Twenty-five VHL patients (13 females, 12 males) underwent resection of 31 CNS hemangioblastomas. Mean age at surgery was 34.9 ± 10.0 y (range 18–51 y). All patients had germline mutations of the VHL gene. MRI revealed 244 craniospinal hemangioblastomas distributed in the cerebellum (111 tumors; 45%), brainstem (14 tumors; 6%), and spinal cord (115 tumors; 47%). There were four (2%) supratentorial hemangioblastomas in the region of the pituitary stalk (three tumors) and gyrus rectus (one tumor). Hemangioblastomas that were removed and studied were from the cerebellum (11 tumors; 35% of tumors), brainstem (five tumors; 16%), or spinal cord (15 tumors; 48%) ().
Distribution of Hemangioblastomas in the Central Nervous Systems of Study Patients
Identification of Tumor Cell of Origin
All tumors were histologically confirmed to be hemangioblastomas by routine staining. Immunofluorescence microscopy demonstrated that the hemangioblastoma neoplastic stromal cells expressed the mesodermal marker brachyury. Brachyury, a founding member of the T-box family of transcription factors, has a conserved role in mesoderm differentiation in vertebrates. Developmental studies indicate that early specification of posterior mesoderm and formation of the notochord is regulated by brachyury [16
]. Coexpression of brachyury and Flk-1 (VEGF receptor-2) confirmed that the neoplastic cells were derived from the hemangioblast subset of mesodermal cells (). Flk-1 (VEGF receptor-2) is a type 3 receptor tyrosine kinase that is believed to play a critical role in developmental angiogenesis and hematopoiesis [17
]. Expression of Scl, which is essential for both primitive and definitive hematopoiesis, was demonstrated in the tumors (). Expression of these embryologic markers was confirmed by Western blot analysis ().
Immunofluorescent Characterization of the Neoplastic Stromal Cells in Hemangioblastoma Tissue Sections
Western Blot Analysis Confirms Expression of Hemangioblast Markers in Tumor Cells
To characterize the multipotent capacity of the hemangioblastoma neoplastic cells, we looked for expression of known stem cell markers. Immunofluorescence demonstrated expression of the stem cell marker CD133 (C) and the hematopoietic stem cell marker CD34 in a few scattered cells throughout the hemangioblastomas. Similarly, c-kit and the common myeloid antigen, IL-3 receptor, were expressed in tumor cells in scattered regions throughout the hemangioblastomas. Coexpression of erythropoietin and erythropoietin receptor was demonstrated throughout the tumors (D).
Primary Culture of Neoplastic Cells
At concentrations of 50 U/ml of erythropoietin or more, hemangioblast cells were sustained and expanded in culture (). The identity of the cultured hemangioblasts, which was confirmed by morphology and immunofluorescence, demonstrated coexpression of erythropoietin and erythropoietin receptor. At culture concentrations of erythropoietin at 100 U/ml, two other populations of nucleated cells (nonadherent and adherent) appeared. The nonadherent cells were identified by Giemsa staining as nucleated erythrocyte or granulocyte progenitors in various stages of differentiation (). These hematopoietic progenitor cells expanded over two weeks when the expansion stopped and the absolute number of cells remained stable. Adherent cells were identified as endothelial cells morphologically (flat and elongated) and by expression of endothelial markers Tie-2 and CD31. These cells expanded beginning at two weeks and continued to expand while in culture (at least 90 d).
Hematopoietic Progenies Are Derived from Hemangioblastoma Tumor Cells
Origin of Hematopoietic and Endothelial Progeny
To determine their origin, VHL gene deletion analyses were performed on microdissected portions of uncultured tumors, cultured tumor cells, cultured hematopoietic progeny, and cultured endothelial progeny. Deletion of the wild-type VHL allele (loss of heterozygosity) occurred in the uncultured tumor specimens, cultured tumor cells, cultured hematopoietic progeny, and cultured endothelial progeny, confirming their neoplastic origin (). Peripheral blood–derived mononuclear cells from patients served as a negative control ().
Development and Expansion of Mature Hematopoietic Progeny
To determine if the tumor-derived nonadherent immature hematopoietic progenitors could be expanded into mature erythrocytic or granulocytic progeny, we developed a culture environment designed to expand immature hematopoietic cells ex vivo that exploited the characterized receptor expression (erythropoietin, IL3, and c-kit). In this environment, expansion of mature enucleated erythrocytes and granulocytes occurred by day 3 with maximal proliferation occurring by day 7. In vitro senescence of mature hematologic progeny occurred beyond day 7. The mature erythrocytes displayed characteristics similar to that of normal erythrocytes, including a mean cell volume of 80.5 fl, a mean cell hemoglobin of 29.7 pg, and a mean corpuscular hemoglobin concentration of 37 g/dl. The mature granulocytes displayed characteristic morphology and the presence of CD13 mRNA by RT-PCR.
Expansion of Endothelial Progeny
To expand the tumor-derived immature endothelial progeny, we used a culture media designed to expand immature endothelial cells ex vivo that exploited the characterized receptor expression (erythropoietin, IL3, c-kit, and Flk-1 [VEGF receptor-2]). The adherent endothelial cells continued to proliferate while in culture (at least 30 d). These expanded cells were adherent and morphologically similar to endothelial cells (flat and elongated). Consistent with an endothelial lineage, these cells expressed the markers Tie-2 and CD31 ().
Endothelial Progeny Derive from Hemangioblastoma Tumor Cells