stemdiff tm trilineage differentiation kit  (STEMCELL Technologies Inc)

Journal: Stem cell research

Article Title: Generation of two induced pluripotent stem cell lines to model and investigate diseases affecting Pacific Islanders

doi: 10.1016/j.scr.2025.103668

Figure Lengend Snippet: Generation and characterization of lines. (A) Brightfield microscopy demonstrates that iPSCs exhibit morphology consistent with iPSC colony formation, including circumscribed regions with defined borders and tightly neighboring cells. (passage 25; same magnification for all images). (B) Using Karyotype analysis, iPSCs lines were demonstrated to have normal karyotype as evaluated by KaryoStat assay. (C) RT-qPCR demonstrated loss of Sendai viral vector genome and reprogramming transgene as compared to the control, an iPSC line at passage 9. (D) Generated lines demonstrate expression of pluripotent markers, SOX2, NANOG, and OCT3/4 as demonstrated by immunocytochemistry (same magnification for all images). (E) RT-qPCR was used to confirm that generated lines express pluripotent genes, SOX2 and NANOG. A previously published iPSC-EC line was used as a negative control. (F) iPSC lines are capable of trilineage differentiation, as confirmed by expression of endoderm (SOX17, FOXA2), mesoderm (BRACHYURY, TBX6), and ectoderm (OTX2, NESTIN) markers (same magnification for all images).

Article Snippet: iPSC lines were cultured until passage 25 and then differentiated into the three germ layers per instruction of the STEMdiff TM Trilineage Differentiation Kit (Stem Cell Technologies).

Techniques: Microscopy, Quantitative RT-PCR, Plasmid Preparation, Control, Generated, Expressing, Immunocytochemistry, Negative Control

Journal: bioRxiv

Article Title: Erosion of X-Chromosome Inactivation in female hiPSCs is heterogeneous and persists during differentiation

doi: 10.1101/2024.03.15.585169

Figure Lengend Snippet: A. Illustration of the experimental design used for trilineage commitment of the three XIST+/XIST-isogenic hiPSC pairs (F7cl15 & F7cl4; 2041cl13 & 2041cl2; 2042cl9 & 2042cl5) into Ectoderm, Mesoderm and Endoderm. The inactive X chromosome (Xi) is marked in red, the active X chromosome (Xa) is marked in green and the eroded X chromosome (Xe) is marked in purple. B. RT-qPCR analysis for NANOG (pluripotency marker), PAX6 (neuronal marker) and XIST (Xi marker) normalized to GAPDH housekeeping gene in F7cl15, F7cl4, 2041cl13, 2041cl2, 2042cl9 and 2042cl5 hiPSCs and after Ectoderm differentiation. Barplots represent the mean relative expression of n=2 for all samples, except 2042cl9 and 2042cl5 in both hiPSCs and Ectoderm (n=1). C. Representative images of XIST RNA-FISH and respective percentages of cells expressing XIST (red dots) in F7cl15, 2041cl13, 2042cl9 and 2041cl2 at D0 (hiPSCs) and D7 (differentiated ectodermal cells). The nuclei are counterstained with DAPI (blue). Scale bars represent 15 μ m. Number of cells counted: hiPSCs-F7cl15: 281, 2041cl13: 173, 2042cl9: 194, 2041cl2: 94. Ectoderm cells-F7cl15: 324, 2041cl13: 326, 2042cl9: 367, 2041cl2: 381. The values represent 1 independent experiment. D. Allelic expression assayed by RT-PCR followed by Sanger sequencing resourcing to informative SNPs to distinguish the two alleles. The chromatograms represent illustrative examples of the allelic expression of heterozygous X-linked genes in each XIST+/XIST-isogenic hiPSC pair at D0 (hiPSCs) and at D7 (differentiated ectodermal cells): SHROOM2 and APOO gene for the F7cl15 and F7cl4, SHROOM2 and CHRDL1 gene for 2041cl13 and 2041cl2, and ATRX and CHRDL1 gene for the 2042cl9 and 2042cl5. CHRDL1 is not expressed in mesoderm cells. E. Minor allele frequency in prevalent X-linked genes between naive (D0) and differentiated (D7) cell states in F7cl15 (XIST+) and F7cl4 (XIST-) cell lines. Heatmap shows genes containing common SNPs in both states, ordered by their position along the X chromosome (gray lines in the ideogram).

Article Snippet: Trilineage differentiation of F7cl15, F7cl4, 2041cl13, 2041cl2, 2042cl9 and 2042cl5 hiPSCs was performed using STEMdiff TM Trilineage Differentiation Kit (#05230, Stem Cell Technologies) according to manufacturer’s instructions.These experiments were conducted with at least one or two replicates ( ; Fig. S5).

Techniques: Quantitative RT-PCR, Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing