Human dental stem cell derived transgene‐free iPSCs generate functional neurons via embryoid body‐mediated and direct induction methods

Abstract

Induced pluripotent stem cells (iPSCs) give rise to neural stem/progenitor cells, serving as a good source for neural regeneration. Here, we established transgene-free (TF) iPSCs from dental stem cells (DSCs) and determined their capacity to differentiate into functional neurons in vitro. Generated TF iPSCs from stem cells of apical papilla and dental pulp stem cells underwent two methods-embryoid body-mediated and direct induction, to guide TF-DSC iPSCs along with H9 or H9 Syn-GFP (human embryonic stem cells) into functional neurons in vitro. Using the embryoid body-mediated method, early stage neural markers PAX6, SOX1, and nestin were detected by immunocytofluorescence or reverse transcription-real time polymerase chain reaction (RT-qPCR). At late stage of neural induction measured at Weeks 7 and 9, the expression levels of neuron-specific markers Nav1.6, Kv1.4, Kv4.2, synapsin, SNAP25, PSD95, GAD67, GAP43, and NSE varied between stem cells of apical papilla iPSCs and H9. For direct induction method, iPSCs were directly induced into neural stem/progenitor cells and guided to become neuron-like cells. The direct method, while simpler, showed cell detachment and death during the differentiation process. At early stage, PAX6, SOX1 and nestin were detected. At late stage of differentiation, all five genes tested, nestin, βIII-tubulin, neurofilament medium chain, GFAP, and Nav, were positive in many cells in cultures. Both differentiation methods led to neuron-like cells in cultures exhibiting sodium and potassium currents, action potential, or spontaneous excitatory postsynaptic potential. Thus, TF-DSC iPSCs are capable of undergoing guided neurogenic differentiation into functional neurons in vitro, thereby may serve as a cell source for neural regeneration.

5-Aza Exposure Improves Reprogramming Process Through Embryoid Body Formation in Human Gingival Stem Cells

“…In conclusion, this study demonstrates a simple and efficient method to develop EBs from oral cavity derived stem cells, according to the study showing that DPSCs are able to form embryoid-body-like structures in vitro containing cells derived from all three embryonic germ layers when injected in nude mice ( Yan et al, 2010 ). Moreover, it has been demonstrated that oral derived stem cells possess higher reprogramming efficiency than dermal fibroblasts, thus prove that these cells are more amenable for reprogramming ( Pisal et al, 2018 ) and that transgene-free-DSC iPSCs are capable to undergo toward neurogenic differentiation till to functional neurons stage in vitro ( El Ayachi et al, 2018 ).…”

Epigenetic Regulation of Dental Pulp Stem Cell Fate

“…When cultured in neuronal inductive conditions for an extended period of time, DPSCs exhibit a neuronal morphology and express neuronal-specific markers such as PSA-NCAM, β -III tubulin, neurofilament-M, and nestin, showing the ability to generate a sodium current consistent with functional neuronal cells [ 70 ]. Moreover, DPSCs transplanted in vitro can generate functional neurons and improve nerve regeneration [ 71 , 72 ]. In addition, several studies also showed that DPSCs exhibit the capacity to acquire the phenotype of endothelial cells and generate vascular-like structures [ 73 – 76 ].…”

Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation

“…These OSCs are heterogeneous population of cells expressing typical MSC markers 13,15 . The above mentioned isolation method routinely gives rise to clonogenic MSCs capable of expansion with robust proliferation in cultures [23][24][25][26] . The size and morphology of DPSCs and SCAP in subconfluent cultures were very similar.…”

“…The staining followed a protocol reported previously 23,26,29 , or with modified approaches as follows. Cell cultures were fixed in 100% ice cold methanol for 10 min, followed by permeabilization with 0.1% Triton-X 100 (Sigma-Aldrich) in PBS for 15 min at room temperature.…”