Establishment of Cerebral Organoid‐like Structures from Dental Pulp Stem Cells (DPSCs) to Model Neurodegenerative Diseases.

Abstract Background 3D‐cell cultures are closer to in vivo conditions than the monolayers. There are different 3D‐cell culture models, the most relevant is the Cerebral Organoids (CO) model (Lancaster et al. 2014), it can show complex processes and proved to show molecular and structural changes when modeling Alzheimer´s disease (AD), and other diseases. One of the main limitations of using this model is the requirement for Induced Pluripotent Stem Cells (iPSC) or Human Embryonic Stem Cells (hESC) for the CO generation; because iPSC could undergo epigenetic changes during the process of reprogramming; moreover, hESC are controversial because of their ethical implications. We proposed to use DPSCs instead of iPSC or hESC to generate organoid‐like structures due to their capability to differentiate into the neuronal lineage, lack of pluripotency, and its easy access. Method The DPSCs were obtained by dissociating the dental pulp of a healthy 46 years old female with a colagenase‐dispase mix at 37°C, isolated and cultured. The cells phenotype was characterized by Western‐Blot, RT‐PCR and immunofluorescence. We cultured the DPSC into low‐adherence wells for 6 days in a DMEM/F12 medium + thiazovivin 50 mM, bFGF 4ng/mL, BFS 20%, Glutamax® 200x, MEM‐NEAA 1:100, 2‐mercaptoethanol 100 µm and murine LIF 10 µg/mL until cell aggregation into embryoid‐like structures. After that, neural induction was carried out. Result We successfully characterized DPSC phenotype by detecting pluripotential markers OCT4, Actin, NanoG, mesenchymal markers CD105, CD73, and the neuronal marker β‐III Tubulin which is always present in the DPSCs lineage, as well as the lack of hematopoietic markers CD14, CD34, and CD45. We obtained Cerebral Organoids‐like structures from de DPSCs after 35 days of the differentiation protocol and detected NeuN, MAP2, β‐III Tubulin, and tau, all neural markers that claim a neuronal phenotype. Conclusion Our results suggest a potential use of this approach to the CO generation using DPSCs instead of iPSC or hESC with the benefits of a shorter time and a less expensive protocol likewise, DPSCs could resemble the molecular aging changes. Further studies are needed to validate the use of this CO to model neurodegenerative diseases such as AD.