Fig 1: Addition of cAMP during VM patterning mimicks the effect of FGF17. A. Experimental outline for bulk and scRNAseq. FGF8- or FGF17-patterned d9 VM DA progenitors were (1) collected at 15 minutes, 1 hour, 4 hours, or 24 hours after FGF addition for bulk RNAseq (n = 3 per condition), or (2) differentiated until d16 for scRNAseq, n = 4. B. PCA plot comparing bulk RNAseq of FGF8 and FGF17 treated VM DA progenitors at all four timepoints, n = 3. C. Volcano plot of DEGs at 15 minutes, 1 hour, 4 hours, and 24 hours between FGF8- and FGF17-treated VM DA progenitors, cutoffs = P_val_adj < 0.01 and log2FoldChange ± 0.4. D. mRNA levels of early response genes EGR4 and FOSB after addition of FGFs. A paired t-test was used to test differences for each time point, n = 5-6. E. UMAP projection of scRNAseq data from FGF8 and FGF17 d16 VM DA progenitors. F. scRNAseq volcano plot of DEGs day 16, cutoffs = P_val_adj < 0.05 and log2FoldChange ± 0. Dots above the dashed line mark significantly expressed genes. G. Violin plots of top DEGs between FGF8 and FGF17 cells in the scRNAseq data. H. Outline of in vitro differentiation strategy to test target pathways uncovered by RNAseq. I. Quantitative RT-PCR of key VM markers in d16 progenitors treated with either PDE8i or cAMP together with FGF8 from days 9 to 16. One-way ANOVA for paired data followed by Dunnet’s multiple comparisons test: *P < .05, **P < .01, ***P < .001, ****P < .0001, ns: non-significant, n = 11. J. Immunocytochemistry showing co-staining of OTX2, LMX1A, and FOXA2 in d16 VM DA progenitors treated with PDE8i or cAMP, scalebar: 100 μM.
Fig 2: Functionality of FGF17-patterned DA neurons upon transplantation to Parkinsonian rats. A. Nude rats (NRs) and Sprague-Dawley rats (SDRs) were unilaterally lesioned with 6-OHDA and then transplanted with cryopreserved d16 FGF17-patterned cells. The functionality of the transplanted cells was evaluated at 16-, 20-, 24-, and 27-week post-transplantation through amphetamine-induced rotations. SDRs were immunosuppressed with Cyclosporin upon transplantation. B-C. Representative images of immunohistochemical stainings for hNCAM (B) and TH (C) in transplanted rat brains, showing dense human fiber innervation and TH-rich grafts, scalebars = 50 µM. D. Mean net turns per min in amphetamine-induced rotation test from rats transplanted with FGF17-patterned cells. n = 7 (week 0), n = 4 (week 16, 20, 24), n = 3 (week 27). A Welch t-test was used to determine differences between weeks 0 and 27 (P-value = 0.0175). E. Representative images of FGF17 graft at 27 weeks, stained for TH, FOXA2, and ALDH1A1. Triple-positive cells are indicated by triangles. Overview scale bar = 200 μm, zoom-in scalebar = 40 μm. F. Quantified total yield of TH + cells per 1e5 cells transplanted in FGF17 grafts at 27 weeks compared to the clinical cell product STEM-PD (STEM-PD data obtained from Kirkeby et al., 2023). n = 8 (FGF17), n = 18 (STEM-PD). Statistical significance determined by t-test, P = 0.378.
Fig 3: FGF subfamily members are capable of ventral midbrain induction. A. Expression of key MHB genes in the E11.5 mouse embryo, from the Allen Brain Atlas.30 B. Spatial transcriptomic data of MHB genes in the developing human postconceptional week (pcw) 5.5 fetus.28 C. ScRNAseq data from an in vitro human neural tube model (MiSTR) at d14 (16). D. Schematic of FGF8 subfamily members expressed at the MHB, involved in the induction of caudal (cVM) vs rostral (rVM) VM patterning, and schematic of the in vitro protocol used for hESC differentiation. E. mRNA expression of EN1 in d16 VM progenitors treated with FGFs from d9-16. Analyzed by Brown-Forsythe ANOVA followed by Dunnet’s multiple comparisons. *P < .05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant, n = 8 (no FGF), n = 10 (FGF8, FGF17), n = 9 (FGF18). F. Immunolabeling of FOXA2 and LMX1A in d16 hESC-derived VM DA progenitors, patterned with FGFs from d9-16. G. Paired analysis of mRNA expression of key VM DA progenitor markers at d16, comparing FGF8 and FGF17. Data points from 1E (EN1, n = 10) are also included in these graphs. **P < 0.01, ***P < 0.001, EN1 P = 0.11, ns: non-significant, analyzed with a paired t-test, n = 19. H. Immunolabeling of FOXA2, LMX1A, and MAP2 in d42 hESC-derived DA neurons. All scalebars: 100 μM.
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