Kasem Kulkeaw

Zebrafish erythropoiesis and the utility of fish as models of anemia

Erythrocytes contain oxygen-carrying hemoglobin to all body cells. Impairments in the generation of erythrocytes, a process known as erythropoiesis, or in hemoglobin synthesis alter cell function because of decreased oxygen supply and lead to anemic diseases. Thus, understanding how erythropoiesis is regulated during embryogenesis and adulthood is important to develop novel therapies for anemia. The zebrafish, Danio rerio, provides a powerful model for such study. Their small size and the ability to generate a large number of embryos enable large-scale analysis, and their transparency facilitates the visualization of erythroid cell migration. Importantly, the high conservation of hematopoietic genes among vertebrates and the ability to successfully transplant hematopoietic cells into fish have enabled the establishment of models of human anemic diseases in fish. In this review, we summarize the current progress in our understanding of erythropoiesis on the basis of zebrafish studies and highlight fish models of human anemias. These analyses could enable the discovery of novel drugs as future therapies.

Variation in Hematopoietic Potential of Induced Pluripotent Stem Cell Lines

Induced pluripotent stem (iPS) cells were originally generated from somatic cells by ectopic expression of four transcription factor genes: Oct3/4, Sox2, Klf4 and c-Myc. Currently, iPS cell lines differ in tissue origin, the combination of factors used to construct them, the method of gene delivery and expression of pluripotency markers. Thus to evaluate iPS cells for haematotherapy, the hematopoietic potential among iPS lines should be compared. Here, we compare differentiation capacity of six iPS lines into mesodermal cells and hematopoietic cells (HCs) through embryoid body (EB) formation. We show that the mouse embryonic fibroblast (MEF)-derived iPS lines 20D17 and 178B5 resemble CCE ES cells in terms of morphology in culture, number and size of EBs and differentiation capacity into mesodermal cells compared to iPS cells derived from adults, although all iPS lines could form EBs. The number of mesodermal cells differentiated from MEF-derived iPS cell lines showed a 3.9–407-fold increase compared to that from iPS lines derived from adults. Furthermore, 178B5 iPS cells generated Ter119+ erythroid cells (3.35%) efficiently in culture. We conclude that hematopoietic potential differs among the six lines and that MEF-derived 20D17 and 178B5 iPS cells generate HCs more efficiently than adult–derived iPS cells.

Hepatoblasts comprise a niche for fetal liver erythropoiesis through cytokine production.

In mammals, definitive erythropoiesis first occurs in fetal liver (FL), although little is known about how the process is regulated. FL consists of hepatoblasts, sinusoid endothelial cells and hematopoietic cells. To determine niche cells for fetal liver erythropoiesis, we isolated each FL component by flow cytometry. mRNA analysis suggested that Dlk-1-expressing hepatoblasts primarily expressed EPO and SCF, genes encoding erythropoietic cytokines. EPO protein was detected predominantly in hepatoblasts, as assessed by ELISA and immunohistochemistry, and was not detected in sinusoid endothelial cells and hematopoietic cells. To characterize hepatoblast function in FL, we analyzed Map2k4(-/-) mouse embryos, which lack hepatoblasts, and observed down-regulation of EPO and SCF expression in FL relative to wild-type mice. Our observations demonstrate that hepatoblasts comprise a niche for erythropoiesis through cytokine secretion.

Embryonic regulation of the mouse hematopoietic niche

Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells (HCs) (such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages) and also undergo self-renewal to sustain hematopoiesis throughout an organisms lifetime. HSCs are currently used clinically as transplantation therapy in regenerative medicine and are typically obtained from healthy donors or cord blood. However, problems remain in HSC transplantation, such as shortage of cells, donor risks, rejection, and graft-versus-host disease (GVHD). Thus, increased understanding of HSC regulation should enable us to improve HSC therapy and develop novel regenerative medicine techniques. HSC regulation is governed by two types of activity: intrinsic regulation, programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from so-called “niche cells” surrounding HSCs. Here, we focus on the latter and discuss HSC regulation with special emphasis on the role played by niche cells.

Cold exposure down-regulates zebrafish hematopoiesis.

Erythropoiesis is regulated such that a sufficient number of mature erythrocytes is produced. Down-regulation of erythropoiesis causes various types of anemia. Although some anemia-related genes have been identified, there are several types of anemic disease for which the molecular mechanisms are yet unclear, suggesting that unidentified genes in addition to the classical cytokine pathways play important roles in anemia. To address this issue, a new animal model for anemia is required. We established a reversible anemic model in zebrafish by keeping fish at 17 degrees C, a low water temperature. In zebrafish kidney marrow, expression of several genes encoding hematopoietic transcription factors (Runx1, scl, c-myb and GATA-2) and particularly erythropoiesis-related factors (klfd, hbaa1, ba1, GATA-1, EPO, and EPOr) was down-regulated, whereas myelopoiesis-related factors (csf1a and csf3) was up-regulated in low temperature conditions. We propose that this zebrafish model is useful to identify novel genes for hematopoiesis, particularly erythropoiesis.

TGF-beta-1 up-regulates extra-cellular matrix production in mouse hepatoblasts.

Fetal liver is the major embryonic hematopoietic organ and is extrinsically colonized by circulating hematopoietic stem cells (HSCs). Integrin beta-1 expression on HSCs is crucial for colonization, suggesting that interaction of Integrin beta-1 with extra-cellular matrix (ECM) factors promotes HSC adherence to fetal liver. However, little is known about how ECM production is regulated in fetal liver. Here we used flow cytometry to sort fetal liver compartments and detected ECM gene and protein expression predominantly in sorted hepatoblasts. mRNA and protein analysis suggested that TGF-beta-1 expressed by hepatoblasts, sinusoid endothelial cells and hematopoietic cells, binds to the TGF-beta receptor type-2 expressed on hepatoblasts to stimulate ECM production. Intra-cardiac injection of TGF-inhibitors into mouse embryos dramatically decreased fetal liver ECM gene expression. Taken together, our observations suggest that hepatoblasts predominantly produce ECM factors under control of TGF-beta-1 in fetal liver.

Cold exposure down-regulates zebrafish pigmentation.

Vertebrates use adaptive mechanisms when exposed to physiologic stresses. However, the mechanisms of pigmentation regulation in response to physiologic stresses largely remain unclear. To address this issue, we developed a novel pigmentation model in adult zebrafish using coldwater exposure (cold zebrafish). When zebrafish were maintained at 17 °C, the pigmentation of their pigment stripes was reduced compared with zebrafish at 26.5 °C (normal zebrafish). In cold zebrafish, gene expression levels of tyrosinase and dopachrome tautomerase, which encode enzymes involved in melanogenesis, were down‐regulated, suggesting that either down‐regulation of melanin synthesis occurred or the number of melanophores decreased. Both regular and electron microscopic observation of zebrafish skin showed that the number of melanophores decreased, whereas aggregation of melanosomes was not changed in cold zebrafish compared with normal zebrafish. Taken together, we here show that cold exposure down‐regulated adult zebrafish pigmentation through decreasing the number of melanophores and propose that the cold zebrafish model is a powerful tool for pigmentation research.

APOA-1 is a Novel Marker of Erythroid Cell Maturation from Hematopoietic Stem Cells in Mice and Humans

The mechanism that regulates the terminal maturation of hematopoietic stem cells into erythroid cells is poorly understood. Therefore, identifying genes and surface markers that are restricted to specific stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identified 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identified and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression of the Apoa-1 gene, an apolipoprotein family member, significantly increased as hematopoietic stem cells differentiated into mature erythroid cells in the mouse FL. The Apoa-1 protein was more abundant in mature erythroid cells than hematopoietic stem and progenitor cells in the mouse FL by ELISA. Moreover, APOA-1 gene expression was detected in mature erythroid cells from human peripheral blood. We conclude that APOA-1 is a novel marker of the terminal erythroid maturation of hematopoietic stem cells in both mice and humans.

Induction of intrinsic apoptosis in leukaemia stem cells and in vivo zebrafish model by betulonic acid isolated from Walsura pinnata Hassk (Meliaceae)

BACKGROUND Leukaemia stem cells (LSC) have been associated with disease relapse and chemotherapy resistance. Betulonic acid (BA), a pentacyclic lupane-type triterpenoid, was reported to exhibit cytotoxicity toward various cancer cells and to be capable of inducing intrinsic apoptosis in solid tumours. However, the in vitro and in vivo apoptotic effects of BA against LSC remain unknown. HYPOTHESIS/PURPOSE We aimed to determine whether BA isolated from bark of Walsura pinnata Hassk (Meliaceae) has pro-apoptotic effects on LSC in in vitro and in vivo models. STUDY DESIGN/METHODS The population of high purity LSC was isolated from the Kasumi-1 cell line using magnetic sorting and characterised by flow cytometry. Cell viability was assessed using the MTS assay to examine dose- and time-dependent effects. The colony formation assay was performed in MethoCult® H4435 enriched media. Apoptosis was analysed using Annexin-V and propidium iodide staining, mitochondrial transmembrane potential was studied using JC-1 staining, and expression of apoptosis related genes (BAX, Bcl-2 and survivin) was evaluated by real time-polymerase chain reaction (RT-PCR). Caspase 3/7 and 9 activities were monitored through Promega Caspase-Glo® over a period of 24h. The in vivo antileukaemia activity was evaluated using LSC xenotransplanted zebrafish, observed for DNA fragmentation from apoptosis by TUNEL assay. RESULTS BA maintained its potency against the LSC population in comparison to parental Kasumi-1 cells (fold differences ≤ 1.94) over various treatment time points and significantly inhibited the formation of colonies by LSC. Apoptosis was triggered by BA through the upregulation of BAX and suppression of Bcl-2 and survivin genes with the loss of mitochondrial transmembrane potential, leading to the activation of caspase 9 followed by downstream caspase 3/7. BA was able to suppressed leukaemia formation and induced apoptosis in LSC xenotransplanted zebrafish. CONCLUSIONS The results demonstrate that BA inhibited the proliferative and colonogenic properties of LSC. BA induced apoptosis in LSC through the mitochondria pathway and was effective in the in vivo zebrafish model. Therefore, BA could be a lead compound for further development into a chemotherapy agent against LSC.

Localized SCF and IGF-1 secretion enhances erythropoiesis in the spleen of murine embryos

Fetal spleen is a major hematopoietic site prior to initiation of bone marrow hematopoiesis. Morphologic analysis suggested erythropoietic activity in fetal spleen, but it remained unclear how erythropoiesis was regulated. To address this question, we performed flow cytometric analysis and observed that the number of spleen erythroid cells increased 18.6-fold from 16.5 to 19.5 days post-coitum (dpc). Among erythropoietic cytokines, SCF and IGF-1 were primarily expressed in hematopoietic, endothelial and mesenchymal-like fetal spleen cells. Cultures treated with SCF and/or IGF-1R inhibitors showed significantly decreased CD45−c-Kit−CD71+/−Ter119+ erythroid cells and downregulated Gata1, Klf1 and &bgr;-major globin expression. Administration of these inhibitors to pregnant mice significantly decreased the number of CD45−c-Kit−CD71+/−Ter119+ cells and downregulated &bgr;-major globin gene expression in embryos derived from these mice. We conclude that fetal spleen is a major erythropoietic site where endothelial and mesenchymal-like cells primarily accelerate erythropoietic activity through SCF and IGF-1 secretion.