Journal: Communications Biology
Article Title: Generation of human iPSC-derived phrenic-like motor neurons to model respiratory motor neuron degeneration in ALS
doi: 10.1038/s42003-024-05925-z
Figure Lengend Snippet: a Heatmap of the 10 most differentially expressed genes (DEGs) identified by single-cell RNA sequencing as up-regulated in cluster #6 (corresponding to phMNs) compared to cluster #4 (corresponding to LMC MNs). Each bar corresponds to a cell and is coloured by the expression of the delineated gene relative to that in the other cluster. b Proportion of IGDCC3-positive and IGDCC3-negative cells isolated by flow cytometry from phMN ENRICHED cultures generated from CS29- and CS52 hiPSC lines ( C9orf72 -mutated (ALS) or isogenic). N = 5 biologically independent cultures for Isogenic, N = 4 biologically independent cultures for ALS. Error bars are means ± standard error of means (SEM) of the average. c Representative images of IGDCC3-positive cells, 3 days after FACS sorting. Cultures were co-stained for the pan-MN marker HB9/ISL1 (red), LHX3 (blue), and SCIP (green). Scale bars = 50 µm. d Quantification of phMNs, identified as ISL1 + /HB9 + SCIP + LHX3 − , as percentages of the number of ISL1 + /HB9 + cells in generic , phMN ENRICHED or phMN ISOLATED cultures from CS29 Isogenic (purple) or ALS (red) iPSCs after 25 days of differentiation. Kruskal-Wallis non parametric test and Dunn’s post-hoc multiple comparisons test; * = p < 0.05; ** = p < 0.005; N = 4 biologically independent cultures for generic and phMN ENRICHED ; N = 3 biologically independent cultures for phMN ISOLATED (with > 500 cells in random fields for each culture). Error bars are means ± standard error of means (SEM) of the average. e , f Quantification of MNs identified as CHAT + cells, as percentages of the total number of cells ( e ), and phMNs identified as CHAT + /LHX3 − /SCIP + , as percentages of the number of CHAT + cells ( f ), two-weeks after FACS sorting of phMN ENRICHED cultures from ALS CS52 iPSCs (D32). Wilcoxon signed rank test; no significant difference; N = 3 biologically independent cultures (with > 500 cells in random fields for each culture). Error bars are means ± standard error of means (SEM) of the average. LMC Lumbar motor column.
Article Snippet: The following primary antibodies were used: mouse anti-OLIG2 (1/100; Millipore Corp.; Billerica, MA, USA; Cat. No. MABN50); goat anti-OLIG2 (1/200; R&D Systems; Cat. No. AF2418); rabbit anti-PAX6 (1/500; Covance; Emeryville, CA, USA; Cat. No. PRB-278P); mouse anti-PAX6 (1/150; Millipore Corp.; Cat. No. MAB5554); rat panTLE antibody (1/10) ; rabbit anti-HOMEOBOX A5 (HOXA5) (1/150; kindly provided by Dr. Jeremy Dasen, New York University School of Medicine, New York, NY); mouse anti-NK2 HOMEOBOX 2 (NKX2.2) (1/100; DSHB; Cat. No. 74.5A5-c); mouse anti-HOMEOBOX PROTEIN HB9 (HB9) (1/30; DSHB; Cat. No. 81.5C10-c); mouse anti-ISLET1 (ISL1) (1/30; DSHB; Cat. No. 39.4D5-c); rabbit anti-LIMB HOMEOBOX CONTAINING 3 (LHX3) (1/100; Abcam; Toronto, ON, Canada; Cat. No. ab14555); goat anti-FORKHEAD BOX PROTEIN 1 (FOXP1) (1/100; R&D Systems; Cat. No. AF4534); mouse anti-neurofilament protein (2H3) (1/35; DSHB; Cat. No. 2H3-c); goat anti-CHAT (1/100; Millipore; Cat. No. MAB144P); and guinea pig anti-SCIP (1/16,000; kindly provided by Dr. Jeremy Dasen).
Techniques: RNA Sequencing, Expressing, Isolation, Flow Cytometry, Generated, Staining, Marker
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