Abstract: BACKGROUND: Cell therapy based on human adipose-derived stem cells holds promise for neurological diseases. However, establishing an efficient, stable, and gene-modification-free directed differentiation protocol remains a major obstacle to clinical application. 
OBJECTIVE: To establish a two-stage, chemically defined protocol using small molecule combinations to achieve the differentiation of human adipose-derived stem cells into motor neuron-like cells, thereby providing a potential cell source for cell-based therapies for neurological diseases.  
METHODS: Adipose-derived stem cells were isolated from adipose tissue, cultured, and characterized. A two-stage induction strategy using small molecule cocktails was employed. In stage one, adipose-derived stem cells were induced to transdifferentiate into neural progenitor cells using a combination of Dorsomorphin (a bone morphogenetic protein inhibitor), CHIR99021 (a Wnt agonist), and SB431542 (a transforming growth factor-β inhibitor). In stage two, the resulting neural progenitor cells were further differentiated into motor neuron-like cells with a combination of CHIR99021, retinoic acid, and Purmorphamine (a specific activator of the Shh signaling pathway). The expression of neural progenitor cell markers (PAX6, SOX1) and motor neuron markers (tubulin β3, ISL1, HB9, and choline acetyltransferase) was dynamically assessed by morphological observation, immunofluorescence staining, western blot assay, and RT-qPCR. 
RESULTS AND CONCLUSION: After 4 days of induction in stage one, the cells formed typical neural rosette-like structures. Both protein and mRNA levels of PAX6 and SOX1 were significantly upregulated (P < 0.001), with positivity rates reaching 85.33% and 83.12%, respectively, indicating the successful transdifferentiation of human adipose-derived stem cells into neural progenitor cells. Following 12 days of induction in stage two, the cells exhibited typical motor neuron morphology. The expression of tubulin β3, ISL1, HB9, and choline acetyltransferase was significantly elevated (P < 0.001). Double immunofluorescence labeling confirmed that 78.56% of the cells co-expressed tubulin β3/ISL1, and 75.04% co-expressed HB9/choline acetyltransferase, confirming their differentiation into motor neuron-like cells. In conclusion, by mimicking in vivo neural developmental signaling (inhibiting the bone morphogenetic protein/transforming growth factor β pathway and activating the Wnt pathway in stage one, and activating the Wnt/Shh/retinoic acid pathway in stage two), this two-stage small molecule compound combination strategy can specifically induce human adipose-derived stem cells to differentiate into motor neuron-like cells, providing a cell source with significant clinical translational potential for cell therapy of neurological diseases. 

Key words: human adipose-derived stem cells, neural differentiation, neural progenitor cells, motor neurons, small molecule compounds, cell therapy