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First published online 18 March 2003
doi: 10.1242/jcs.00376

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Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation

Fina Liu¹, Luc Malaval^1,* and Jane E. Aubin^1,2,

¹ Department of Anatomy & Cell Biology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
² Department of Molecular & Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
^* Present address: INSERM Unit 403, Hôpital E. Herriot, Lyon, 69437, France

View larger version (19K): [in a new window]   Fig. 1. Plating efficiencies/colony forming efficiencies of bone and non-bone colonies in controls (Co), master dishes (M) and replica cloths (R) under different replication conditions. Bars are means and standard deviations of colony counts from a range of 4-34,100 mm dishes depending on the condition. The days indicated correspond to when the replica cloths were placed over/removed from the colonies in master dishes as described in Materials and Methods.

View larger version (25K): [in a new window]   Fig. 4. Expression levels for each probe within categories of population. The means and standard deviations (bars on the left) of corrected (standardized against total cDNA) expression levels were calculated and subjected to the Welch t-test for statistically significant changes as cells differentiate (table on the right). Categories were defined as COLL-I-/ALP- colonies, COLL-I+/ALP- colonies, COLL-I+/ALP+ colonies, and OB colonies (COLL-I+/ALP+/BSP+/OCN+) (nt, not testable).

View larger version (73K): [in a new window]   Fig. 2. Phosphorimage cDNA expression profiles of osteoprogenitor/preosteoblast (OP/Pre-OB) colonies. OP/Pre-OB colonies were identified by the replica plating technique, and early and mature osteoblast colonies were identified morphologically by the cuboidal shape of osteoblasts and deposition of osteoid (OB) and mineralization of osteoid (Mature OB), respectively. The mRNA from each sample was reverse transcribed into cDNA and amplified by the poly(A)-PCR method. The resulting cDNA amplified from each colony was then probed for the expression of total cDNA (a control for the amplification procedure), the various bone-related proteins (COLL-I, ALP, OPN, BSP and OCN, PTH1R, PTHrP), and cytokine receptor messages (FGF-R1 and PDGF-R) as specified. Each vertical lane is the cDNA from the same colony. The exposure is the same for all colonies for each probe. These colonies are in random order within each category.

View larger version (35K): [in a new window]   Fig. 3. Rank order profiles of normalized cDNA expression by the colonies characterized in Fig. 2. Phosphorimage signal intensities for each probe were quantified and standardized against total cDNA. Immature osteoprogenitor colonies identified by replica plating that did not express detectable levels of ALP were classed as immature or primitive osteoprogenitors (OP) compared to more mature osteoprogenitors/preosteoblasts (Pre-OB) that did express low levels of ALP message; OBs and Mature OBs are defined as in Fig. 2. All colonies were rank-ordered according to the paradigm outlined in the Results.

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