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.     

Differential Gene Expression of Human CD34+ Hematopoietic Stem and Progenitor Cells Before and After Culture

Ex vivo expanded CD34⁺ hematopoietic stem and progenitor cells (HSPCs) have compromised homing and engraftment capacities. To investigate underlying mechanisms for functional changes of expanded HSPCs, we compared gene expression profiling of cultured and fresh CD34⁺ cells derived from cord blood using SMART-PCR and cDNA array: 20 genes were up-regulated while 25 genes were down-regulated in cultured CD34⁺ HSPCs. These differentially expressed genes are involved primarily in proliferation, differentiation, apoptosis, and homing. Revisions requested 27 September 2005; Revisions received 14 December 2005     

Role of Reactive Oxygen Species in the Radiation Response of Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem/progenitor cells (HSPCs), which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS). The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34⁺ cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34⁺ HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34⁺ HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34⁺ HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34⁺ HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34⁺ HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.     

Unlimited in vitro expansion of adult bi‐potent pancreas progenitors through the Lgr5/R‐spondin axis

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt‐agonistic R‐spondins (RSPOs). Intestinal, stomach and liver Lgr5⁺ stem cells grow in 3D cultures to form ever‐expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1‐based cultures, and develop into budding cyst‐like structures (organoids) that expand five‐fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi‐potentiality.     

Effective expansion of umbilical cord blood hematopoietic stem/progenitor cells by regulation of microencapsulated osteoblasts under hypoxic condition

The expansion of hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) with the support of microencapsulated osteoblasts under hypoxia environment was investigated. The expansion of HSPCs was evaluated through the total number of UCB mononuclear cells (MNCs) produced, their repopulating potential with the colony-forming unit assay (CFU-Cs) and CD34⁺ phenotypic analysis with flow cytometry. At the end of 7 days of culture, the UCB-MNCs, CFU-Cs and CD34⁺ cells had achieved 18.7 ± 1.6, 11.6 ± 0.9 and 23.4 ± 2-fold expansions, respectively, in the test groups. These were significantly different from those in control groups. Microencapsulated osteoblasts under hypoxia conditions had therefore a significant effect on the expansion potential of HSPCs in vitro.     

The pharmacological activation of adenosine A1 and A3 receptors does not modulate the long- or short-term repopulating ability of hematopoietic stem and multipotent progenitor cells in mice

This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A₁ and A₃ receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N ⁶-cyclopentyladenosine (CPA), an adenosine A₁ receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N ⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), an adenosine A₃ receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.     

Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67‐RFP allele

Cycling Lgr5⁺ stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5⁺ cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67^RFP knock-in allele that identifies dividing cells. Using Lgr5-GFP;Ki67^RFP mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5^high stem cells are continuously in cell cycle, while a fraction of Lgr5^low progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5^low Ki67⁻ cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment.     

Lgr5+ amacrine cells possess regenerative potential in the retina of adult mice

Current knowledge indicates that the adult mammalian retina lacks regenerative capacity. Here, we show that the adult stem cell marker, leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (Lgr5), is expressed in the retina of adult mice. Lgr5⁺ cells are generated at late stages of retinal development and exhibit properties of differentiated amacrine interneurons (amacrine cells). Nevertheless, Lgr5⁺ amacrine cells contribute to regeneration of new retinal cells in the adult stage. The generation of new retinal cells, including retinal neurons and Müller glia from Lgr5⁺ amacrine cells, begins in early adulthood and continues as the animal ages. Together, these findings suggest that the mammalian retina is not devoid of regeneration as previously thought. It is rather dynamic, and Lgr5⁺ amacrine cells function as an endogenous regenerative source. The identification of such cells in the mammalian retina may provide new insights into neuronal regeneration and point to therapeutic opportunities for age‐related retinal degenerative diseases.     

Delineating spatiotemporal and hierarchical development of human fetal innate lymphoid cells

Whereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA^– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2^– CCR9⁺ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.Subject terms: Innate immunity, Haematopoietic stem cells     

TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis

In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutant^cas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1^cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1^cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1^cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.     

Kit ligand has a critical role in mouse yolk sac and aorta–gonad–mesonephros hematopoiesis

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.     

Evaluation of a clinical-grade,cryopreserved, ex vivo-expanded stem cell product from cryopreserved primary umbilical cord blood demonstrates multilineage hematopoietic engraftment in mouse xenografts

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34⁺ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP).MethodscGMP VPA-mediated expansion was initiated with CD34⁺ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts.ResultsThe authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products.ConclusionsThe authors’ results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34⁺ cells for clinical utilization.     

Bmi-1 over-expression in neural stem/progenitor cells increases proliferation and neurogenesis in culture but has little effect on these functions in vivo

The polycomb gene Bmi-1 is required for the self-renewal of stem cells from diverse tissues, including the central nervous system (CNS). Bmi-1 expression is elevated in most human gliomas, irrespective of grade, raising the question of whether Bmi-1 over-expression is sufficient to promote self-renewal or tumorigenesis by CNS stem/progenitor cells. To test this we generated Nestin-Bmi-1-GFP transgenic mice. Analysis of two independent lines with expression in the fetal and adult CNS demonstrated that transgenic neural stem cells formed larger colonies, more self-renewing divisions, and more neurons in culture. However, in vivo, Bmi-1 over-expression had little effect on CNS stem cell frequency, subventricular zone proliferation, olfactory bulb neurogenesis, or neurogenesis/gliogenesis during development. Bmi-1 transgenic mice were born with enlarged lateral ventricles and a minority developed idiopathic hydrocephalus as adults, but none of the transgenic mice formed detectable CNS tumors, even when aged. The more pronounced effects of Bmi-1 over-expression in culture were largely attributable to the attenuated induction of p16^Ink4a and p19^Arf in culture, proteins that are generally not expressed by neural stem/progenitor cells in young mice in vivo. Bmi-1 over-expression therefore has more pronounced effects in culture and does not appear to be sufficient to induce tumorigenesis in vivo.     

Pannexin-1 channel “fuels” by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1⁺ and CD11b⁺ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

     

CD74 is a regulator of hematopoietic stem cell maintenance

Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment.

Hematopoietic stem and progenitor cells (HSPCs) can self-renew and differentiate into all blood cell lineages, making them useful for clinical transplantations and regenerative medicine. This study shows that blocking the MIF receptor CD74 increases the accumulation of HSPCs and could improve the efficacy of bone marrow transplantation protocols.     

miR-155 Is Associated with the Leukemogenic Potential of the Class IV Granulocyte Colony-Stimulating Factor Receptor in CD34+ Progenitor Cells

Granulocyte colony-stimulating factor (G-CSF) is a major regulator of granulopoiesis on engagement with the G-CSF receptor (G-CSFR). The truncated, alternatively spliced, class IV G-CSFR (G-CSFRIV) has been associated with defective differentiation and relapse risk in pediatric acute myeloid leukemia (AML) patients. However, the detailed biological properties of G-CSFRIV in human CD34⁺ hematopoietic stem and progenitor cells (HSPCs) and the potential leukemogenic mechanism of this receptor remain poorly understood. In the present study, we observed that G-CSFRIV–overexpressing (G-CSFRIV⁺) HSPCs demonstrated an enhanced proliferative and survival capacity on G-CSF stimulation. Cell cycle analyses showed a higher frequency of G-CSFRIV⁺ cells in the S and G2/M phase. Also, apoptosis rates were significantly lower in G-CSFRIV⁺ HSPCs. These findings were shown to be associated with a sustained Stat5 activation and elevated miR-155 expression. In addition, G-CSF showed to further induce G-CSFRIV and miR-155 expression of peripheral blood mononuclear cells isolated from AML patients. A Stat5 pharmacological inhibitor or ribonucleic acid (RNA) interference–mediated silencing of the expression of miR-155 abrogated the aberrant proliferative capacity of the G-CSFRIV⁺ HSPCs. Hence, the dysregulation of Stat5/miR-155 pathway in the G-CSFRIV⁺ HSPCs supports their leukemogenic potential. Specific miRNA silencing or the inhibition of Stat5-associated pathways might contribute to preventing the risk of leukemogenesis in G-CSFRIV⁺ HSPCs. This study may promote the development of a personalized effective antileukemia therapy, in particular for the patients exhibiting higher expression levels of G-CSFRIV, and further highlights the necessity of pre-screening the patients for G-CSFR isoforms expression patterns before G-CSF administration.     

Gene Targeting Study Reveals Unexpected Expression of Brain-expressed X-linked 2 in Endocrine and Tissue Stem/Progenitor Cells in Mice

Identification of genes specifically expressed in stem/progenitor cells is an important issue in developmental and stem cell biology. Genome-wide gene expression analyses in liver cells performed in this study have revealed a strong expression of X-linked genes that include members of the brain-expressed X-linked (Bex) gene family in stem/progenitor cells. Bex family genes are expressed abundantly in the neural cells and have been suggested to play important roles in the development of nervous tissues. However, the physiological role of its individual members and the precise expression pattern outside the nervous system remain largely unknown. Here, we focused on Bex2 and examined its role and expression pattern by generating knock-in mice; the enhanced green fluorescence protein (EGFP) was inserted into the Bex2 locus. Bex2-deficient mice were viable and fertile under laboratory growth conditions showing no obvious phenotypic abnormalities. Through an immunohistochemical analysis and flow cytometry-based approach, we observed unique EGFP reporter expression patterns in endocrine and stem/progenitor cells of the liver, pyloric stomach, and hematopoietic system. Although Bex2 seems to play redundant roles in vivo, these results suggest the significance and potential applications of Bex2 in studies of endocrine and stem/progenitor cells.