Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes.
A replication-defective recombinant retrovirus containing the human papilloma virus E6/E7 genes (LXSN-16 E6E7) was used to immortalize stromal cells from human marrow. The E6/E7 gene products interfere with the function of tumor-suppressor proteins p53 and Rb, respectively, thereby preventing cell cycle arrest without causing significant transformation. Twenty-seven immortalized clones designated HS-1 to HS-27 were isolated, four of which are characterized in this report. Two cell lines, HS-5 and HS-21, appear to be fibroblastoid and secrete significant levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF), Kit ligand (KL), macrophage-inhibitory protein-1 alpha, interleukin-6 (IL-6), IL-8, and IL-11. However, only HS-5 supports proliferation of hematopoietic progenitor cells when cocultured in serum-deprived media with no exogenous factors. Conditioned media (CM) from HS-5 promotes growth of myeloid colonies to significantly greater extent than a cocktail of recombinant factors containing 10 ng/mL of IL-1, IL-3, IL-6, G-CSF, GM-CSF, and KL and 3 U of erythropoietin (Epo). Two additional clones, HS-23 and HS-27, resemble "blanket" cells, with an epithelioid morphology, and are much larger, broader, and flatter when compared with HS-5 and HS-21. These lines secrete low levels of growth factors and do not support proliferation of isolated progenitor cells in cocultures. CM from HS-23 and HS-27 also fail to support growth of myeloid colonies. Both HS-23 and HS-27 express relatively high levels of VCAM-1, yet HS-27 is the only line that supports the formation of "cobblestone" areas by isolated CD34+38lo cells. We hypothesize that HS-5, HS-21, HS-23, and HS-27 represent functionally distinct components of the marrow microenvironment.
Program in Transplantation Biology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
B A
BA
Roecklein
B
B
Torok-Storb
D017667:Adipocytes / Q000166:cytology / Q000187:drug effects; D000328:Adult; D000469:Alkaline Phosphatase / Q000032:analysis; D015703:Antigens, CD / Q000032:analysis; D015139:Blotting, Southern; D001854:Bone Marrow Cells; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D004247:DNA / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D003907:Dexamethasone / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics*; D010641:Phenotype; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology
D015703:Antigens, CD; D004279:DNA, Viral; D007328:Insulin; D009856:Oncogene Proteins, Viral; D003907:Dexamethasone; D004247:DNA; D000469:Alkaline Phosphatase; D006854:Hydrocortisone