microenvironment are thought to promote entry of HSC into the cell cycle. However, it has been cumbersome to assess cycle status of viable HSC and thus explore unique features associated with division. In this study, we show that expression of endothelial cell-selective adhesion molecule (ESAM) can be a powerful indicator of HSC activation. ESAM levels clearly mirrored the shift of HSC between quiescence and activation, and it was prominent in comparison with other HSC-related Ags. ESAM(hi) HSC were actively dividing, but had surprisingly high long-term reconstituting capacity. Immunohistochemical analyses showed that most ESAM(hi) HSC were located near vascular endothelium in the BM after 5-fluorouracil treatment. To determine the importance of ESAM in the process of BM recovery, ESAM knockout mice were treated with 5-fluorouracil and their hematopoietic reconstruction was examined. The ESAM deficiency caused severe and prolonged BM suppression, suggesting that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis. With respect to intracellular regulators, NF-κB and topoisomerase II levels correlated with the ESAM upregulation. Thus, our data demonstrate that the intensity of ESAM expression is useful to trace activated HSC and to understand molecular events involved in stem cell states.

Safety and immunogenicity of BK-SE36/CpG malaria vaccine in healthy Burkinabe adults and children: a phase 1b randomised, controlled, double-blinded, age de-escalation trial.

Background: BK-SE36/CpG is a recombinant blood-stage malaria vaccine candidate based on the N-terminal Plasmodium falciparum serine repeat antigen5 (SE36), adsorbed to aluminium hydroxide gel and reconstituted, prior to administration, with synthetic oligodeoxynucleotides bearing CpG motifs. In healthy Japanese adult males, BK-SE36/CpG was well tolerated. This study assessed its safety and immunogenicity in healthy malaria-exposed African adults and children. Methods: A double-blind, randomised, controlled, age de-escalating clinical trial was conducted in an urban area of Ouagadougou, Burkina Faso. Healthy participants (n=135) aged 21-45 years (Cohort 1), 5-10 years (Cohort 2) and 12-24 months (Cohort 3) were randomised to receive three vaccine doses (Day 0, 28 and 112) of BK-SE36/CpG or rabies vaccine by intramuscular injection. Results: One hundred thirty-four of 135 (99.2%) subjects received all three scheduled vaccine doses. Vaccinations were well tolerated with no related Grade 3 (severe) adverse events (AEs). Pain/limitation of limb movement, headache in adults and fever in younger children (all mild to moderate in intensity) were the most frequently observed local and systemic AEs. Eighty-three of BK-SE36/CpG (91%) recipients and 37 of control subjects (84%) had Grade 1/2 events within 28 days post vaccination. Events considered by the investigator to be vaccine related were experienced by 38% and 14% of subjects in BK-SE36/CpG and control arms, respectively. Throughout the trial, six Grade 3 events (in 4 subjects), not related to vaccination, were recorded in the BK-SE36/CpG arm: 5 events (in 3 subjects) within 28 days of vaccination. All serious adverse events (SAEs) (n=5) were due to severe malaria (52-226 days post vaccination) and not related to vaccination. In all cohorts, BK-SE36/CpG arm had higher antibody titres after Dose 3 than after Dose 2. Younger cohorts had stronger immune responses (12-24-month-old > 5-10 years-old > 21-45 years-old). Sera predominantly reacted to peptides that lie in intrinsically unstructured regions of SE36. In the control arm, there were no marked fold changes in antibody titres and participants' sera reacted poorly to all peptides spanning SE36. Conclusion: BK-SE36/CpG was well-tolerated and immunogenic. These results pave the way for further proof-of-concept studies to demonstrate vaccine efficacy. Clinical trial registration: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1921, PACTR201701001921166.

NAD-dependent histone deacetylase, SIRT1, plays essential roles in the maintenance of hematopoietic stem cells.

Sir2 has been shown to be essential for transcriptional silencing and longevity provided by calorie restriction in Saccharomyces cerevisiae and Caenorhabditis elegans. In this study, we investigated the role for its mammalian homologue, SIRT1, in hematopoietic cells. SIRT1 inhibitor, nicotinamide (NA), promoted and its activator, resveratrol, inhibited the differentiation of murine bone marrow c-Kit(high)Sca-1(+)Lineage(-) (KSL) cells during the culture system ex vivo. To further clarify the roles of SIRT1 in hematopoietic cells, we isolated KSL cells from fetal liver of SIRT1 knockout (KO) mice and cultured them for 5days, because SIRT1 KO mice die shortly after the delivery. In agreement with the results from the experiments using NA and resveratrol, KSL cells isolated from SIRT1 KO mice more apparently differentiated and lost the KSL phenotype than those from wild-type (WT) mice. Furthermore, in each of colony assay, replating assay, or serial transplantation assay, SIRT1 KO KSL cells lost earlier the characteristics of stem cells than WT KSL cells. In addition, we found that SIRT1 maintains prematurity of hematopoietic cells through ROS elimination, FOXO activation, and p53 inhibition. These results suggest that SIRT1 suppresses differentiation of hematopoietic stem/progenitor cells and contributes to the maintenance of stem cell pool.

Pharmacokinetics, Safety, and Tolerability of NPC-21, an Anti-Cytomegalovirus Monoclonal Antibody, in Healthy Japanese and White Adult Men: A Randomized, Placebo-Controlled, First-in-Human Phase 1 Study.

NPC-21 (EV2038) is a fully human monoclonal antibody that targets the antigenic domain 1 of glycoprotein B on the human cytomegalovirus (hCMV) envelope. NPC-21 has been shown to have broadly neutralizing activity and to inhibit cell-to-cell transmission of hCMV in preclinical studies. It is currently in development for the prophylactic or preemptive treatment of hCMV in patients receiving a solid-organ transplant or hematopoietic stem cell transplant. A first-in-human phase 1 study was conducted to assess the pharmacokinetics, safety, and tolerability of NPC-21 in healthy adult men. Forty participants (Japanese, n = 32; White, n = 8) were randomly assigned to receive a single intravenous dose of NPC-21 1, 3, 10, or 20 mg/kg or placebo. Six Japanese participants were included in each dose group and six White participants received a 10-mg/kg dose. The placebo group included 8 Japanese participants and 2 White participants. All 40 participants completed the study. Serum concentration, maximum serum concentration, area under the plasma concentration-time curve from time 0 to the last measurable concentration, and area under the plasma concentration-time curve from time 0 to infinity increased dose dependently; dose proportionality was linear. NPC-21 demonstrated a biphasic elimination pattern, with an estimated half-life between 612 and 790 hours. NPC-21 was safe and well tolerated up to 20 mg/kg. All adverse events were mild, and none led to treatment discontinuation or were considered related to the study drug. There were no differences in pharmacokinetics or safety between Japanese and White participants. These results support further investigation of NPC-21.

The effects of continuous exposure to low-dose chlorine dioxide gas on the characteristics of induced pluripotent stem cells.

Background: Recently, various regenerative therapies have been developed based on induced pluripotent stem (iPS) cells. However, hygienic control strategies have not been established at the manufacturing facilities. We aimed to evaluate the safety and effects of continuous exposure to low-dose chlorine dioxide (ClO2) gas on cell fates, and to determine the optimum dose for safe usage of this disinfectant. Methods: We cultured an iPS cell line in the absence or presence of various doses of ClO2 gas. We evaluated cell proliferation, cell death, the maintenance of undifferentiated state, and cell senescence. Results: We found that iPS cell proliferation was not affected by 0.05 or 0.1 ppmv ClO2 gas in the atmosphere. Although 0.1 ppmv ClO2 slightly affected apoptosis, it was not a significant effect. Moreover, neither at 0.05 nor 0.1 ppmv ClO2 gas significantly affected the characteristics of iPS cells. Discussion and conclusion: Continuous exposure to 0.05 or 0.1 ppmv ClO2 gas did not affect the fate of iPS cells. These results may contribute to the development of new strategies for hygiene control in cell processing facilities.

BCR-ABL but not JAK2 V617F inhibits erythropoiesis through the Ras signal by inducing p21CIP1/WAF1.

BCR-ABL is a causative tyrosine kinase (TK) of chronic myelogenous leukemia (CML). In CML patients, although myeloid cells are remarkably proliferating, erythroid cells are rather decreased and anemia is commonly observed. This phenotype is quite different from that observed in polycythemia vera (PV) caused by JAK2 V617F, whereas both oncogenic TKs activate common downstream molecules at the level of hematopoietic stem cells (HSCs). To clarify this mechanism, we investigated the effects of BCR-ABL and JAK2 V617F on erythropoiesis. Enforced expression of BCR-ABL but not of JAK2 V617F in murine LSK (Lineage(-)Sca-1(hi)CD117(hi)) cells inhibited the development of erythroid cells. Among several signaling molecules downstream of BCR-ABL, an active mutant of N-Ras (N-RasE12) but not of STAT5 or phosphatidylinositol 3-kinase (PI3-K) inhibited erythropoiesis, while N-RasE12 enhanced the development of myeloid cells. BCR-ABL activated Ras signal more intensely than JAK2 V617F, and inhibition of Ras by manumycin A, a farnesyltransferase inhibitor, ameliorated erythroid colony formation of CML cells. As for the mechanisms of Ras-induced suppression of erythropoiesis, we found that GATA-1, an erythroid-specific transcription factor, blocked Ras-mediated mitogenic signaling at the level of MEK through the direct interaction. Furthermore, enforced expression of N-RasE12 in LSK cells derived from p53-, p16(INK4a)/p19(ARF)-, and p21(CIP1/WAF1)-null/wild-type mice revealed that suppressed erythroid cell growth by N-RasE12 was restored only by p21(CIP1/WAF1) deficiency, indicating that a cyclin-dependent kinase (CDK) inhibitor, p21(CIP1/WAF1), plays crucial roles in Ras-induced suppression of erythropoiesis. These data would, at least partly, explain why respective oncogenic TKs cause different disease phenotypes.

Ectonucleotidase CD39 is highly expressed on ATLL cells and is responsible for their immunosuppressive function.

Adult T-cell leukemia/lymphoma (ATLL) patients have an extremely poor prognosis, partly due to their immunosuppressive state. The majority of ATLL patients have leukemic cells with phenotype similar to Tregs, prompting suggestions that ATLL cells themselves have immunosuppressive functions. In this study, we detected CD39 expression on ATLL cells, particularly frequent on aggressive subtypes. CD39 and CD73 convert extracellular adenosine triphosphate (ATP) into adenosine, a key player in Tregs' immunosuppression. In vitro culture, both CD39+ ATLL cells and normal Tregs converted rapidly extracellular ATP to AMP, which was disturbed by CD39 inhibitors, and was negated in the CD39 knockout MJ cell line. The proliferation of cocultured CD4+/CD8+ normal T cells was suppressed by CD39+ MJ cells, but not by CD39 knockout MJ cells. Supplemented ATP was exhausted by an EG7-OVA T-cell line with stable CD39 induction, but not by mock. When these cell lines were subcutaneously transplanted into murine flanks, Poly(I:C) peritoneal administration reduced tumor size to 1/3 in mock-transplanted tumors, but not in CD39 induced tumors. Overall, we found that ATLL cells express CD39 at a high rate, and our results suggest that this helps ATLL cells escape antitumor immunity through the extracellular ATPDase-Adenosine cascade. These findings will guide future clinical strategies for ATLL treatment.

Effects of continuous exposure to low concentration of ClO2 gas on the growth, viability, and maintenance of undifferentiated MSCs in long-term cultures.

INTRODUCTION: Hygienic management is more important in the manufacturing of cell products than in the production of chemical agents, because cell material and final product cannot be decontaminated. On the other hand, especially in the selection of hygienic agent, the adverse effects on the cells must be considered as well as the decontamination effect. ClO2 is a potent disinfectant, which is now expected as a safe and effective hygienic agent in the field of cell production. In this study, we investigated the effects of low dose ClO2 gas in the atmosphere of CO2 incubator on the characteristics of MSCs cultured in it. METHODS: First, we installed a ClO2 generator to a CO2 incubator for cell culture in which a constant level of ClO2 can be maintained. After culturing human cord derived MSCs in the CO2 incubator, the characteristics of cells were analyzed. RESULTS: Continuous exposure to 0.05 ppmv of ClO2 gas did not affect cell proliferation until at least 8th passage. In the FACS analysis, antigens usually expressed on MSCs, CD105, CD90, CD44, CD73 and CD29, were positively observed, but differentiation markers, CD11b and CD34, were little expressed on the MSCs exposed to 0.05 ppmv or 0.1 ppmv of ClO2 gas just as on the control cells. Also in the investigation for cell death, 0.05 ppmv and 0.1 ppmv of ClO2 gas little affected the viability, apoptosis or necrosis of MSCs. Furthermore, we assessed senescence using SA-ß-gal staining. Although the frequency of stained cells cultured in 0.1 ppmv of ClO2 gas was significantly increased than that of not exposed cells, the stained cells in 0.05 ppmv were rare and their frequency was almost the same as that in control. CONCLUSIONS: All these results indicate that, although excessive concentration of ClO2 gas induces senescence but neither apoptosis nor cell differentiation, exposure to 0.05 ppmv of ClO2 gas little affected the characteristics of MSCs. In this study we demonstrate that continuous exposure to appropriate dose of ClO2 gas can be safely used as decontamination agent in cell processing facilities.

First-in-human randomised trial and follow-up study of Plasmodium falciparum blood-stage malaria vaccine BK-SE36 with CpG-ODN(K3).

BACKGROUND: BK-SE36 is blood-stage malaria vaccine candidate that is undergoing clinical trials. Here, the safety and immunogenicity of BK-SE36 with a novel adjuvant, CpG-ODN(K3) (thus, BK-SE36/CpG) was assessed in a phase 1a trial in Japan. METHODS: An investigator-initiated, randomised, single-blind, placebo-controlled, dose-escalation study was conducted at Osaka University Hospital with 26 healthy malaria naïve Japanese male adults. The trial was conducted in two stages: Stage/Group 1, half-dose (n = 7 for BK-SE36/CpG and n = 3 for control) and Stage/Group 2, full-dose (n = 11 for BK-SE36/CpG and n = 5 for control). There were two intramuscular vaccinations 21 days apart for both half-dose (0.5 ml: 50 µg SE36 + 500 µg aluminum + 500 µg K3) and full-dose (1.0 ml: 100 µg SE36 + 1000 µg aluminum + 1000 µg K3). A one-year follow-up was done to monitor changes in autoimmune markers and vaccine-induced antibody response. RESULTS: BK-SE36/CpG was well tolerated. Vaccination site reactions were similar to those observed with BK-SE36. During the trial and follow-up period, no subject had clinical evidence of autoimmune disease. The full-dose group had significantly higher titres than the half-dose group (Student's t-test, p = 0.002) at 21 days post-second vaccination. Antibody titres remained above baseline values during 12 months of follow-up. The vaccine induced antibody was mostly composed of IgG1 and IgM, and recognised epitopes close to the polyserine region located in the middle of SE36. CONCLUSIONS: BK-SE36/CpG has an acceptable safety profile. Use of CpG-ODN(K3) greatly enhanced immunogenicity in malaria naïve Japanese adults when compared to BK-SE36 alone. The utility of BK-SE36/CpG is currently under evaluation in a malaria endemic setting in West Africa. TRIAL REGISTRATION: JMACCT Clinical Trial Registry JMA-IIA00109.

Regulation of human B lymphopoiesis by the transforming growth factor-beta superfamily in a newly established coculture system using human mesenchymal stem cells as a supportive microenvironment.

OBJECTIVES: To characterize and evaluate the validity of a novel coculture system for studying human B-lymphocyte developmental biology. MATERIALS AND METHODS: We developed a long-term culture system to produce B lymphocytes from human CD34(+) cells purified from umbilical cord blood using human mesenchymal stem cells (hMSC) as stroma. We evaluated the effects of several low molecular weight inhibitors, recombinant proteins, and neutralizing antibodies (Abs) as potential regulators of B-lymphocyte development. RESULTS: Our cocultures of 2000 CD34(+) cells in the presence of stem cell factor and Flt3-ligand produced 1-5 x 10(5) CD10(+) cells after 4 weeks of culture. Surface IgM(+) immature B cells began to appear after 4 weeks. We evaluated the negative-regulatory effects of the transforming growth factor (TGF)-beta superfamily on human B lymphopoiesis, and found that adding an anti-activin A antibody enhanced generation of CD10(+) cells two- to three-fold. As well, the proportion of CD10(+) cells in the generated cells increased markedly, indicating that activin A downregulated B lymphopoiesis more efficiently than myelopoiesis. Addition of TGF-beta1 suppressed B-lymphocyte production by 20% to 30%, while addition of an anti-bone morphogenetic protein (BMP)-4 antibody or recombinant BMP-4 had no effect. Therefore, the strength of ability to suppress human B lymphopoiesis seemed to be activin A > TGF-beta1 > BMP-4. None of these three factors influenced the emergence of IgM(+) cells. CONCLUSIONS: hMSC coculture supported human B lymphopoiesis. Activin A selectively suppressed B lymphocyte production.

Understanding the formation and behaviors of droplets toward consideration of changeover during cell manufacturing.

Preventing the contamination of processed cells is required for achieving reproducible manufacturing. A droplet is one of the potential causes contamination in cell manufacturing. The present study elucidates the formation mechanism and characteristics of droplets based on the observation and detection of droplets on the base surface of the biological safety cabinet (BSC) where cell processing is conducted under unidirectional airflow. Pouring fluorescent solution into the vessel using a measuring pipette was conducted to visualize the formation of droplets by videos as well as visual detection by blacklight irradiation on the base surface of the BSC. The experiments revealed that airborne and non-airborne droplets emerged from bursting bubbles, which formed when the entire solution was pushed out of the measuring pipette. Therefore, the improving procedure of pouring technique when entire solution was not pushed out of the pipette realized no formation of the droplets due to the prevention of emergence of bubble. In addition, an alternative procedure in which the entire solution was poured into the deep point of the test tube prevented the flying of non-airborne droplets outside the tube, while airborne droplets that escaped the tube rode the airflow of BSC. These results suggested a method for the prevention of the droplet formation, as well as the deposit control of droplets onto the surface in BSC, leading to cleanup area in the BSC for changeover with environment continuity.

Unexpected Complication of R-CHOP Chemotherapy: Rapidly Progressive Bronchiolitis Obliterans Syndrome.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the term used for the progressive obliteration of small airways before the patient has had a confirmatory lung biopsy. It is also recognized as a transplant-related complication. There have been no reports of BOS during initial standard chemotherapy. CASE PRESENTATION: A 50-year-old woman with newly diagnosed follicular lymphoma grade 2, stage 3A, presented with hypoxia and progressive dyspnoea after the fifth cycle of R-CHOP. High-resolution computed tomography showed air trapping enhanced at the end-expiratory phase. Pulmonary function testing revealed severe obstructive and restrictive failure without bronchodilator response. We diagnosed BOS based on current criteria and treated the patient with glucocorticoids and cyclosporin. She was discharged home on oxygen therapy. However, soon after discharge, her respiratory symptoms deteriorated and she was hospitalized in a palliative care unit. She died of respiratory failure within a year of symptom onset. CONCLUSIONS: This is the first case report to describe rapidly progressive BOS in a patient undergoing R-CHOP treatment, which strongly suggests the condition was caused by the chemotherapy. Although a pathological diagnosis was not obtained, the clinical diagnosis of BOS was important so that the patient could receive appropriate treatment and palliative care based on the prognosis of this incurable condition. LEARNING POINTS: R-CHOP chemotherapy may cause rapidly progressive bronchiolitis obliterans syndrome (BOS).When it is difficult to obtain a pathological diagnosis, a clinical diagnosis of BOS is important for early intervention and appropriate palliative care.

Endothelial Cell-Selective Adhesion Molecule Contributes to the Development of Definitive Hematopoiesis in the Fetal Liver.

Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker of hematopoietic stem cells (HSCs). Although we previously elucidated the functional importance of ESAM in HSCs in stress-induced hematopoiesis in adults, it is unclear how ESAM affects hematopoietic development during fetal life. To address this issue, we analyzed fetuses from conventional or conditional ESAM-knockout mice. Approximately half of ESAM-null fetuses died after mid-gestation due to anemia. RNA sequencing analyses revealed downregulation of adult-type globins and Alas2, a heme biosynthesis enzyme, in ESAM-null fetal livers. These abnormalities were attributed to malfunction of ESAM-null HSCs, which was demonstrated in culture and transplantation experiments. Although crosslinking ESAM directly influenced gene transcription in HSCs, observations in conditional ESAM-knockout fetuses revealed the critical involvement of ESAM expressed in endothelial cells in fetal lethality. Thus, we showed that ESAM had important roles in developing definitive hematopoiesis. Furthermore, we unveiled the importance of endothelial ESAM in this process.

Variable SATB1 Levels Regulate Hematopoietic Stem Cell Heterogeneity with Distinct Lineage Fate.

Hematopoietic stem cells (HSCs) comprise a heterogeneous population exhibiting self-renewal and differentiation capabilities; however, the mechanisms involved in maintaining this heterogeneity remain unclear. Here, we show that SATB1 is involved in regulating HSC heterogeneity. Results in conditional Satb1-knockout mice revealed that SATB1 was important for the self-renewal and lymphopoiesis of adult HSCs. Additionally, HSCs from Satb1/Tomato-knockin reporter mice were classified based on SATB1/Tomato intensity, with transplantation experiments revealing stronger differentiation toward the lymphocytic lineage along with high SATB1 levels, whereas SATB1- HSCs followed the myeloid lineage in agreement with genome-wide transcription and cell culture studies. Importantly, SATB1- and SATB1+ HSC populations were interconvertible upon transplantation, with SATB1+ HSCs showing higher reconstituting and lymphopoietic potentials in primary recipients relative to SATB1- HSCs, whereas both HSCs exhibited equally efficient reconstituted lympho-hematopoiesis in secondary recipients. These results suggest that SATB1 levels regulate the maintenance of HSC multipotency, with variations contributing to HSC heterogeneity.

ESAM is a novel human hematopoietic stem cell marker associated with a subset of human leukemias.

Reliable markers are essential to increase our understanding of the biological features of human hematopoietic stem cells and to facilitate the application of hematopoietic stem cells in the field of transplantation and regenerative medicine. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel functional marker of hematopoietic stem cells in mice. Here, we found that ESAM can also be used to purify human hematopoietic stem cells from all the currently available sources (adult bone marrow, mobilized peripheral blood, and cord blood). Multipotent colony-forming units and long-term hematopoietic-reconstituting cells in immunodeficient mice were found exclusively in the ESAM(High) fraction of CD34(+)CD38(-) cells. The CD34(+)CD38(-) fraction of cord blood and collagenase-treated bone marrow contained cells exhibiting extremely high expression of ESAM; these cells are likely to be related to the endothelial lineage. Leukemia cell lines of erythroid and megakaryocyte origin, but not those of myeloid or lymphoid descent, were ESAM positive. However, high ESAM expression was observed in some primary acute myeloid leukemia cells. Furthermore, KG-1a myeloid leukemia cells switched from ESAM negative to ESAM positive with repeated leukemia reconstitution in vivo. Thus, ESAM is a useful marker for studying both human hematopoietic stem cells and leukemia cells.

Cell cycle regulation in hematopoietic stem/progenitor cells.

Hematopoietic stem cells (HSCs) are characterized by pluripotentiality and a capacity for self-renewal. In order to both maintain a supply of mature blood cells and not to exhaust HSCs throughout the lifespan of the organism, most HSCs remain quiescent and only a limited number enter the cell cycle. In HSCs, the cell cycle is crucially regulated by external factors such as cytokines and interactions with stromal cells and the extracellular matrix (ECM) in the bone marrow (BM) microenvironment. In addition, intrinsic transcription factors expressed in HSCs, including c-Myb, GATA-2, HOX family proteins, and Bmi-1, also control their growth through their effect on gene transcription. In terms of the particular roles in regulation of the cell-cycle, p21WAF1 (p21) and p27KIP1 (p27) were shown to maintain the quiescence of HSCs and of progenitor cells, respectively, thereby governing their available pool sizes. Also, p16INK4A (p16) and p15INK4B (p15) are thought to act as tumor suppressors, since their inactivation and/or deletion are observable in various types of hematologic malignancies. These results make evident that appropriate cell cycle control, particularly at the early stage of stem/progenitor cells, is required for maintaining normal hematopoiesis.