• Optimized Transgene-Free Reprogramming of Human Fibroblasts into Functional Neurons using Small Molecules
  • Sahba Shahbazi,1,* Mehran Habibi Rezaee,2
    1. Protein Biotechnology Research Lab (PBRL), Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran.
    2. Protein Biotechnology Research Lab (PBRL), Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran.


  • Introduction: Neuronal transdifferentiation plays a vital role in the progress of regenerative medicine and the modeling of neurodegenerative diseases. Current methods utilize transcription factors, which raise safety concerns and limit their clinical use. Our study addresses these challenges by introducing an optimized transgene-free method that directly converts human fibroblasts into functional neurons using a small molecule cocktail, bypassing the neural progenitor state. In this study, we reprogrammed human dermal fibroblasts obtained from the skin biopsy of a 67-year-old Alzheimer's patient into mature neurons.
  • Methods: To generate neuronal cells from human fibroblasts, 5000 fibroblasts were seeded per well in Nunc® Lab-Tek® II chambered coverglass coated with PLO/Lam and maintained in a fibroblast culture medium for one day. To promote cell differentiation and survival, conditioned media from mixed glial cell cultures (mGCM) and neural conditioned medium (NCM) were employed. Primary microglia were isolated from mixed glial cell cultures of neonatal mouse brain tissue to prepare these media, and neural stem cells were isolated from the postnatal cerebellum. Neural induction medium consisting of a 1:1:1 mixture of NCM, mGCM, and Neurobasal supplemented with N-2, B-27, db-cAMP, and bFGF, along with a small molecule cocktail containing Valproic acid, CHIR99021, Repsox, Forskolin, SP600125, GO6983, and Y-27632. This cocktail was refreshed every four days for 8 days. Subsequently, cells were switched to a neuronal maturation medium containing the same basal components supplemented with BDNF, GDNF, NT3, AA, db-cAMP, N-2, and B-27 and cultured for an additional two weeks.
  • Results: Finally, we successfully obtained human chemical-induced neuronal cells (hciNs) that expressed neuronal markers, including Tau and Tuj1. After fine-tuning the technique, we managed to generate functional neurons without introducing exogenous genes.
  • Conclusion: Our findings offer a safer, more efficient platform for personalized medicine and disease modeling in neurodegenerative diseases.
  • Keywords: Regenerative Medicine, Primary Culture, Conditioned Medium, Transdifferentiation, Neurodegenerative