HEMANGIOBLAST STEM CELLS

Brief History, Introduction:
      Studies on stem cells is not new, but began decades ago as scientists began to investigate novel methods of therapies for injuries. In 1961, Till and McCulloch performed the monumental experiment where they were able to reconstitute the immune system of a lethally irradiated mouse through bone marrow transplants from healthy donor 13. These studies contributed to the hypothesis that precursor cells are responsible for immune and circulatory system. One such hypothesis pointed to the hemangioblasts 12. P.D.F. Murray first studied hemangioblasts in 1932 when he coined the term “hemangioblasts” through work with chick embryos where the precursor was identified that produce both blood and blood vessels 18.
     Embryogenesis results in the development of three germ layers. Hemangioblasts are derived from differentiation of mesodermal layer. Since endothelial and hematopoietic stem cells are generated in parallel, it is hypothesized that they share a common progenitor 3. Differentiation of the ventral mesoderm yields hemangioblasts that generates blood vessels and immune/blood cells by a complex pathway that is involves bone morphogenic protein (BMP-4) 4. 

Basic Biology:
      Hemangioblasts can be found in the earliest developmental areas of the embryo, and are found at high levels in the placenta and the aorta-gonad-mesonephros (AGM) on E9.5-10.5 mice, while on E8.5-9.5 they are found in the para-aortic splanchopleura, where hematopoiesis was also observed to be high 12, 17. The cells proceed from the AGM to the fetal liver and fetal bone marrow, and finally to the adult bone marrow for immune system education and differentiation. There is evidence that they function throughout the postnatal life, since they might act as reservoirs of hematopoietic and endothelial cells 15. Hemangiopoietin is the human hemangioblast growth factor that regulates hematopoietic and endothelial cell proliferation and survival at the level of primitive hematopoiesis 18.
     Hemangioblasts give rise to primitive and definitive hematopoiesis, as well as endothelial precursor cells (Figure 1). Hematopoiesis begins immediately after gastrulation of the blood islands in the extraembryonic yolk sac 3. The blood island is a cluster of endothelial precursor cells or angioblasts and hematopoietic stem cells. The outer cells differentiate into endothelial cells for the blood vessels, while the inner cell mass differentiates into hematopoietic cells for the blood cells 6. Thus, megakaryocytes are a direct result of the differentiation of hemangioblast precursors from the gastrulation of the blood island 3. Vascular endothelial growth factor (VEGF) and stem cell factor (SCF) are required for hemangioblast development in the yolk sac. Since hemangioblasts occur in vivo in developing embryos, its functional and morphological correlate is used in vitro, the hemangioblast colony-forming cell (BL-CFC) 5.
     The allantois is involved in respiration, excretion, nutrient exchange, ion reabsorption, and development of bone 14. It has been implicated as a site for hemangioblasts in birds. This is confirmed by the expression of VEGF-R2, and transcription factors Gata-2, SCL/tal-1, and Gata-1; the morphology of the blood island was similar to what is found in mice and zebrafish 7. Grafts between chick and quail embryos provide additional evidence to the allantois as housing progenitors for endothelial and hematopoietic cells, since erythrocytes were produced along with the proper vascular network 7.
Hemangioblast differentiation is studied based on markers found on endothelial cells and hematopoietic cells. Endothelial cells markers, such as VEGF-R2/Flk-1 and VE cadherin are used to study the development of CD45+ hematopoietic cells. Thus, hemangioblasts are characterized as cells that are Scl+/Flk-1+ since the combined expression of both factors is required for the proper development of hematopoietic, endothelial, and smooth muscle cells 11. Endothelial cells and hematopoietic cells share many of the same markers, and studies of mutations and deletions in zebrafish and mouse embryos showed that both cell types were affected 12.

      The primitive embryo is composed of three layers of pluripotent cells: the ectodermal, the mesodermal and the endodermal. Mesodermal differentiation in the embryo leads to the development of organ systems in the fetus. Hemangioblasts start differentiating in the primitive streak of the mesoderm upon gastrulation of the blood island. The blood island is composed of endothelial progenitor cells or angioblasts and hematopoietic stem cells. Hemangioblasts are the progenitors for primitive and definitive hematopoiesis. Primitive hematopoiesis yields primitive cells of the erythroid lineage, while definitive hematopoiesis yields cells of all lineages other than the erythroid lineage 11. Angioblasts are also known as endothelial precursor cells that give rise to cells of the blood vessels and intestinal lining. Figure 1 at the end of the paper summarizes the developmental schema of hemangioblasts as observed in vitro. Thrombopoietin (Tpo) is also postulated to be involved in the differentiation of hemangioblasts to secondary hematopoiesis, specifically platelet production, and endothelial cells. When Tpo was administered with anti-VEGF antibody into mice, it did not affect BL-CFC differentiation; Tpo does not require VEGF for function 5.
      Several genes are responsible for the development of hemangioblasts, such as Gata2, Runx1, Stem cell leukemia (scl) factor, and Flk1. As the mesoderm differentiates into the hemangioblast stem cells around E2.5, there is a high level of expression in the hemangioblast regulatory genes. The second wave of high gene expression is correlated with differentiation towards the hematopoietic fate or angioblast fate 4.
Scl and Runx1 transcription factors along with VEGF/ Flk-1 are required for the development of hemangioblasts from the mesoderm and differentiation of hemangioblast to yield hematopoietic and endothelial cells 1. Flk-1 deficiency causes defects in the blood island and the associated blood vessels 11. Scl and Flk-1 effects were seen in Scl-/Flk-1- knockouts of zebrafish, cloche mutants, where overexpression of Scl rescued defects in hematopoiesis and endothelial cells 17. Hex gene of the homeobox family of genes is not required for the formation of hemangioblasts or for occurrence of primitive hematopoiesis. However, it is required for endodermal organ formation, and definitive hematopoiesis 16. Hex -/- mice were deficient in many progenies of definitive hematopoiesis, such as burst-forming unit erythroid (BFU-E) cells, colony-forming-unit granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) 1. Smad1 is another novel transcription factor thought to be involved in the formation and development of hemangioblast 4. Smad levels are initially upregulated as the mesoderm begins to differentiate towards the hemangioblast fate, and then downregulated during definitive hematopoiesis 4. Brachyury is an essential gene for the proper differentiation of the mesoderm 11.  Notch and delta genes are required for regulation of cell fates through intercellular mechanisms. There was no observable notch or delta activity in the yolk sac blood island, so lineage restriction must occur very early in development 14.
      The microenvironment of hemangioblasts involves regulation by levels of oxygen. It has been shown that in vitro, BL-CFC differentiate and expand under hypoxic conditions, as reported by Ramirez-Bergeron et. Al8. This is consistent with the propensity for hematopoietic stem cells to home to areas of low oxygen levels; the characteristic is conserved through the differentiation of hemangioblasts. Hematopoietic stem cells have also shown hemangioblast activity by differentiating into endothelial cells and all the hematopoietic lineages to promote wound healing in response to injury 10. Nitric oxide (NO) can also modulate the activity of progenies of hemangioblasts, specifically endothelial precursor cells (EPC), by regulating blood vessel formation during development and wound healing 9. Neovascularization was seen in iNOS-/- (inducible nitric oxide synthase) mice engrafted with wild type hemangioblasts, while wild type mice injected with eNOS (endothelial NO synthase) showed poor blood vessel branching and poor blood perfusion 9.

Methods:
      There are a variety of methods for studying the multitudes of stem cells present in the human body. Some of the standard procedures involve RT-PCR, tissue cultures, immunohistochemistry, fluorescence activated cell sorting, flow cytometry, as well as a host of assays specific to the stem cell under consideration. Transmission electron microscopy (TEM) and photography of stem cells are also done as evidence. The fluorescence can best be seen through the TEM, allowing the cells to be distinguished based on color.
Hemangioblasts are transient cells in the course of embryonic development, making their long-term study very difficult. Thus, in vitro correlates of hemangioblasts, hemangioblast colony forming cells (BL-CFC) are studied applying similar microenvironment conditions. The embryonic stem (ES) cell/ embryoid body (EB) system is used to derive BL-CFCs in vitro, and the hemangioblast equivalent cells start to appear during the EB stage 4.  Pelosi et al used the extended long-term cell (ELTC) culture technique to evaluate multiple generations of hemangioblasts colonies for the developmental genes and factors.
The Hemangioblast assay was used by Tober et. Al3, to plate embryonic stem cells on methylcellulose with the appropriate batch of nutrients (glutamine, monothialglycerol, transferring) and growth factors (LIF, VEGF, IGF, Tpo) on semisolid media designed for hemangioblast expansion. The colonies were then plucked out from the plates and examined further for morphology, gene expression, and functionality. Similar conditions could be used to study the progenies of hemangioblasts.

      Hemangioblasts could be used as diagnostic cells for multiple diseases involving hyper/ hypoproliferation of hematopoietic and endothelial cells. Young patients with Down’s syndrome tend to develop myeloproliferative disorders that progress into adult megakaryocytic leukemia. These disorders are caused by the overexpression of the unfinished version of Gata-1 (Gata-1s), a transcription factor that causes hyperproliferation of megakaryocytes 3. Pharmaceutical agents could be developed or viruses could be transfected into target cells to target Gata-1s, reducing its expression, thereby reducing the negative side effects.
      Severe thrombocytopenia is another disorder that results from hypoproliferation of the hematopoietic cells. Patients with such a disorder have very low platelet counts; they can survive gestation and birth, but postnatal survival is rare due to hemorrhages 3. Hemangioblasts could be studied in developing embryos to examine proper differentiation, so that prospective diagnoses of such disorders can be made possible in the future. Thus far only retrospective diagnosis has been possible based on testing of patient’s peripheral blood and bone marrow.
      Hemangiopoietin could be used in cases of HSC transplantation and vasculopathic conditions 18. Injections of hemangiopoietin would ensure proper differentiation of the hemangioblasts into target tissues for neovascularization and stimulate blood synthesis. Anti-angiogenic therapy could be used to treat disorders, such as breast cancer and systemic sclerosis, characterized by hyperproliferation of endothelial precursor cells. Therapeutic angiogenesis could used to treat hypoproliferation in cases of would healing, heart attacks, and stroke. Another novel therapy has been proposed that involves harvesting endothelial precursor cells and expanding them in vitro for infusion into patients as modes of site-specific cell therapy 18.       Hemangioblasts not only have therapeutic uses, but also are responsible for disorders. Hemangioblastomas are tumors of the central nervous system (CNS) whose origin has not yet been elucidated. They can be the disorder itself or be a side affect from another, such as von Hippel-Lindau disease 2. The cancerous hemangioblastic cells are made of stromal cells that cause abnormal angiogenesis, while expressing the normal set of proteins of normal stromal cells 2. Hemangioblastomas are hypothesized to be activated through an autocrine stimulatory loop involving the Tie-2 receptor and the hypoxia-inducible factor (HIF) that targets Ang-1, to promote angiogenesis 2. The exact mechanism for the activation of the autocrine loop is not yet understood, but further research into this topic would yield treatment options for brain and spinal tumors.
      There are naturally occurring cases of neovascularization in the body that are beneficial and some that are harmful. The advantageous instances of vascularization occur during menstruation, would healing, myocardial infarction, and cerebrovascular accident 18. There are even greater numbers of instances where it is harmful for vascularization to occur, such as cancer, diabetic retinopathy, rheumatoid arthritis, hereditary hemorrhage telangectasia, arteriosclerosis, sickle- cell anemia, systemic sclerosis, lymphoma, and breast cancer 18. Diabetic retinopathy is one of the leading causes of blindness in the world today. In vivo experiments on small animal models, it was shown that blocking stromal derived-factor (SDF)-1 expression would prevent blindness, since vitreous SDF-1 expression increases with severity of retinopathy 18.            

References:

1. Chan RJ, Hromas R, Yoder MC. The role of Hex in hemangioblast and hematopoietic development. Meth Mol Biol 2006;330:123-33.
2. Glasker S, Li J, Xia JB, Okamoto H, Zeng W, Lonser RR, Zhuang Z, Oldfield EH, Vortmeyer AO. Hemangioblastomas share protein expression with embryonal hemangioblast progenitor cell. Cancer Res 2006;66:4167-72.
3. Tober JM, Koniski A, McGrath KE, Vemishetti R, Emerson R, de Mesy-Bentley KK, Waugh R, Palis J. The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis. Blood 2007;109:1433-41.
4. Zafonte BT, Liu S, Lynch-Kattman M, Torregroza I, Benvenuto L, Kennedy M, Keller G, Evans T. Smad1 expands the hemangioblast population within a limited developmental window. Blood 2006;109:516-23.
5. Perlingeiro RC, Kyba M, Bodie S, Daley GQ. A Role for Thrombopoietin in Hemangioblast Development. Stem Cells 2003;21:272-280
6. Choi K. The Hemangioblast:  a common progenitor of hematopoietic and endothelial cells. J Hematother Stem Cell Res 2002;11:91-101
7. Caprioli A, Minko K, Drevon C, Eichmann A, Dieterlen-Lievre F, Jaffredo T. Hemangioblast commitment in the avian allantois: cellular and molecular aspects. Dev Biol 2001;238:64-78.
8. Ramirez-Bergeron DL, Runge A, Dahl KD, Fehling HJ, Keller G, Simon MC. Hypoxia affects mesoderm, and enhances hemangioblasts specification during early development. Dev 2004;131:4623-34
9. Guthrie SM, Curtis LM, Mames RN, Simon GG, Grant MB, Scott EW. The nitric oxide pathway modulates hemangioblast activity of adult hematopoietic stem cells. Blood 2005;105:1916-22.
10. Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8:607-12.
11. Park C, Ma YD, Choi K. Evidence for the hemangioblast. Exp Hematol 2005;33:965-70.
12. Bollerot K, Pouget C, Jaffredo T. The embryonic origins of hematopoietic stem cells: a tale of hemangioblast and hemogenic endothelium. Acta Path Micro et Immuno Scand 2005;113:790-803.
13. Till JE, McCulloch E. A direct measurement of radiation sensitivity of normal mouse bone marrow cells. Radiat Res 1961;14:213-22
14. Jaffredo T, Bollerot K, Sugiyama D, Gautier R, Drevon C. Tracing the hemangioblast during embryogenesis: developmental relationships between endothelial and hematopoietic cells. Int J Dev Biol 2005;49:269-77.
15. Pelosi E, Valtieri M, Coppola S, Botta R, Gabbianelli M, Lulli V, Marziali G, Masella B, Muller R, Sgadari C, Testa U, Bonanno G, Peschle C. Identification of the hemangioblast in postnatal life. Blood 2002;100:3203-8.
16. Guo Y, Chan R, Ramsey H, Li W, Xie X, Shelley WC, Martinez-Barbera JP, Bort B, Zaret K, Yoder M, Hromas R. The homeoprotein Hex is required for hemangioblast differentiation. Blood 2003;102:2428-35.
17. Forrai A, Robb L. The hemangioblast--between blood and vessels. Cell Cycle 2003;2:86-90.
18. Cogle CR & Scott EW. The Hemangioblast: Cradle to clinic. Exp Hematol 2004;32:885-90.

Acknowledgements:
This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences (Fall 2006), University of Medicine and Dentistry of New Jersey:

Shweta Rane, Nidhi Shah (in alphabetical order)

Teaching Assistant: Katherine Liu