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

Abstract

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K(+) but not outward Na(+) currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.

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 (n = 4 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 2^−ΔΔCT method (n = 3, 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, ^∗∗ p < 0.01. Scale bars = 100 μm.

Figure 2

Only high nestin-expressing mDPSC clones possess the ability to differentiate into neuronal-like cells. (a) Representative phase contrast images of high (clone 2) and low (clone 3) nestin-expressing clones prior to and following 15 days of neuronal differentiation demonstrating a more neuronal-like morphology in high nestin-expressing clones with small refractive cell somas extending multiple interconnecting processes. (b) Immunocytochemical staining identified the presence of microtubule-associated protein 2 (Map2) and NF-l in high, but not low, nestin-expressing mDPSC clones following 15 days of neuronal differentiation. (c) Changes in mRNA expression of mesenchymal and neural markers during neuronal differentiation of high nestin-expressing mDPSC clones (clone 2). Expression levels of target genes were normalized against GAPDH and the 2^−ΔΔCT method for qPCR analysis used to calculate fold change in expression relative to predifferentiation cells on day 0 ± SEM (n = 3 independent differentiation experiments). One-way ANOVA with Dunnett multiple comparisons posttest to identify significant increases/decreases in expression compared to day 0 cells: ^∗ p < 0.05 and ^∗∗ p < 0.01. Scale bars = 100 μm.

Figure 3

Neuronally differentiated high nestin-expressing mDPSCs show immature electrophysiology properties. Current density-voltage relationships of transmembrane K⁺ currents of undifferentiated (a) and neuronally differentiated (b) high nestin-expressing mDPSCs (clone 2) in the absence (○, inlet 1 illustrates exemplar trace of currents, n = 14  and  17 cells, resp.) and presence of TEA (1 mM) (■, inlet 2 illustrates exemplar trace of currents, n = 9 for each differentiation condition). (c) Current density-voltage relationships of transmembrane K⁺ current (○) and Na⁺ currents (▼) of mSTM neurons (n = 23 cells). Inlet illustrates exemplar trace of currents. The mean ± SEM current densities at +80 mV of undifferentiated (6.4 ± 1.5 pA/pF) and neuronally differentiated mDPSCs (7.3 ± 1.4 pA/pF) showed a significant difference in comparison with primary cultured mSTM neurons (155.4 ± 10.1 pA/pF): p < 3.0E − 13 and p < 6.0E − 15, respectively. (d) Comparison of capacitances of all three cell types. The mean values ± SEM (□) of undifferentiated mDPSCs (62.0 ± 10.2) and neuronally differentiated high nestin-expressing mDPSCs (30.7 ± 4.0), as well as undifferentiated mDPSCs and P0 mSTM neurons (20.9 ± 6.6), were considered as significantly different: p < 0.005 and p < 0.002, respectively. ● and ○ are maximal and minimal values, respectively.

Figure 4

Only high nestin-expressing mDPSC clones display the ability to differentiate into oligodendrocyte-like cells. (a) Representative phase contrast images of high (clone 2) and low (clone 3) nestin-expressing clones following 15 days of oligodendrocyte-like differentiation. Clones with higher levels of nestin mRNA were found to adopt a more highly branched oligodendrocyte-like morphology compared to lower nestin-expressing clones. (b) Immunocytochemical staining identified the presence of myelin basic protein (MBP) and the oligodendrocyte transcription factors Olig1 and Olig2 in high, but not low, nestin-expressing mDPSC clones following 10 days of differentiation. β-actin staining was performed to demonstrate highly branched morphology. Scale bars = 100 μm.