Naslov
Heterogeneity of bone cell differentiation in MSF and ROS 17/2.8 cell culture

Autori
Dacic, Sanja ; Visnjic, Dora ; Rowe, David

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
J Bone Miner Res 12 Suppl 1, S185, T333, 1997 / - , 1997

Skup
Annual Meeting of ASBMR

Mjesto i datum
Cincinnati (OH), Sjedinjene Američke Države, 10.09.1997. - 14.09.1997

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
bone; differentiation

Sažetak
Cell culture models of the osteoblastic phenotype are implicitly regarded as a uniform population of cells at a certain stage of differentiation even though heterogeneity of cell differentiation is widely appreciated. We have been using COL1A1 promoter transgenes to distinguish cells that have attained the osteoblastic status from preosteoblastic cells and cells of the non-osteoblastic lineage. Immunofluorescence of the marker gene (CAT) is obtained in fixed cells after light proteolysis of the proteoglycan layer that overlies the MSF culture. MSF derived from the ColCAT3.6 mouse uniformly express the transgene in the cell monolayer early in culture, and within and between bone nodules late in culture. Similar cultures derived from the ColCAT2.3 mouse are inactive for the first 10-12 days of culture, and then activate only in nodules at the same time that BSP mRNA is detectable in the total culture extract. Cells between the nodules are inactive. The heterogeneity of the culture can be better appreciated when the entire dish is examined in Fluorimager. The total nodule number (EtBr+), osteoblastic nodules (CAT+) and mineralized nodules (calcein+) can be quantitated to indicate the proportion of nodules which entered the osteoblastic lineage and progressed to full mineralization. Analysis of cell differentiation in real time can be assesed with the same promoter driving derivatives of the autofluorescent protein GFP. Stably transfected ROS cells demonstrate cluster of positive cells that increase in size but not number with increasing time in culture. These cells can be FACS sorted, and the active and inactive populations re-expanded in culture. Sorted GFP positive cells become inactive and then begin to form clusters of positive cells no differently than cells which were initially isolated as GFP negative. This suggests that in ROS cells a relatively small proportion of cells commit to a osteoblastic lineage irrespective of their prior history of osteoblastic differentiation. We are now examining ROS cells transfected with a ColTK1.68 gene as a way to specifically target a cell type within the lineage and enrich for a subpopulation of cells within the lineage when gancyclovir is added to the culture medium.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA