Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Schematic diagram of the 4 DIV CNP differentiation protocol. b , Representative immunostaining images of H9 and GBX2 -/- CNPs at 4 DIV showing OTX2 - /CDX2 + cells. A few 4X CNPs were positive for OTX2, but no cells were positive for CDX2. Scale bars, 20 µm. c-d , qPCR analysis of OTX2 (c) and CDX2 (d) expression in H9, GBX2 -/- and 4X CNPs at 4 DIV. The data are presented as the mean ± SD; n= 3. One-way ANOVA showed statistical significance, and then an unpaired t-test comparing two groups was performed. *P< 0.05; **P< 0.01. e , Heatmap of the expression of pluripotent and neural genes representing the anterior, midbrain, hindbrain and spinal cord regions in H9, GBX2 -/- and 4X CNPs at 4 DIV. f , The top 10 downregulated (blue) and upregulated (red) genes (and additional selected genes in bold) between cultured 4X and H9 cells, cultured GBX2 -/- and H9 cells and cultured 4X and GBX2 -/- cells at 4 DIV. The threshold bar (white line) indicates a fold change of ±2. g , Schematic diagram of the 11 DIV CNP differentiation protocol. h , RNA expression analysis of the midbrain genes (orange) OTX2, EN1 and PAX8 ; the hindbrain genes (gray) MAFB, EGR2, HOXA2, HOXB1, HOXA3, HOXB2 , and HOXA4 ; and the spinal cord genes (purple) HOXB8 and HOXC10 . The data are presented as the mean ± SD; n= 3. One-way ANOVA followed by Tukey’s multiple comparisons test. *P< 0.05; **P< 0.01. i , Representative immunohistochemical analysis of OTX2/EN1 double-positive cells among 11 DIV 4X cells. No OTX2/EN1 double-positive cells were detected among H9 cells. Scale bars, 10 µm. For (b) and (i), DAPI was used as a nuclear stain. Caudal neural progenitor: CNP.

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Immunostaining, Expressing, Cell Culture, RNA Expression, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Schematic diagram of the 11 DIV CNP differentiation protocol using different concentrations of GSK3i ranging from 0.7 µM to 3 µM. b , Flow cytometry analysis of the percentage of OTX2-positive cells among H9, GBX2 -/- and 4X CNPs at 11 DIV. The data are presented as the mean ± SD; n= 3. Two-way ANOVA followed by Tukey’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. **P< 0.01. c , Schematic diagram of the 16 DIV caudal midbrain differentiation protocol using different concentrations of GSK3i ranging from 0.5 µM to 1 µM. d , Quantification of OTX2-positive cells among H9 and 4X cells at 16 DIV after administration of GSK3i at concentrations ranging from 0.5 µM to 1 µM. The data are presented as the mean ± SD; n= 4-8. Two-way ANOVA followed by Sidak’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. **P< 0.01. e , Expression of OTX2, EN1, LMX1A, CNPY1 , and HOXA2 in H9 and 4X cells treated with a range of GSK3i concentrations at 16 DIV. The data are presented as the mean ± SD., n= 3. Two-way ANOVA followed by Sidak’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. *P< 0.05; **P< 0.01. f , Representative immunohistochemical analysis of OTX2, EN1, FOXA2, and LMX1A expression in H9 and 4X cells treated with GSK3i at concentrations of 0.6 µM and 1 µM on 16 DIV. DAPI was used as a nuclear stain. Scale bars, 50 µm. Caudal neural progenitor: CNP.

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Flow Cytometry, Concentration Assay, Expressing, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , UMAP of cultured 4X and H9 cells at 16 DIV and a graph of the percentage of cells that each cell line contributed to each cluster (n = 10 4X cell spheres; n = 10 H9 cell spheres; total of 4,682 cells). b , Heatmap of the expression of selected genes illustrating the identity of the clusters. c, Percentage of 4X and H9 cells expressing OTX2 and EN1 . d , Feature plot of the contribution of each cell line to each cluster and feature plot of gene expression levels of OTX2, EN1, LMX1A, FOXA2, FGF8, HOXA/B family members and STMN2 . e , UMAP of cultured 4X and H9 cells at 62 DIV and a graph of the percentage of cells that each cell line contributed to each cluster (n = 4 4X cell cultures; n = 4 H9 cell cultures; total of 6,804 cells). f , Heatmap of the expression of selected genes illustrating the identity of the clusters. g , Feature plot of the contribution of each cell line to each cluster and feature plot of the gene expression levels of STMN2, DCX, TH, LMX1A, EN1, SHH and COL3A1 .

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Cell Culture, Expressing

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Overview of the in vivo study. Unilateral 6-OHDA-induced MFB lesions were generated (week -4) and confirmed 3 weeks later by the cylinder and amphetamine-induced rotation tests. The animals were subdivided into 3 groups with similar average scores on the rotation test. Four weeks after lesioning (week 0), two of these subgroups were transplanted with 250,000 cells (H9 or 4X cells), and the third group did not undergo transplantation (6-OHDA lesion group). The rotation test was repeated at weeks 8 and 18 posttransplant, and the cylinder test was repeated at week 18. The animals were killed at week 19 posttransplantation (23 week after lesioning) for histological analysis. b , Amphetamine-induced rotational asymmetry. Two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; time: F(1.689, 35.46) =19.50, P<0.0001; treatment: F (2, 21) = 15.23 P< 0.0001. ** P< 0.01 and ****P< 0.0001 vs. the 4X cell-transplanted group at the same time point. §§ P< 0.01 and §§§§P<0.0001 vs. the same group at week -1. c , The use of each forelimb (contra or ipsi) and both forelimbs in the cylinder test was analyzed by two-way repeated measures ANOVA followed by Sidak’s multiple comparison test with time and group as variables. Time x group: both: F (2, 22) = 5.785, P=0.009; ipsi: F (2, 22) = 8.800, P=0.001; contra: F (2, 22) = 4.642, P=0.021. *P<0.05 and **P<0.01 vs. the same group at -1 week. $P<0.05 and $$P<0.01 vs. the 6-OHDA lesion group at the same time point. £P<0.05 and ££P<0.01 vs. the H9 cell-transplanted group at the same time point. The data in (b) and (c) are presented as the mean ± SEM. n= 7 rats in the 6-OHDA lesion group, n = 9 rats in the 4X cell-transplanted group, and n=8 rats in the H9 cell-transplanted group). d , Representative photos of coronal sections from all three groups immunostained for TH. Higher magnification images of the areas in the frame are shown on the right. Scale bars, 50 µm for all three photos in the column. The graphs on the right show (e) the estimated numbers of TH-positive cells in the grafts, (f) the yield of TH-positive neurons per 100,000 grafted cells and (g) the volume of the TH-positive graft (see Methods for details). h-i , Representative photomicrographs showing HNA-positive and TH-positive cells within H9 cell (h) and 4X cell (i) grafts. The squares in h-i and h’-i’ indicate the magnified areas shown in h’-i’ and h’’-i’’ , respectively. DAPI was used as a nuclear stain. Scale bars, 200 μm (h-i) and 50 μm ( h’-i’) . j-n , Representative immunofluorescence images of cells double-positive for TH/FOXA2 (j) , TH/LMX1A (k) , TH/EN1 (l) , TH/GIRK2 (m) and TH/CALB1 (n) within 4X cell grafts. (j’-n’) High-power images of j-n highlighting the graft composition. Scale bar, 50 μm. o , Quantitative analysis of the immunofluorescence data in m and n, showing the percentages of GIRK2/TH and CALB1/TH double-positive cells within 4X cell grafts. The data are presented as the mean percentage ± SD (n=9 rats).

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: In Vivo, Generated, Transplantation Assay, Staining, Immunofluorescence