a–c Representative immunofluorescence micrograph showing staining of hCOL1A1 in TH-rich hESC-derived grafts from three additional grafting experiments where the transplant was generated from VM-patterned RC17 (a), H9 (b)25, and HS980 hESC lines (c)4. d TH/hCOL1A1 immunofluorescence staining of a VM-patterned iPSC-transplant sorted for IAP expression prior to transplantation25. e, f TH/hCOL1A1 immunofluorescence staining of VM-patterned hESC-derived grafts generated from cryopreserved cells (e) or fresh cells (f). g–i Representative micrographs of TH/hCOL1A1 double immunostaining in terminally differentiated hESC in vitro cultures derived by three different clinically relevant VM-patterning differentiation protocols: the protocol used in this study (g), a protocol developed in the Studer lab that uses CHIR boost instead of FGF8 for proper caudalization (described in https://patents.justia.com/patent/20180094242) (h), and a protocol developed by the Takahashi lab where the cells are sorted based on Corin prior to grafting12,26 (i)). Nuclei were counterstained with DAPI in all three cultures. j hCOL1A immunostaining in a self-organized midbrain patterned organoid. Scale bars, 200 µM (a–f) and 100 µM (g–j).