Clonal Heterogeneity in the Neuronal and Glial Differentiation of Dental Pulp Stem/Progenitor Cells

Figure 1

In vitro expansion and heterogeneity in the expression of developmental markers by clonal mDPSC cultures. (a) Single cell-derived clones, each expanded from a separate pulpal extraction, proliferated steadily for up to 240 days of culture reaching 50+ population doublings ( clones). Traces represent continuous culture growth from day of primary isolation, and cryopreserved cells continued to proliferate beyond the population doublings indicated. (b) Double immunostaining of clonal cultures for neural progenitor markers nestin and musashi. (c) RNA extracted from each clone between 20 and 40 population doublings was used in RT-PCR to identify clonal differences in the expression of RNA transcripts for CD90, stem cell antigen 1 (SCA1), glutamate aspartate transporter (GLAST), Sox2, Pax6, myelin transcription factor 1-like (Myt1l), P75, musashi, neurofilament light chain (NF-l), and CD133. (d) qPCR analysis of nestin mRNA expression by four mDPSC clones. Clones 1 and 2 were each individually found to express significantly higher levels of nestin than both clones 3 and 4. DPSC cultures were subsequently divided into strongly nestin-positive clones (clone 1 and clone 2) and weakly nestin-positive clones (clone 3 and clone 4). Nestin expression was calculated as a relative percentage of GAPDH ± SEM using the method (, RNA samples extracted from three separate passages per clone between 16 and 40 population doublings). One-way ANOVA with Tukey-Kramer posttest: n.s. = not significant, . Scale bars = 100 μm.

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