[Chemical reprogramming of human embryonic fibroblasts into neural progenitor cells in vitro]

Objective: To establish a protocol for reprogramming human embryonic fibroblasts (HEFs) into chemically induced neural progenitor cells (ciNPCs).

Methods: In the two-staged reprogramming of HEFs, the intermediate compact cell colonies were first chemically induced in KSR medium containing small-molecule compounds (VCR) for 15 days in normoxia, followed by the lineage-specific induction stage, in which the compact cell colonies were digested with 0.25% trypsin and the cells were cultured in low adhesion plates. After formation of a large number of free-floating neurospheres 2 days later, the ciNPCs were labeled with CM-DiI and transplanted into rat models of Parkinson's disease (PD)to observe the survival, migration and differentiation of the cells in PD brain.

Results: After induction with VCR for 10 days under normoxic condition, compact cell colonies occurred in HEF cultures (approximately 40 colonies in each well containing 1×10⁵ HEFs), and most of the colonies expressed high levels of alkaline phosphatase. A large number of free-floating neurospheres formed 2 days after passage and were defined as P1 ciNPCs. These ciNPCs exhibited typical neurosphere-like structures and expressed NPC-specific markers (nestin, Sox2, and Pax6). Under neuronal or glial differentiation condition, the ciNPCs expressed the neuron-specific marker Tuj1 and the astrocyte-specific marker GFAP. These ciNPCs could differentiate into Tuj1⁺, GFAP⁺, TH⁺ and GABA⁺ cells 4 weeks after transplantation into the brain of PD rats.

Conclusion: HEFs can be directly reprogrammed into ciNPCs using smallmolecule compounds without the need of introducing exogenous genes. This success may provide a solution to the shortage of donor materials for neuroscience research and treatment of neurodegenerative diseases.