A method of manufacturing a multilayered cell sheet of neural crest stem cells (NCSCs), includes: (1) isolating and culturing NCSCs from peripheral nerves; (2) embedding the cultured NCSCs in a hydrogel; (3) culturing the hydrogel comprising the NCSCs embedded therein under stressed culture conditions in which a physical support is applied; and (4) culturing the resulting hydrogel of step (3) under non-stressed culture conditions in which a physical support is removed.