Journal: Nature biomedical engineering

Article Title: Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder

doi: 10.1038/s41551-019-0381-8

Figure Lengend Snippet: a , Schematic of LSB2i differentiation using H9 SOX10::eGFP reporter hESCs. b , Immunofluorescence for eGFP and TUJ1 demonstrate SOX10+ SCPs in association with TUJ1+ neurons (bar = 50 μm). c , Flow cytometry demonstrates substantial overlap between the CD49d+ population and SOX10::eGFP expression. d , Real time PCR for Schwann cell lineage markers in CD49d+ putative SCPs, CD49d- non- SCPs, and unsorted cells. Data expressed as mean +/− SD ( n = 6, independent samples) and p -values calculated by one-way ANOVA. e , Heat map representation of supervised hierarchical clustering of OCT4::eGFP+ hESCs, SOX10::eGFP+ neural crest, and CD49d+ putative SCPs, based on all differentially expressed genes with p <0.001. f , Validation of microarray results with qRT-PCR for select SCP genes, normalized to GAPDH . Data expressed as mean +/− SD ( n = 3, independent samples) and p -values calculated by one-way ANOVA.

Article Snippet: Briefly, SCPs were purified with Fluorescence Activated Cell Sorting (FACS) after 21–23 days of differentiation using a PE-conjugated antibody to the alpha4 integrin, CD49d (R&D Systems, FAB1354P).

Techniques: Immunofluorescence, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Biomarker Discovery, Microarray, Quantitative RT-PCR

Journal: Nature biomedical engineering

Article Title: Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder

doi: 10.1038/s41551-019-0381-8

Figure Lengend Snippet: Gene ontology and pathway analysis with upregulated gene list (CD49d positive cells over hESCs)

Article Snippet: Briefly, SCPs were purified with Fluorescence Activated Cell Sorting (FACS) after 21–23 days of differentiation using a PE-conjugated antibody to the alpha4 integrin, CD49d (R&D Systems, FAB1354P).

Techniques:

Journal: Nature biomedical engineering

Article Title: Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder

doi: 10.1038/s41551-019-0381-8

Figure Lengend Snippet: a , Schematic of the reprogramming methods to generate CMT1A patient hiPSC derived SCPs from CMT1A patient fibroblasts. b , FACS purification of LSB2i treated CMT1A and control hiPSCs reveals similar yields of CD49d+ SCPs. Data expressed as mean +/− SD (control n = 36, CMT1A n = 9, independent samples) and p -values calculated by unpaired t-test. c , Post-FACS evaluation of Schwann cell lineage gene enrichment in CD49d+ hiPSC-SCPs versus matched CD49d- non-SCPs, by real time PCR. Data expressed as mean +/− SD (control n = 11 for CDH19 and n = 15 for MPZ , CMT1A n = 28 for CDH19 and n = 37 for MPZ , independent samples) and p -values calculated by unpaired t-test. d , Real time PCR for PMP22 in CMT1A and control CD49d+ hiPSC-SCPs. Data expressed as mean +/− SD (control n = 29, CMT1A n = 76, independent samples) and p -values calculated by unpaired t-test. b-d , Analysis includes five control hPSC lines representing four genotypes and seven CMT1A hiPSC lines representing four genotypes. e , Volcano plot representation of microarray array expression profiling of four biologically independent CMT1A SCP samples and four biologically independent control hPSC-SCP samples. Select genes of interest highlighted and designated for validation with qRT-PCR. f , Results from DAVID gene ontology and Ingenuity Pathway Analysis of differentially expressed genes. Data expressed as p -value of each pathway. g , Real time PCR validation of microarray results in biologically independent samples from five hiPSC clones representing three CMT1A patients. Data expressed as ratio of gene expression in CMT1A versus control hiPSC-derived SCPs. Data from independent clones are displayed as different colors, dots of same color are independent assays from same clone. Data expressed as ratio of gene expression in CMT1A SCPs versus matched control (05148#1 n = 4, 05148#4 n = 4, 05165#7 n = 5, 05167#5 n = 2, 05167#8 n = 2, independent samples) and p -values calculated by ratio paired t-test. h , Cytokine array profiling of cell conditioned medium from Day 10 post-FACS CMT1A and control SCPs. Data expressed as ratio of protein expression in CMT1A SCPs versus matched control (05148#1 n = 4, 05165#5 n = 2, independent samples) and p -values calculated by ratio paired t-test. Data from independent differentiations are displayed as different colors, dots of same color are from same differentiation.

Article Snippet: Briefly, SCPs were purified with Fluorescence Activated Cell Sorting (FACS) after 21–23 days of differentiation using a PE-conjugated antibody to the alpha4 integrin, CD49d (R&D Systems, FAB1354P).

Techniques: Derivative Assay, Purification, Control, Real-time Polymerase Chain Reaction, Microarray, Expressing, Biomarker Discovery, Quantitative RT-PCR, Clone Assay, Gene Expression

Journal: Nature biomedical engineering

Article Title: Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder

doi: 10.1038/s41551-019-0381-8

Figure Lengend Snippet: a , Schematic of the method to generate CMT1A PGD-hESC derived SCPs from CMT1A blastocysts. b , FACS purification of LSB2i treated CMT1A and control PGD-hESCs reveals similar yields of CD49d+ PGD-hESC- SCPs. Data expressed as mean +/−SD (control n = 27, CMT1A n = 13, independent samples) and p -values calculated by unpaired t-test. c , Post-FACS evaluation of Schwann cell lineage gene enrichment in CD49d+ SCPs versus matched CD49d- non-SCPs, by real time PCR. Data expressed as mean +/−SD (control n = 9 for CDH19 and n = 9 for MPZ , CMT1A n = 7 for CDH19 and n = 9 for MPZ , independent samples) and p -values calculated by unpaired t-test. d , Real time PCR for PMP22 in CMT1A and control CD49d+ SCPs. Data expressed as mean +/− SD (control n = 32, CMT1A n = 25, independent samples) and p -values calculated by unpaired t-test. b-d , Analysis includes two control hESC lines representing one genotype and two CMT1A PGD-hESC lines representing two genotypes. e , Schematic of the method to directly convert CMT1A patient fibroblasts into CMT1A hiNC-Schwann cells. f , FACS purification of CMT1A and control hiNCs by using CD34 antibody. Data expressed as mean +/−SD (control n = 12, CMT1A n = 24, independent samples) and p -values calculated by unpaired t-test. g , Real time PCR for PMP22 in CMT1A and control CD34+ Schwann cells. Data expressed as mean +/− SD (control n = 19, CMT1A n = 22, independent samples) and p -values calculated by unpaired t-test. f-g . Analysis includes two control fibroblast lines representing two control patients and three CMT1A fibroblast lines representing three CMT1A patients. h , Representative immunofluorescence for GFAP and S100b in hiNC-Schwann cells from two control fibroblasts and two CMT1A fibroblasts. (bar = 50 μm).

Article Snippet: Briefly, SCPs were purified with Fluorescence Activated Cell Sorting (FACS) after 21–23 days of differentiation using a PE-conjugated antibody to the alpha4 integrin, CD49d (R&D Systems, FAB1354P).

Techniques: Derivative Assay, Purification, Control, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: Nature biomedical engineering

Article Title: Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder

doi: 10.1038/s41551-019-0381-8

Figure Lengend Snippet: a , TALEN-based gene editing results. Targeting sequences of TALEN-based exon editing for PMP22 (upper). Sequencing result of TALEN-based 2nd exon inactivation indicated target sequences harboring start codon were eliminated (lower). Efficiency of TALEN-based exon targeting with no off-target mutations in 16 predicted regions (47% clones with modifications and 12% clones with two copies of wild type and one copy of knock out alleles). b , Representative FACS plots from purification of CD49d+ Schwann cell precursors from CMT1A hiPSC (CS67iCMT-n4) and its isogenic control hiPSC (CS67iCMT-n4.ISOC4). c , Efficiency of differentiation from both lines. Data expressed as mean +/−SD (CMT1A n = 5, isogenic control n = 5, independent samples) and p -values calculated by paired t-test. d , Relative level of mRNA transcription of PMP22 in CMT1A- and isogenic control Schwann cell precursors. Data expressed as mean +/−SD (CMT1A n = 3, isogenic control n = 3, independent samples) and p -values calculated by unpaired t-test. e-f , Protein expression level of PMP22 in undifferentiated hiPSCs and hiPSC-Schwann cells from the CMT1A hiPSC line and its isogenic control ( e and ), and validation of protein band intensity ( f ). GAPDH was used as an internal control. Data expressed as mean +/−SD (CMT1A n = 3, isogenic control n = 3, independent samples) and p -values calculated by paired t-test. g , Transcription level of MCP1 and CXCL1 in CMT1A hiPSC-derived- and isogenic control Schwann cells were validated by quantitative PCR. Data expressed as mean +/−SD (CMT1A n = 3, isogenic control n = 3, independent samples) and p -values calculated by paired t-test. h , Chemotaxis assay with THP1 monocytes was performed with media control, CMT1A-Schwann cells, CMT1A-Schwann cells with antibody neutralization, and isogenic control Schwann cells. Representative image of monocytes migrated into the Schwann cell side of the chamber. Data expressed as mean +/−SD (CMT1A n = 6, isogenic control n = 3, independent samples) and p -values calculated by one-way ANOVA. i , Protein expression level of CXCL1 in CMT1A and control patient nerve biopsies. Data expressed as mean +/− SD of cytokine dot array intensity ( n = 4 for each sample, independent biopsies) and p -values calculated by one-way ANOVA. j , Protein expression level of MCP1 in CMT1A and control patient nerve biopsies. Data expressed as mean +/− SD of cytokine dot array intensity ( n = 4 for each sample, independent biopsies) and p -values calculated by one-way ANOVA. k , CD68 staining (a marker for monocytes/macrophages, and immune-labelled cells appear as brown stained.) in intercostal nerve biopsy from a CMT1A patient (scale bar = 100 μm).

Article Snippet: Briefly, SCPs were purified with Fluorescence Activated Cell Sorting (FACS) after 21–23 days of differentiation using a PE-conjugated antibody to the alpha4 integrin, CD49d (R&D Systems, FAB1354P).

Techniques: Sequencing, Clone Assay, Knock-Out, Purification, Control, Expressing, Biomarker Discovery, Derivative Assay, Real-time Polymerase Chain Reaction, Chemotaxis Assay, Neutralization, Staining, Marker

Journal: bioRxiv

Article Title: Gene-Centric Functional Dissection of Human Genetic Variation Uncovers Regulators of Hematopoiesis

doi: 10.1101/500579

Figure Lengend Snippet: (A) Representative FACS plots of alternate erythroid cell surface markers CD49d (α4 integrin) and CD235a (Glycophorin A) expression at various time points during erythroid differentiation. (B) May-Grunwald Giemsa staining showing more differentiated erythroid cells after TFR2 knockdown at day 18 of erythroid culture. (C) Western blot showing downregulation of TFR2 in UT7/EPO cells. (D) Time dependent absolute value of MFI of STAT5 in UT7/Epo cells after TFR2 knockdown.

Article Snippet: The following antibodies were used anti-human CD235a-APC (eBioscience, Clone HIR2), anti-human CD71-FITC (eBioscience, Clone OKT9), anti-human CD71-PEcy7 (eBioscience, Clone OKT9), ant-human CD49d-PE (Miltenyi, Clone MZ18-24A9), anti-human CD41a-PE (eBioscience, Clone HIP8), anti-human CD11b-PE (eBioscience, Clone ICRF44), anti-mouse Ter119-APC (eBioscience, Clone TER119), anti-mouse CD71-PE (eBioscience, Clone R17217) and Alexa Fluor-647 anti-phospho STAT5 (pY694) (BD Bioscience Cat#: 612599).

Techniques: Expressing, Staining, Knockdown, Western Blot