Optimized Transdifferentiation of Fibroblasts: A Robust Approach to Alzheimer's Disease Modeling
Optimized Transdifferentiation of Fibroblasts: A Robust Approach to Alzheimer's Disease Modeling
Sahba Shahbazi,1Mehran Habibi Rezaie,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: Transdifferentiation of human adult fibroblasts into mature neurons offers a promising and efficient method for generating patient-specific neuronal cells for neurodegenerative diseases like Alzheimer's. This approach is precious for disease modeling, mechanistic studies, and drug screening. Unlike induced pluripotent stem cells (iPSCs), which undergo rejuvenation and lose aging characteristics, transdifferentiated neurons retain the donor's aging signatures, providing a more accurate model of age-related diseases. Although transdifferentiation can be challenging, especially with increasing passage numbers, it avoids the need for an intermediate pluripotent stage, making it faster and more cost-effective. Transdifferentiated cells also maintain genetic diversity, reflecting the natural mosaicism of the original fibroblast population.
Methods: In this study, we optimized existing protocols to efficiently generate mature neurons from fibroblasts of elderly Alzheimer's patients, regardless of passage number. These neurons retained age-related characteristics and exhibited functional capabilities similar to native neurons. For this purpose, human dermal fibroblasts were obtained from skin biopsies of six Alzheimer's patients. Lenti-X 293 cells were transfected with a lentiviral vector containing Ascl1 and Brn2 transcription factors, along with shRNA against REST and packaging vectors pMD2.G and psPAX2. Three days post-transduction, human dermal fibroblasts were cultured in an induction medium containing dual SMAD inhibition factors, growth factors, and VPA. On day 12, cells were passaged using ROCKi and accutase onto plates coated with PO/FN/Lam. Seven days later, a secondary induction medium containing LM-22A4, GDNF, NT3, AA, and db-cAMP was applied.
Results: By day 25, a significant yield of neurons, accounting for approximately 50% of the total cells, was achieved. Western blot, real-time PCR, and immunocytochemistry analyses confirmed the expression of neuronal markers such as MAP2 and TAU, confirming their mature neuronal identity. The conversion process was consistent across different cell lines, including those from older donors and those that had undergone multiple passages.
Conclusion: This protocol offers an efficient method for generating induced neurons (iNs) from human adult dermal fibroblasts, regardless of passage number. Our results demonstrate the feasibility of producing neurons from individuals aged 50-79 with high efficiency, providing a valuable tool for Alzheimer's disease research.