Differential SOD2 and GSTZ1 profiles contribute to contrasting dental pulp stem cell susceptibilities to oxidative damage and premature senescence

Abstract

Background Dental pulp stem cells (DPSCs) are increasingly being advocated as viable cell sources for regenerative medicine-based therapies. However, significant heterogeneity in DPSC expansion and multi-potency capabilities are well-established, attributed to contrasting telomere profiles and susceptibilities to replicative senescence. As DPSCs possess negligible human telomerase (hTERT) expression, we examined whether intrinsic differences in the susceptibilities of DPSC sub-populations to oxidative stress-induced biomolecular damage and premature senescence further contributed to this heterogeneity, via differential enzymic antioxidant capabilities between DPSCs. Methods DPSCs were isolated from human third molars by differential fibronectin adhesion, and positive mesenchymal (CD73/CD90/CD105) and negative hematopoietic (CD45) stem cell marker expression confirmed. Isolated sub-populations were expanded in H2O2 (0–200 μM) and established as high or low proliferative DPSCs, based on population doublings (PDs) and senescence (telomere lengths, SA-β-galactosidase, p53/p16INK4a/p21waf1/hTERT) marker detection. The impact of DPSC expansion on mesenchymal, embryonic, and neural crest marker expression was assessed, as were the susceptibilities of high and low proliferative DPSCs to oxidative DNA and protein damage by immunocytochemistry. Expression profiles for superoxide dismutases (SODs), catalase, and glutathione-related antioxidants were further compared between DPSC sub-populations by qRT-PCR, Western blotting and activity assays. Results High proliferative DPSCs underwent > 80PDs in culture and resisted H2O2−induced senescence (50–76PDs). In contrast, low proliferative sub-populations exhibited accelerated senescence (4–32PDs), even in untreated controls (11-34PDs). While telomere lengths were largely unaffected, certain stem cell marker expression declined with H2O2 treatment and expansion. Elevated senescence susceptibilities in low proliferative DPSC (2–10PDs) were accompanied by increased oxidative damage, absent in high proliferative DPSCs until 45–60PDs. Increased SOD2/glutathione S-transferase ζ1 (GSTZ1) expression and SOD activities were identified in high proliferative DPSCs (10–25PDs), which declined during expansion. Low proliferative DPSCs (2–10PDs) exhibited inferior SOD, catalase and glutathione-related antioxidant expression/activities. Conclusions Significant variations exist in the susceptibilities of DPSC sub-populations to oxidative damage and premature senescence, contributed to by differential SOD2 and GSTZ1 profiles which maintain senescence-resistance/stemness properties in high proliferative DPSCs. Identification of superior antioxidant properties in high proliferative DPSCs enhances our understanding of DPSC biology and senescence, which may be exploited for selective sub-population screening/isolation from dental pulp tissues for regenerative medicine-based applications.

Dental Pulp Stem Cell Heterogeneity: Finding Superior Quality “Needles” in a Dental Pulpal “Haystack” for Regenerative Medicine-Based Applications

“…However, there is currently no specific marker for hDPSCs, and although expression of a wide range of other mesenchymal, embryonic, neural crest, and other cell surface markers has been extensively examined, the heterogeneous nature of hDPSC subpopulations within dental pulp tissues and their distinct immunophenotypic characteristics have led to considerable inconsistencies and diversity being displayed in their marker expression profiles [9,11,48]. Nonetheless, in addition to CD73, CD90, and CD105, hDPSCs have been reported to express numerous other MSC surface markers, such as CD13 (aminopeptidase N), CD29 (β 1 -integrin), CD44, CD166 (activated-leucocyte cell adhesion molecule), and CD271 (low-affinity nerve growth factor receptor, LANGFR/p75) [9,11,[49][50][51][52][53][54][55][56]. Consistent with their proposed location within the perivascular niche [37][38][39][40][41][42][43][44], hDPSCs have also been demonstrated to positively express perivascular cell (STRO-1 (stromal precursor antigen 1), STRO-3, and PDGFR-β (platelet-derived growth factor receptor-β)), endothelial cell (CD106, vascular cell adhesion molecule-1; CD146, melanoma cell adhesion molecule), smooth muscle cell (α-smooth muscle actin (αSMA)), and pericyte (3G5, ribosomal protein S14; NG2, neuron-glial antigen 2) markers, with hDPSCs predominantly presenting a pericyte-associated phenotype [6, 9, 12, 43, 44, 50-53, 56, 57].…”

“…Consistent with their proposed location within the perivascular niche [37][38][39][40][41][42][43][44], hDPSCs have also been demonstrated to positively express perivascular cell (STRO-1 (stromal precursor antigen 1), STRO-3, and PDGFR-β (platelet-derived growth factor receptor-β)), endothelial cell (CD106, vascular cell adhesion molecule-1; CD146, melanoma cell adhesion molecule), smooth muscle cell (α-smooth muscle actin (αSMA)), and pericyte (3G5, ribosomal protein S14; NG2, neuron-glial antigen 2) markers, with hDPSCs predominantly presenting a pericyte-associated phenotype [6, 9, 12, 43, 44, 50-53, 56, 57]. Analysis of embryonic stem cell markers has revealed varying levels of OCT4 (octamer-binding transcription factor-4), NANOG (homeobox transcription factor), SOX2 (SRY-(sex determining region Y-) box 2), SSEA4 (stagespecific embryonic antigen-4), and Slug expression in hDPSCs, which regulate stem cell properties such as selfrenewal, multi/pluripotency, and mesenchymal lineage commitment [9,11,49,50,53,[58][59][60]. Furthermore, hDPSCs exhibit positive gene expression for self-renewal and multipotency marker, BMI-1 [53,61,62].…”

“…Analysis of embryonic stem cell markers has revealed varying levels of OCT4 (octamer-binding transcription factor-4), NANOG (homeobox transcription factor), SOX2 (SRY-(sex determining region Y-) box 2), SSEA4 (stagespecific embryonic antigen-4), and Slug expression in hDPSCs, which regulate stem cell properties such as selfrenewal, multi/pluripotency, and mesenchymal lineage commitment [9,11,49,50,53,[58][59][60]. Furthermore, hDPSCs exhibit positive gene expression for self-renewal and multipotency marker, BMI-1 [53,61,62]. Based on their neural crest origins, expression of various neural lineage markers has also been identified in hDPSCs, including CD117 (c-Kit), CD271, Nestin, glial fibrillary acidic protein (GFAP), β-III tubulin, S100, Notch 1, musashi-1, synaptophysin, microtubule-associated protein 2 (MAP-2), and oligodendrocyte-associated CNPase [11,12,49,50,52,53,[63][64][65].…”

“…Furthermore, hDPSCs exhibit positive gene expression for self-renewal and multipotency marker, BMI-1 [53,61,62]. Based on their neural crest origins, expression of various neural lineage markers has also been identified in hDPSCs, including CD117 (c-Kit), CD271, Nestin, glial fibrillary acidic protein (GFAP), β-III tubulin, S100, Notch 1, musashi-1, synaptophysin, microtubule-associated protein 2 (MAP-2), and oligodendrocyte-associated CNPase [11,12,49,50,52,53,[63][64][65].…”

“…However, low proliferative hDPSCs (2-10PDs) exhibited inferior SOD-, catalase-, and glutathione-related antioxidant expression and activities overall. As mitochondria are the principle cellular source of ROS during senescence, mitochondrial SOD2 and mitochondrial/cytosolic GSTZ1 are likely candidates as the principle protective enzymic antioxidant defence mechanisms against oxidative stress in highly proliferative hDPSC subpopulations, preventing mitochondrial damage and hDPSC senescence and leading to the extended maintenance of proliferative, stemness, multipotency, and other cellular characteristics [52,53,62,96,[104][105][106][107]. Thus, such telomere length, senescence, oxidative stress, and antioxidant characteristics may be utilised as predictors of hDPSC proliferative and multipotency qualities for future regenerative medicine exploitation [108].…”