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Hypoxia is considered a key factor in cellular differentiation and proliferation, particularly during embryonic development; the process of early neurogenesis also occurs under hypoxic conditions. Apart from these developmental processes, hypoxia preconditioning or mild hypoxic sensitization develops resistance against ischemic stroke in deteriorating tissues. We therefore hypothesized that neurons resulting from hypoxia-regulated neuronal differentiation could be the best choice for treating brain ischemia, which contributes to neurodegeneration. In this study, infrapatellar fat pad (IFP), an adipose tissue present beneath the knee joint, was used as the stem cell source. IFP-derived stem cells (IFPSCs) are totally adherent and are mesenchymal stem cells. The transdifferentiation protocol involved hypoxia preconditioning, the use of hypoxic-conditioned medium, and maintenance in maturation medium with α-lipoic acid. The differentiated cells were characterized using microscopy, reverse transcription PCR, real time PCR, and immunocytochemistry. To evaluate the epigenetic reprogramming of IFPSCs to become neuron-like cells, methylation microarrays were performed. Hypoxia preconditioning stabilized and allowed for the translocation of hypoxia inducible factor 1α into the nucleus and induced achaete-scute homologue 1 and doublecortin expression. Following induction, the resultant cells expressed neuronal markers neuron-specific enolase, neurofilament-light chain, growth associated protein 43, synaptosome associated protein 25, and β-III tubulin. The differentiated neural-lineage cells had functional gene expression pertaining to neurotransmitters, their release, and their receptors. The molecular signaling mechanisms regulated developmental neurogenesis. Furthermore, the in vitro physiological condition regulated neurotransmitter respecification or switching during IFPSC differentiation to neurons. Thus, differentiated neurons were fabricated against the ischemic region to treat neurodegenerative diseases.

Table S1. Cytotoxicity IC50 values of ethyl acetate extracts of C. nigricolor grown using different media on HeLa cells. The fungus C. nigricolor was grown in the above-mentioned media for 21 days, harvested, extracted using ethyl acetate and evaluated for its cytotoxic activity. Cytotoxicity was determined by the MTT assay using human cervical cancer cell line, HeLa. IC50 values are mean ± SD calculated from results obtained from triplicate of two independent experiments (n = 6). Fig. S1 Cytotoxicity of EA and hexane extracts of C. nigricolor grown using M-1D medium tested on HEK 293 T (Non cancerous cells). The fungus C. nigricolor was grown in the above-mentioned medium as a liquid culture for 21 days, harvested, extracted using ethyl acetate or hexane and evaluated for its cytotoxic activity on HEK 293 T cells using the MTT assay. Percentage of dead cells on treatment with different concentrations of A) EA extract, B) EA mycelial extract C) EA culture filtrate extract D) Hexane culture filtrate extract are represented as bar graphs. Mean ± SD calculated from results obtained from triplicate of two independent experiments (n = 6). (DOCX 107 kb)