Hemangioblast Differentiation Research Articles

MiRNA-7145-cuedc2 axis controls hematopoiesis through JAK1/STAT3 signaling pathway

Hematopoiesis ensures tissue oxygenation, and remodeling as well as immune protection in vertebrates. During embryogenesis, hemangioblasts are the source of all blood cells. Gata1a and pu.1 are co-expressed in hemangioblasts before hemangioblasts are differentiated into blood cells. However, the genes that determine the differentiation of hemangioblasts into myeloid or erythroid cell lineages have not been fully uncovered. Here we showed that miRNA-7145, a miRNA with previously unknown function, was enriched in erythrocytes at the definitive wave, but not expressed in myeloid cells. Overexpression and loss-of-function analysis of miRNA-7145 revealed that miRNA-7145 functions as a strong inhibitor for myeloid progenitor cell differentiation while driving erythropoiesis during the primitive wave. Furthermore, we confirmed that cuedc2 is one of miRNA-7145 targeted-genes. Overexpression or knock-down of cuedc2 partially rescues the phenotype caused by miRNA-7145 overexpression or loss-of-function. As well, overexpression and loss-of-function analysis of cuedc2 showed that cuedc2 is required for myelopoiesis at the expense of erythropoiesis. Finally, we found that overexpression of zebrafish cuedc2 in 293 T cell inhibits the JAK1/STAT3 signaling pathway. Collectively, our results uncover a previously unknown miRNA-7145-cuedc2 axis, which regulate hematopoiesis through inhibiting the JAK1/STAT3 signaling pathway.

Gfi1aa/Lsd1 Facilitates Hemangioblast Differentiation Into Primitive Erythrocytes by Targeting etv2 and sox7 in Zebrafish.

The first wave of hematopoiesis is the primitive hematopoiesis, which produces embryonic erythroid and myeloid cells. Primitive erythrocytes are thought to be generated from bipotent hemangioblasts, but the molecular basis remains unclear. Transcriptional repressors Gfi1aa and Gfi1b have been shown to cooperatively promote primitive erythrocytes differentiation from hemangioblasts in zebrafish. However, the mechanism of these repressors during the primitive wave is largely unknown. Herein, by functional analysis of zebrafish gfi1aa smu10 , gfi1b smu11 , gfi1ab smu12 single, double, and triple mutants, we found that Gfi1aa not only plays a predominant role in primitive erythropoiesis but also synergizes with Gfi1ab. To screen Gfi1aa downstream targets, we performed RNA-seq and ChIP-seq analysis and found two endothelial transcription factors, etv2 and sox7, to be repressed by Gfi1aa. Genetic analysis demonstrated Gfi1aa to promote hemangioblast differentiation into primitive erythrocytes by inhibiting both etv2 and sox7 in an Lsd1-dependent manner. Moreover, the H3K4me1 level of etv2 and sox7 were increased in gfi1aa mutant. Taken together, these results suggest that Gfi1aa/Lsd1-dependent etv2/sox7 downregulation is critical for hemangioblast differentiation during primitive hematopoiesis by inhibition of endothelial specification. The different and redundant roles for Gfi1(s), as well as their genetic and epigenetic regulation during primitive hematopoiesis, help us to better know the molecular basis of the primitive hematopoiesis and sheds light on the understanding the Gfi1(s) related pathogenesis.

Synthesis of Novel Class of <i>N</i>-Alkyl-isatin-3-iminobenzoic Acid Derivatives and Their Biological Activity in Zebrafish Embryos and Human Cancer Cell Lines

Isatin (1H-indole-2,3-dione) and many of its derivatives are reported to have pharmacological properties. In this study, we report the synthesis and biological activity of a new class of N-alkyl-isatin-3-iminobenzoic acid derivatives prepared via the condensation of N-alkyl isatin with 4-aminobenzoic acid by conventional, microwave, and ultrasonic methods. Microwave irradiation yielded the products in a shorter reaction time with higher yields and purities. The compounds were screened in zebrafish embryos, and also in three human cancer cell lines (MCF7, HepG2, and Jurkat) and one normal human cell line i.e., human foreskin cell line (HFF-1). Two compounds (3c, 3f) were found to be highly effective against hematopoiesis in live zebrafish embryo at 10 µM concentration. The developmental stage-dependent treatment indicated that these compounds interfered with the differentiation of hemangioblasts to hematopoietic cells in zebrafish embryos. The comparative screening of semaxanib (SU5416) (a known isatin derivatives), to compounds synthesized in this study, revealed the contrasting effects of these two classes of isatin derivatives on zebrafish hematopoiesis. Most of the N-alkyl-isatin-3-iminobenzoic acid derivatives were toxic on cancer and non-cancer tested human cells lines, however, the compounds 3c and 3f specifically affected the cell viability of Jurkat cells (human hematological cell line) with least IC50 values of 16.5 and 7.8 µM. The structure-activity relationship (SAR) analysis indicated that the substitution pattern of the isatin at the 5-position was vital for activity. The in vivo and in vitro biological activities of these compounds suggested their potential use as pharmaceutical compounds for human leukemia treatment.

Directing human embryonic stem cells towards functional endothelial cells easily and without purification.

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.

Well-defined differentiation of hESC-derived hemangioblasts by embryoid body formation without enzymatic treatment.

Human hemangioblasts exist only during the early embryonic developmental stage thereby limiting the adult cellular source from which to obtain such cells for study. To overcome this, hemangioblast studies have focused on utilizing human embryonic stem cell (hESC) derivatives but current methods are cell-line dependent. Single cell dissociation of a hESC colony quickly led to cell death in most hESC lines due to enzyme treatment which, in turn, reduced induction potential and hemangioblast differentiation efficiency. Therefore, we sought to effectively improve the process of cell dissociation that is adaptable to various hESC lines and increase the initial induction potential of embryoid body (hEB). As a result, we determined an effective cell dissociation method through a comparison study involving various reagents which demonstrated successful dissociation regardless of cell line and enhanced hemangioblast differentiation efficiency.

3D microcarrier system for efficient differentiation of human induced pluripotent stem cells into hematopoietic cells without feeders and serum

Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential, a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation, the medium for 3D microcarriers was directly switched to EB medium. hESCs on 3D microcarriers maintained pluripotency and formed EBs, which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating, EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore, this 3D system can also be adapted to human induced pluripotent stem cells, which generate functional hemangioblasts with high efficiency. This 3D serum- and stromal-free microcarrier system is important for future clinical applications, with the potential of developing to a GMP-compatible scalable system.

Effect of LMO2 overexpression in hemangioblast on its proliferation and hematopoietic differentiation

To investigate function of the Lim-only protein(LMO2) in hemangioblast generated from murine embryonic stem cells differentiation to hematopoietic cells. The hemangioblast-specific expression vector with lmo2 or green fluorescence protein gene was constructed, respectively. The murine embryonic stem cells were transfected by the hemangioblast-specific expression vectors. The neomycin-resistance ES cell clones were obtained after having been screened by G418. The cell clones were spontaneously differentiated into embryo bodies(EB) containing hemangioblast.Expression of the hematopoietic genes was investigated by real-time reverse transcription-ploymerase chain reaction during EB differentiation.For the EB cells, blast-cloning forming cells analysis and blood-colony forming unit analysis were then performed, respectively. The numbers of the blasts were counted during hematopoietic differentiation. The hemangioblast-specific expression vector with lmo2 or green fluorescence protein was transfected into ES cells.The neomycin-resistance ES cells generated EBs from 2.5 days to 10 days.Real time reverse transcription-ploymerase chain reaction analysis indicated that overexpression of lmo2 increased the expression of hematopoietic genes(gata1, tal1, Β-h1, and Β-major globin) during EB formation.Blast-cloning forming cells analysis showed that the numbers of the blasts generated by ES/lmo2 was 2-or 3-fold than those in the controls.The total numbers of the blood-colony forming unit or the numbers of the erythrocyte colony-forming unit generated by ES/lmo2 were 2.5 times or 3 times, respectively, when compared with the controls. LMO2 enhances the proliferation and differentiation of hemangioblasts.

Endothelio-hematopoietic relationship: getting closer to the beginnings

The close association between hematopoietic and endothelial cells during embryonic development led to the proposal that they may originate from a common ancestor - the hemangioblast. Due to a lack of unique specific markers for in vivo cell fate tracking studies, evidence supporting this theory derives mainly from in vitro differentiation studies. Teixeira and colleagues describe a novel enhancer that drives specific eGFP expression in blood islands of the electroporated chick embryo, thereby presenting a tool potentially suitable for analysis of hemangioblast differentiation and development of blood islands.See research article: http://www.biomedcentral.com/1471-213X/11/76

Efficient Differentiation of Common Marmoset Embryonic Stem Cells Into Hemangioblast-Like Cells by the Inhibition of PI3K-AKT Pathway

Abstract 2206Recently various kinds of functional cells differentiated from embryonic stem cells and induced pluripotent stem cells (ESCs/iPSCs) are expected to be utilized for cell therapy in clinical medicine. Among the transplantable functional cells differentiated from ESCs/iPSCs, endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) are considered to be strong candidate cells for regenerative medicine to cure various diseases such as ischemic disease and hematopoietic malignancy. Although the transplantation of EPCs and HSCs derived from human bone marrow, mobilized peripheral blood, and umbilical cord blood is commonly conducted in clinical settings, their availability for clinical use has often been hampered by both the lack of HLA compatible donor and the insufficient number of the cells. As the in vitro expansion of EPCs and HSCs derived from above sources is very difficult using current technology, it may be easier to expand EPCs and HSCs derived from ESCs/iPSCs in vitro.Hemangioblasts have the ability to differentiate into both EPCs and HSCs. Thus the technology to differentiate hemangioblast from ESCs/iPSCs that possess indefinite proliferative capacity is strongly expected. Differentiation of ESCs/iPSCs to hemangioblasts is best exemplified in recent studies that have used two step procedures to enhance hemangioblast differentiation with embryoid body (EB) formation and blast colony forming cell (BL-CFC) assay (Lu SJ et al., Nat Methods 4: 501–509, 2007). However the efficiency of hemangioblast differentiation by this method was quite low (approximately 0.35 ± 0.01%).PI3K-AKT pathway is well known to regulate various cell functions. In ESCs, PI3K-AKT pathway plays an important role in maintaining the undifferentiated state (Armstrong L et al., Hum Mol Genet 15: 1894–1913, 2006), suggesting that inhibition of PI3K may promote the differentiation of ESCs/iPSCs.Previously, we demonstrated that common marmosets (CM) are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies (Hibino H et al., blood 1: 2839–2848, 1999). To develop the method for the more efficient generation of hemangioblasts from ESCs/iPSCs, we promoted the hemangioblast differentiation by the inhibition of PI3K-AKT pathway with the inhibitor, LY294002. CM-ESCs (Cj11 and CM40) were differentiated by EB formation in the presence of LY294002 for 4 days, and the EBs were trypsinized, and the dissociated individual cells were processed for BL-CFC assay in the methylcellulose medium containing various cytokines without LY294002 for 7 days.The number of blast colonies found in the BL-CFC assay significantly increased (approximately 10-fold; 3.5 ± 0.3%, p < 0.001) with the treatment of LY294002 during EB formation compared with control. The colonies formed in the BL-CFC assay were homogeneous and looked like a tuft of grapes which is one of hemangioblast characters, and expressed hemagioblast markers (FLK1+, VE-cadherin+, CD31+ and CD45−), suggesting that the inhibition of PI3K during EB formation promoted the generation of hemangioblast-like cells from CM-ESCs. To determine endothelial potential of these hemangioblast-like cells derived from CM-ESCs, we grew them as adherent layers on gelatin-coated plates in EGM-2 medium. The adherent cells derived from hemangioblast-like cells expressed endothelial cell markers (CD31 and vWF). Next, we also examined hematopoietic potential of hemangioblast-like cells by colony forming unit (CFU) assay. Unexpectedly no colonies were formed regardless of whether LY294002 was added or not during EB formation, indicating that hemangioblast-like cells derived from CM-ESC might be endothelial progenitors rather than hemangioblasts.Our novel technology is 10-fold more efficient in inducing endothelial differentiation from ESCs than previously reported methods. It should be emphasized that these endothelial progenitors are morphologically homogenous and expressed endothelial cell markers in a defined adherent cell culture condition, suggesting that our novel technology will be useful for an efficient generation of homogeneous EPCs for future regenerative medicine against ischemic diseases. Disclosures:No relevant conflicts of interest to declare.

Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Coordinated Regulation of Embryonic and Adult Hematopoietic Stem Cell Activity by PTPN11/Shp2.

AbstractAbstract 2630Shp2, a tyrosine phosphatase with two SH2 domains, is implicated in malignant blood diseases. Germline dominant active mutations in PTPN11 that codes for Shp2 are found in approximately 50% of Noonan syndrome (NS) patients who have high risk of juvenile myelomonocytic leukemia (JMML). Somatic gain-of-function mutations in PTPN11 have also been detected in nonsyndromic JMML, pediatric AML, B-cell precursor ALL and myelodysplastic syndromes. The majority of leukemia-associated PTPN11 mutations disrupt the auto-inhibitory interactions between its N-terminal SH2 and the phosphatase domain, resulting in elevated catalytic activity. However, the physiological function of Shp2 in normal hematopoiesis in adults remains to be elucidated, although a Shp2 function in embryonic hematopoiesis was documented previously. Mouse embryonic stem cells (mESCs) homozygous for targeted exon 3 deletion are defective in differentiation to all blood cell lineages, suggesting its role in commitment/specification of hematopoietic stem cells (HSCs) during embryogenesis. Indeed, our previous experiments suggested a developmental blockage from mESC to mesoderm and hemangioblast differentiation by the exon 3 deletion. Notably, distinct mechanisms are involved in embryonic and adult hematopoiesis. Despite an essential role for specification of HSC fate from embryonic mesoderm, SCL/tal-1 was found dispensable for HSC maintenance and differentiation in adults. Similar observation was made for transcription factor Runx. We show here that inducible ablation of PTPN11/Shp2 in the hematopoietic compartment leads to severe cytopenia in bone marrow, spleen and periphery. Shp2 removal suppressed the number and function of HSCs and myeloid progenitors. Shp2-deficient HSCs failed to reconstitute irradiated recipient mice, due to combined defects in homing, self-renewal and survival. Therefore, PTPN11/Shp2 indeed plays an essential role in maintenance of a functional HSC pool in adult mammals. This study reveals a critical signaling component conserved in embryonic and adult hematopoiesis. Given the essential role of Shp2 in normal HSCs, developing a chemical that distinguishes wild-type and mutant Shp2 enzymes may be a therapeutic approach for elimination of leukemic stem cells (LSCs) harboring mutant PTPN11/Shp2. Disclosures:No relevant conflicts of interest to declare.

Af9 Regulates gata2 Expression During Early Hemangioblast Specification and Vascular Pattern Formation In Zebrafish.

AbstractAbstract 2600 BackgroundAF9 is a transcription factor that plays an essential role in hematopoiesis and embryonic development. The alteration of AF9 is principally associated in acute myeloid leukemia as fusion partner of human MLL (mixed-lineage leukemia) gene rearrangements. Zebrafish is an excellent model organism to study embryonic development and hematopoiesis. We have previously shown that zebrafish af9 is expressed within the intermediate cell mass (ICM), a site of primitive hematopoiesis in zebrafish. Here we study the loss of af9 in zebrafish development to further understand how af9 modulates early hematopoietic and embryonic development. Methods and resultsTwo morpholino antisense oligos (MOs), designed to block translation and inhibit pre-mRNA splicing of af9, were co-injected in embryos at 1–2 cell stage. To control for off-target effects, two morpholino mismatch oligos were designed and co-injected. Efficacy of MOs was demonstrated by Western blot analysis and RT-PCR in controls and MO-injected embryos (morphants). In vivo monitoring of both morphants and control embryos was carried out by microscopy. Effects of af9 depletion on vasculature and erythropoiesis were evaluated in Tg(fli1:eGFP) and Tg(gata1:DsRed) transgenic lines, respectively. Whole-mount in situ hybridization of known hematopoietic markers was used to decipher the developmental time-points in which af9 regulates blood development. Following injection of two MOs at 1–2 cell stage, we compared the morphological features of the morphants with control embryos at about 24 hours post-fertilization (hpf). The af9 morphants showed small head and eyes, disruption of tail development and pronounced swelling in the posterior ICM. Circulating blood cells were reduced from 26 hpf to later stages of development. At 48 hpf the heart was enlarged, showed a paucity of blood-cells and pericardial edema. Decreased number of blood cells in morphant embryos was further confirmed by o-dianisidine staining at 48 hpf and 72 hpf and in living af9-knockdown gata1:DsRed transgenic animals, suggesting that the differentiation of erythroblasts remains insufficient or impaired. Concordant with this observation, we examined the expression of specific markers for early hematopoiesis (scl, lmo2 and gata2) and primitive erythropoiesis (gata1, hbbe, and band3) using whole-mount in situ hybridization (WISH). At the 5-somite stage, the early hematopoietic precursor marker gata2 was markedly increased while scl and lmo2 remained unaffected in af9 morphants. Interestingly, by 24 hpf gata2 was found to be specifically over-expressed in ICM while no change was observed for scl and lmo2 markers. Besides, the erythroid progenitors and mature erythrocyte markers gata1, band3 and hbbe displayed nearly normal expression. To further confirm the role af9 in early hematopoiesis, we examined its expression in moonshine, a mutant zebrafish with defects in erythroid maturation due to deficiency of tif1γ, a key regulator of hematopoietic gene expression. WISH analysis in moonshine showed loss of af9 expression in the ICM at 24 hpf, suggesting that af9 functions genetically downstream of tif1γ in normal erythroid cell development. To determine the effect of af9 on endothelial and vascular development, we performed knockdown of af9 in fli1:eGFP transgenic line. By 24 hpf, these morphants showed significant increase of fluorescence intensity in the posterior ICM and a clear perturbation in the inter-segmental vessels (ISV) of the trunk at 30 hpf, indicating that af9 is required for early steps in hemangioblast specification and vascular pattern formation in zebrafish. Conclusionaf9 regulates gata2 expression during early hemangioblast specification and vascular pattern formation in zebrafish. af9 may also be involved in caudal segment morphogenesis. Taken together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation. Disclosures:No relevant conflicts of interest to declare.

Large Scale Generation of Functional Megakaryocytes From Human Embryonic Stem Cells (hESCs) Under Stromal-Free Conditions.

Abstract 2540Poster Board II-517Platelets collected from donors have very limited shelf life and are increasingly needed for transfusions. In contrast to donor dependent cord blood or bone marrow CD34+ stem cells, hESCs are a promising alternative source for continuous in vitro production of platelets under controlled conditions. Current procedures for in vitro generation of megakaryocytes/platelets from hESCs are not efficient and require undefined animal stromal cells. We have developed a novel system to generate megakaryocytes (MKs) from human ES cells under serum and stromal-free conditions. In the current system, hESCs are directed towards megakaryocytes through distinct steps including embryoid body formation and hemangioblast development (Lu et al, Nature Methods, 4:501–509, 2007). A transient bi-potential cell population expressing both CD41a and CD235a markers has been identified at the end of hemangioblast culture. These cells are capable of generating both MKs and erythroid cells as demonstrated by FACS sorting and CFU assays. TPO, SCF and IL-11 are used to further direct MK differentiation of hemangioblasts derived from human ES cells in suspension culture. Currently, up to 2.5×107 MKs (CD41a+) can be generated from 1×106 hESCs, which is approximately 10 times more efficient than recently reported methods (Takayama et al Blood, 111(11):5298–5306, 2008). Without further purification, >90% of live cells from the suspension cultures are CD41a+ and the majority of these cells are also CD42b+ (>70%). These in vitro derived MK cells have morphological characteristics of mature, polyploid MKs as shown by Giemsa staining and immunofluorescent staining of vWF in cytoplasmic granules. Importantly, proplatelet forming cells are constantly observed at the late stage of MK culture indicating that MKs generated in this system are able to undergo terminal differentiation under feeder-free conditions. Platelet-like particles are also detected in culture media by FACS. When plated on OP9 cells, these MKs generate functional platelets that are responsive to thrombin stimulation. In summary, we have established a novel system for the generation of platelet-producing MKs from human ES cells that is suitable for scale up and future preclinical and clinical studies. Disclosures:Li:Stem Cell & Regenerative Medicine International: Employment. Lu:Stem Cell & Regenerative Medicine International: Employment. Feng:Stem Cell & Regenerative Medicine International: Employment. Lanza:Stem Cell & Regenerative Medicine International/Advanced Cell Technology, Inc: Employment.

Iron Chelation, Blood Transfusion and Splenectomy Decreases Angiogenesis in Patients with Thalassemia Major

Introduction: Thalassemia major (TM) is an important cause of severe anemia that necessitates regular blood transfusion to prevent the profound weakness and cardiac decompensation caused by the anemia. However, iron overloading is an inevitable consequence of prolonged transfusion therapy. In addition, extramedullary hematopoiesis and hemosiderosis cause spleen, liver and marrow enlargement. In recent years the role of angiogenesis has been investigated in physiologic and pathologic conditions. However, it is known that angiogenetic factors, especially vascular endothelial growth factor (VEGF), cause differentiation of the hemangioblast. The role of angiogenesis has been investigated in different kinds of anemia, such as malignancy related anemia and sickle cell anemia. However, the role of angiogenesis has not been investigated in thalassemia major (TM) patients. In this study the angiogenesis was researched in thalassemic patients by serum VEGF measurement.Material and method: Forty-four consecutive patients with TM were included in this prospective study. TM patients' findings were compared with those of a healthy control group (n=12). Blood samples were analyzed using commercially available ELISA kits for VEGFResults: VEGF levels were not affected by hemoglobin levels, ferritin levels, or chelation type (p>0.05). However, VEGF was positively affected by chelation starting age and negatively affected by yearly blood transfusion rate (p<0.05). In addition, VEGF of patients who underwent splenectomy was higher than those who didn't undergo splenectomy (p<0.05).In conclusion, VEGF causes differentiation of hemangioblasts, however, early starting transfusion age and regular blood transfusion decrease angiogenesis in thalassemic patients. The besides regular blood transfusion and effective chelation therapy, splenectomy decreases angiogenesis in this group of patients.

Human Embryonic Stem Cells-Derived Hemangioblasts Express HLA Antigens and Genes Related to Hematopoeisis and Endopoiesis

The early hematopoietic/endothelial progenitor (hemangioblast) has been recently described; however, HLA antigen expression of this progenitor was not characterized as yet. We have isolated blast-like cells (BLC) from ES/EB-based cultures and characterized their HLA-expression and ability to form hematopoietic and endothelial colonies. We used the two-step differentiation technology to obtain the bipotential blast cells from human embryonic stem cells (hES). This involved short differentiation in embryoid body (EB) system followed by differentiation in semisolid culture medium (Methocult H4436), supplemented with mixture of cytokines (BMP-4, VEGF, SCF, Flt3l, and TPO). First, we have shown that the occurrence of hemangioblast (BL-CFC) during EB differentiation (day 0–6) is transient and peaks on day 3. On this day about 125±35 out of 2400 EB cells form blast colonies. The emergence of this event was associated with the expression of T (early mesoderm gene) and was suppressed by the development of endoderm layer in subsequent days in culture. Similarly, the highest HB occurrence was associated with dramatic increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The similar pattern of expression of Patch1 and Gli1 genes suggests association with hedgehog (Hh) pathway activation during this process. The BL-CFC colonies were composed of 30–50 blast cells on day 6 of culture and had homogenous morphology in Wright-Giemsa stain with big nucleus containing disorganized chromatin and narrow rim of cytoplasm filled with large-size granules. However, as shown by FACS staining, they were quite heterogenous and expressed markers of both immature hematopoietic and endothelial progenitors (CD31, CD34, VE-cadherin, Flt-1) and mature differentiated cells (CD45, CD33, CD146). At least some of them expressed fetal and embryonic globin genes as shown by RT-PCR. Moreover, they could be characterized by high expression of HLA class I molecules by immunofluorescence, when compared to hES and EB cells. The ES cells, EB-derived cells and blast-like cells (BLC) were stained with FITC-labeled anti-HLA-A2 antibody BB7.2 and cells were observed under fluorescent microscope. While undifferentiated ES cells were negative for HLA-A2, the EB cells showed slight expression and the blast-like cells were strongly positive for HLA-A2 expression. These blast-like cells (BLC) could be successfully differentiated to hematopoietic cells in standard CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4±0.8%) containing macrophages, BFU-E (19.5±3.5%) containing nucleated red blood cells and CFU-EM colonies (17.1±2.7%) composed of macrophages and nucleated erythrocytes. The cells of CFU-EM and BFU-E expressed both epsilon- and gamma-globin genes, but not adult-type beta-globin. When in endothelial cell culture conditions, the blast cells differentiated to endothelial cells which had the ability to uptake Dil-Ac-LDL and form complicated vascular networks in Matrigel. Concluding:Hemangioblasts transiently exist in early embryonic development and can form single cell-derived colonies,differentiation of hemangioblasts can be tracked by the use of chosen molecular markersblast-like colonies (BL-CFC) consist of cells having properties of endothelial and hematopoietic precursors, however, the issue if they can maintain dual properties over time needs to be further exploredthese blasts-like cells can be potentially used in regenerative medicine, however, strong expression of HLA molecules may limit their clinical applications in the future.

The Renin-Angiotensin Axis Regulates the Development of a Yolk Sac-Like Hemangioblastic Progenitor of Primitive and Definitive Hematopoiesis from Human Pluripotent Stem Cells.

We have defined serum-free (SF) culture conditions for efficiently generating clonogenic hemangioblasts from pluripotent human embryonic stem cells (hESC). We also report that angiotensin-converting enzyme (ACE), a critical physiologic regulator of blood pressure, angiogenesis, and inflammation, is a novel marker for identifying and purifying hemangioblastic stem-progenitors from differentiating hESC. Using in vitro clonal assays of primitive and definitive hematopoietic potential, we demonstrated that ACE+ human embryoid body (hEB) cell populations contain a common yolk sac (YS)-like progenitor for not only endothelium, but also both primitive and definitive hematopoieses. Thrombopoietin (TPO) and basic fibroblast growth factor (FGF2) were identified as critical growth factors for expanding ACE+ hemangioblasts. ACE+CD34-CD45- human embryoid body (hEB) cells contained the highest number of these progenitors. Further maturation of ACE+ hemangioblasts in a stromal environment produced definitive-type lympho-hematopoietic progenitors, albeit with a late YS, and not AGM phenotype. The developmental kinetics of YS-like hematopoiesis generated from hEB cells was remarkably in parallel to the actual timeline of human YS development, which occurs during weeks 2–6 of human gestation. We also showed that ACE and the renin-angiotensin system (RAS) axis plays a direct role in hESC-derived hemangioblastic differentiation, and is directly regulated via signaling through the two major angiotensin peptide receptors (AGTR1 and AGTR2). Moreover, directed differentiation of hemangioblasts toward either endothelium, or multipotent primitive hematopoietic progenitors was efficiently enhanced via modulation of either AGTR1 or AGTR2 signaling. RAS axis manipulation may therefore be exploited for directing the differentiation of transplantable hESC-derived hemangioblastic stem-progenitors, thus providing novel opportunities for human tissue engineering. The key molecular and cellular events of human hemato-endothelial genesis can now be delineated in a manner that was previously impossible due to inaccessibility of human embryonic tissue. [Display omitted]

Differential Role of the Transcription Factor ZBP-89 in Hemangioblast Fate Determination: ZBP-89 Is a Direct Regulator of SCL.

Several molecular pathways have been identified that regulate distinct stages in the developmental progression from mesoderm to the formation of the hematopoietic and vascular lineages. Our previous work indicated that ectopic expression of the zinc finger transcription factor ZBP-89 promotes hematopoietic lineage development and represses endothelial cell lineage differentiation from hemangioblasts in murine embryonic stem cells. Here we evaluated the functional consequences of stable knockdown of ZBP-89 in embryonic stem cells (ESC) on hematopoietic and vascular development. Stable knock down of ZBP-89 in ESC significantly decreased the number of Blast Colony Forming Cells (BL-CFC) hemangioblasts, as well as primitive and definitive hematopoietic progenitor colonies BFU-E, GM-CFU, G-CFU, M-CFU and GEMM-CFU in vitro. In contrast, sprouting angiogenesis was markedly increased in EB cultures. Flow cytometric analysis of the lineages derived from ZBP-89 deficient EB cultures showed that the early (C-kit+Sca-1+) and definitive (CD45+) hematopoietic stem cells populations were reduced, but the endothelial cell population (CD31+ VE-Cadherin+) was increased. RT-PCR analysis of EB cultures revealed a direct correlation between the expression levels of ZBP-89 and hematopoietic markers (including SCL and Runx1) but an inverse correlation with the vascular marker CD31, with no change in Oct4 expression level. To investigate the mechanism underlying the role of ZBP-89 in hematopoiesis, the effect of ZBP-89 on expression of SCL, a master regulator of hematopoiesis, was examined. The murine SCL promoter transduced into the ZBP-89-expressing MEL cell line drove luciferase gene expression. ZBP-89 knockdown in MEL cells markedly reduced SCL expression. ChIP analysis showed that endogenous ZBP-89 protein bound directly to the murine SCL promoter in MEL cells. Thus ZBP-89 plays a central role in fate determination of hemangioblasts; its induction suppresses angiogenesis but enhances differentiation of hemangioblasts along the hematopoietic pathway, an effect mediated through the regulated expression of SCL.

Wnt2 Coordinates the Commitment of Mesoderm to Hematopoietic, Endothelial, and Cardiac Lineages in Embryoid Bodies

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.

The carnivore pregnancy: The development of the embryo and fetal membranes

The aim of this research was to compare the morphological aspects during the development of pregnancy in dogs and cats, distinguishing features of the fetal membranes, such as yolk sac evolution and differentiation of hemangioblasts, and the degree of elaboration of the amnion and allantois. Canine and feline placentae from 20, 24, 35, 45 and 55 d of pregnancy were perfusion-fixed for histological investigation and vascular corrosion casts were produced. The casts were prepared for scanning electron microscopy (SEM) and the embryo and fetal membrane development was analyzed. The growth patterns of the conceptuses were compared with the organization of the placentation process, and changes of the morphology during pregnancy were recorded. In feline placentae, an incomplete zonary shape was present in 62.5% out of 60 studied cases. This was located distal to the insertion of the umbilical cord. In the lamellar zone, the interhemal membrane or placental barrier resembled endotheliochorial conditions, and the maternal–fetal microvascular blood flow interrelationship was of simple crosscurrent type. Dogs have a zonary placenta, completely surrounding the fetus, and complex lamellar organization of maternal and fetal tissues. At the border, two marginal hematomes with green colouration delimited the central placental girdle. The yolk sac consisted of one large sacculation with an inverted “T” shape and an enormous number of blood vessels; it had hemangioblast cells in contact with the epithelium. The amnion was avascular in early stages, but became vascularized by blood vessels of the internal allantoic membrane in later stages of pregnancy by intrinsic relation.

Gene Expression Profiling of Hematopoietic Stem and Progenitor Cells in Zebrafish.

The differentiation of hematopoietic stem cells (HSCs) into distinct blood lineages is orchestrated by a tight regulation of critical genes during development. To describe the genetic program during the early steps of blood and blood vessel formation in greater detail, we analyzed the expression profiles of zebrafish cloche mutants (clo), in which blood and endothelial cells are completely abrogated, and of isolated hematopoietic and vascular progenitor cell populations from FAC-sorted transgenic lmo2-GFP zebrafish embryos. Consistent with a defect in hemangioblast differentiation, clo mutants at the 5 somite stage were characterized by a prominent downregulation of transcription factors, such as scl and lmo2. As a novel finding, clo mutants at this stage strongly upregulated muscle-related genes, such as myosin, actin, or creatine kinase. FAC-sorted lmo2-GFP cells from 14 somite stage embryos expressed similar hematopoietic transcription factors including scl, lmo2, and gata1. In addition, socs genes, tgfb, fos and jun related genes, and heat shock proteins were also strongly expressed. Comparison with the expression profile of human and mouse fetal liver HSCs revealed a similar regulation between fish and mammals: Expression of 102/168 genes from lmo2-GFP positive cells were also present in mammalian fetal HSCs, indicating an evolutionary highly conserved genetic program of blood formation. In sum, gene expression profiling of zebrafish mutants and of sorted progenitor cells from transgenic zebrafish represent a powerful and versatile tool to define the genetic regulation of vertebrate hematopoietic development.