Abstract: 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 small-molecule 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.

Key words: small molecule compounds; cell reprogramming; human embryonic fibroblasts; neural progenitor cells