Xeno‐free vmDA protocol differentiation to enriched ventral floor plate precursors interrogated with LMX1A‐eGFP reporter line. (A): Study overview detailing novel reporter hPSC lines, fully defined, feeder‐free, xeno‐free differentiation protocol that is scalable and cryopreservable. (B–E): Developing embryo schematic illustrating sagittal (Bi, Ci, Di, Ei) and coronal (Bii, Cii, Dii, Eii) expression of key transcription factors OTX2 (B), FOXA2 (C), LMX1A (D), and merged (E), that in combination are indicative of vmDA precursors. Expression of OTX2, FOXA2, and merged at D11 under xenogeneic differentiation (F–H) and xeno‐free (I–K) protocols. Insets show higher magnification, highlighting the presence of poorly specified progenitors that express OTX2 (white arrows) or FOXA2 only (yellow arrows). (L): Quantification of vmDA precursors cultured under xenogeneic and xeno‐free conditions for hESCs and hiPSCs. Differentiation of LMX1A‐eGFP hESC under xeno‐free conditions confirmed correct specification of vmDA precursors with increasing expression of LMX1A from D11 to D25 by live cell flow cytometry (M–O) and live imaging (P–R). (S–V): Immunostaining at D18 of OTX2 (S), FOXA2 (T), LMX1A‐eGFP (U), and DAPI (V). n = 3 technical and culture replicates, mean ± SEM. ∗∗, p < .01, ∗∗∗, p < .001. Immunofluorescence images are at ×100 magnification. Abbreviations: BP, basal plate; D, day; DAPI, 4′,6‐diamidino‐2‐phenylindole; FB: forebrain; FP, floor plate; GFP, green fluorescent protein; HB, hindbrain; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; MB: midbrain, NPC, neural progenitor cell; vmDA, ventral midbrain dopaminergic.

Differentiation of hPSCs under xeno‐free conditions increases generation of bona fide vmDA neurons at D25. Images of FOXA2 (A, D), TH (B, E), and FOXA2/TH (C, F) coexpression in H9 cells differentiated under xenogeneic (A–C) and xeno‐free (D–F) conditions, showing increased FOXA2/TH colabeled vmDA neurons under xeno‐free conditions. (G): Quantification of subpopulations. (H): Quantification of the temporal expression of FOXA2 reveals maintenance of early fate specification for xeno‐free conditions, yet xenogeneic cultures fail to increase FOXA2 progenitors yields through time. (I–K): Image of xeno‐free differentiated culture expressing NURR1 (I), TH (J), and merge (K). (L–N): Sagittal schematic of the developing embryo illustrating FOXA2 (L), PITX3 (M), and combined FOXA2/PITX3 (N) expression, where vmDA neurons are born. (O–T): Xeno‐free differentiation and quantification of an H9::PITX3‐eGFP reporter line at D25 revealed cultures rich in bona fide vmDA neurons as revealed by FOXA2⁺/TH⁺/PITX3⁺ coexpression. n = 3–5 culture replicates, mean ± SEM. ∗∗, p < .01; ∗∗∗, p < .001. Immunofluorescence images are at ×100 magnification. Abbreviations: D, day; DAPI, 4′,6‐diamidino‐2‐phenylindole; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; n.s., not significant; TH, tyrosine hydroxylase; vmDA, ventral midbrain dopamine.

Residual fibroblasts, at the initiation of vmDA differentiation, influence fate specification. (A, F): Phase contrast image of hPSCs cultured in xenogeneic conditions on MEFs (A) or xeno‐free conditions on laminin‐521 (F). Black arrows: hPSC colonies, white arrows: MEFs. Xenogeneic and xeno‐free hPSCs coexpressed pluripotency markers OCT4 (B, G) and SOX2 (C, H). Analysis of pluripotent surface antigens CD9 and GCTM2 by FACS revealed that xenogeneic cultured hPSCs (D) were indistinguishable from xeno‐free cultures (I). (E, J): Despite depletion, xenogeneic conditions retained a small fraction of CD90.2⁺ MEFs (E), which were absent in xeno‐free conditions (J). (K): Xeno‐free vmDA cultures were spiked at initiation with MEFs to elucidate potential effects on differentiation to 11 day ventral midbrain NPCs. (L, M): Xeno‐free cultures at DIV0 widely expressed OCT4 and were devoid of CD44⁺ fibroblasts. (N, O): The presence of fibroblasts demonstrated in xeno‐free DIV0 spike cultures confirmed by CD44 reactivity. Confirmation of vmDA precursor specification under xeno‐free conditions at DIV11 by immunolabeling for OTX2 (P) and FOXA2 (Q). Cultures spiked with MEFS demonstrated reduced numbers of OTX2‐expressing (R) and FOXA2‐expressing (S) cells. (T): Percentage decrease in the proportion of cells in 11‐day vmDA cultures expressing OTX2, FOXA2, and OTX2/FOXA2 following spiking at D0 with MEFs, compared with standard xeno‐free cultures. Note there is a significant reduction in specification of OTX2/FOXA2 DA precursors following MEF spiking of the cultures. n = 3 culture replicates, data represent mean ± SEM. ∗, p < .05. Phase contrast images at ×40, immunofluorescence at ×100 magnification. Abbreviations: DA, dopamine; DIV, day in vitro; FACS, fluorescence‐activated cell sorting; hPSC, human pluripotent stem cell; NPC, neural progenitor cell; MEF, mouse embryonic fibroblast; vmDA, ventral midbrain dopaminergic.

Scaled differentiation and cryopreservation of xeno‐free vmDA precursors. (A, B): Xeno‐free differentiation in flasks yields high cell quantities enriched for LMX1A‐eGFP at D22. (C, D): Scaled vmDA cultures (C) did not include pluripotent GCTM2/CD9 cells compared with pluripotent controls (Di) and unstained controls (Dii). (E, F): Culture flask expansion had no impact on fate specification as revealed by LMX1A‐eGFP expression (E) or cell survival (F). (G, H): Cryopreservation and thawing also had no impact on LMX1A‐eGFP expression (G) and minimal impact on survival (H). After thawing, LMX1A‐eGFP vmDA progenitors maintained their capacity to differentiate into DA neurons, as revealed by DAPI (I), FOXA2 (J), LMX1A (K), TH (L), and merged (M) expression at D40. n = 3 culture replicates, mean ± SEM. Phase contrast images are at ×40, immunofluorescence at ×200 magnification. Abbreviations: D, day; DA, dopamine; DAPI, 4′,6‐diamidino‐2‐phenylindole; GFP, green fluorescent protein; hPSC, human pluripotent stem cell; TH, tyrosine hydroxylase; vmDA, ventral midbrain dopamine.

Maturation of hPSCs under xeno‐free conditions improved the yield of mature DA neurons and DA metabolism. Neurons displayed electrophysiological properties of classical vmDA neurons. Images of hPSCs differentiated under xenogeneic (A–E) or xeno‐free (F‐J) conditions illustrating the composition of DAPI, FOXA2, and TH labeled neurons. (K): Quantification of subpopulations revealed a significant increase in DA neurons under xeno‐free conditions across lines. (L–Q): Temporal gene expression profile of xenogeneic and xeno‐free cultures for OCT4 (L), FOXA2 (M), LMX1A (N), NURR1 (O), PITX3 (P), and TH (Q). (R–T): High‐performance liquid chromatography revealed a significant increase in dopamine release (R) and dopamine turnover (T) (DA:HVA ratio) in xeno‐free compared with xenogeneic cultures. (S): No change was observed in HVA levels. (U): Extended culture of H9::PITX3‐eGFP reporter vmDA neurons to D60 generated dense networks of DA neurons, as revealed by FOXA2/PITX3‐eGFP/TH coexpression, with extensive axonal fasciculation. (V): Cells were targeted for whole cell patch clamping. (V′): Biocytin‐labeled neuron coexpressing TH after recording. (W): Representative trace of action potential generation at resting membrane potential. (X): All cells exhibited inward rectification in response to hyperpolarization steps and generated action potentials. (Y): Trace recording of vmDA neuron receiving spontaneous excitatory postsynaptic currents, which could be blocked by administration of the AMPA antagonist, NBQX. n = 3 culture replicates (immunocytochemistry and high‐performance liquid chromatography), n = 6 culture replicates (qPCR), n = 21 recorded neurons (electrophysiology). Mean ± SEM. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001. Immunofluorescence, ×100 magnification; electrophysiology images, ×1,000 magnification. Abbreviations: AP, action potential; D, day; DA, dopamine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DIV, day in vitro; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; HVA, homovanillic acid; TH, tyrosine hydroxylase; vmDA, ventral midbrain dopamine.

Transplantation of xeno‐free vmDA progenitors improved the yield of correctly specified cells within grafts of Parkinsonian mice. (A, G): Chromogenic PSA‐NCAM and GFP staining of transplanted H9::LMX1A‐eGFP progenitors differentiated for 25 days under xenogeneic (A) or xeno‐free (G) conditions and analyzed at 1 month after implantation. Note similarity in graft size, shown by PSA‐NCAM and enrichment of LMX1A‐eGFP cells under xeno‐free conditions. DAPI (B, H), OTX2 (C, I), FOXA2 (D, J), LMX1A‐eGFP (E, K), and merged images (F, L) show that xeno‐free derived grafts were enriched for DA progenitors, as shown by increased OTX2/FOXA2/LMX1A‐eGFP coexpression seen in the high‐power images: xeno‐free (L′) compared with xenogenic (F′). (M): Volumetric analysis revealed no difference in graft size between xenogeneic or xeno‐free conditions. (N): Quantification of progenitors within the graft. (O): Chromogenic PSA‐NCAM and GFP staining of transplanted H9::PITX3‐eGFP progenitors, differentiated for 25 days under xeno‐free conditions and analyzed at 1 month after implantation. Grafts show high levels of PITX3‐GFP staining (O, O′) and modest GFP innervation of the host striatum (O′′). Immunohistochemistry for FOXA2 (P), TH (Q), PITX3‐eGFP (R), and merged (S) confirmed that a significant proportion of cells possessed a vmDA phenotype. (P′, S′): Higher magnification images of (P)–(S). Animals grafted with H9::PITX3‐eGFP progenitors (open circles) showed a significant improvement in amphetamine induced rotational asymmetry compared with 6‐OHDA lesioned controls (closed circles). (U): Representative H9::PITX3‐eGFP graft showing integration into the host striatum at 6 months. (U′, U′′): Grafts contained a dense network of cells and reinnervation of the dorso‐lateral striatum (U′′) as well as other vmDA targets, including the overlying cortex. LMX1A‐GFP cell grafts: n = 6 animals per group (xenogeneic and xeno‐free). PITX3‐GFP grafts: n = 3 at 1 month, and n = 6 at 6 months. n = 6 lesion controls. Mean ± SEM. ∗, p < .05. Abbreviations: DA, dopamine; DAPI, 4′,6‐diamidino‐2‐phenylindole; eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; TH, tyrosine hydroxylase; vmDA, ventral midbrain dopamine.