IMMORTALIZATION AND CHARACTERIZATION OF BONE-MARROW STROMAL FIBROBLASTS FROM A PATIENT WITH A LOSS OF FUNCTION MUTATION IN THE ESTROGEN RECEPTOR-ALPHA GENE

A male patient with abnormal postpubertal bone elongation was shown ea rlier to have a mutation in both alleles of the estrogen receptor, res ulting in a nonfunctional gene, Marrow stromal fibroblasts (MSFs) deri ved from this patient were called HERKOs (human estrogen receptor knoc k outs), and in order to obtain continuous HERKO cell lines, they were immortalized using a recombinant adenovirus-origin-minus SV40 virus, MSFs are unique cells because they support hematopoesis and contain a mixed population of precursor cells for bane, cartilage, and fat; Thre e established cell lines (HERKO2, HERKO4, and HERKO7) were characteriz ed and compared with the heterogeneous population of nonimmortalized H ERKOs for their osteogenic potential, We performed Northern analysis o f matrix genes implicated in bone development and metabolism and an in vivo bone formation assay by transplanting the cells subcutaneously i nto immunodeficient mite, All three HERKO lines expressed high amounts of collagen 1A1, osteopontin, osteonectin, fibronectin, decorin, bigl ycan, and alkaline phosphatase, Except for osteopontin, expression of these genes was slightly lower compared with nonimmortalized HERKOs. I n tile in vivo bone formation assay, the heterogeneous population of n onimmortalized HERKOs formed bone with high efficiency, while the HERK O lines induced a high-density, bone-like matrix. Finally, all HERKO c ell types secreted high levels of insulin-like growth factor I and int erleukin-6 into the culture medium relative to tells of normal human s ubjects, In summary, these lines of HERKO cells retain several of the phenotypic traits of MSFs after immortalization, including matrix and cytokine production, and provide a valuable source of a unique human m aterial for future studies involving estrogen action in bone and bone marrow metabolism.