Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma

Kevin C. Hicok, Thierry Thomas, Francesca Gori, David J. Rickard, Thomas C. Spelsberg, B. Lawrence Riggs

Research output: Contribution to journalArticle

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Abstract

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature- sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1α,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10-8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10-3 M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and β-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.

Original languageEnglish (US)
Pages (from-to)205-217
Number of pages13
JournalJournal of Bone and Mineral Research
Volume13
Issue number2
DOIs
StatePublished - Feb 1998

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Osteocalcin
Osteoblasts
Dexamethasone
Alkaline Phosphatase
Bone Marrow
Parathyroid Hormone Receptor Type 1
Cell Line
Lipoprotein Lipase
Collagen Type I
Adipocytes
Temperature
Glycerophosphates
Gene Expression
Messenger RNA
Calcitriol
Viral Tumor Antigens
Reverse Transcriptase Polymerase Chain Reaction
Bone Marrow Cells
Transfection
Rabbits

ASJC Scopus subject areas

  • Surgery

Cite this

Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma. / Hicok, Kevin C.; Thomas, Thierry; Gori, Francesca; Rickard, David J.; Spelsberg, Thomas C.; Riggs, B. Lawrence.

In: Journal of Bone and Mineral Research, Vol. 13, No. 2, 02.1998, p. 205-217.

Research output: Contribution to journalArticle

Hicok, Kevin C. ; Thomas, Thierry ; Gori, Francesca ; Rickard, David J. ; Spelsberg, Thomas C. ; Riggs, B. Lawrence. / Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma. In: Journal of Bone and Mineral Research. 1998 ; Vol. 13, No. 2. pp. 205-217.
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