Journal: bioRxiv

Article Title: A Comprehensive Roadmap of Human Placental Development in vitro

doi: 10.1101/2022.04.07.487558

Figure Lengend Snippet: Overview of the roadmap that differentiated hPSCs into TE and self-renewing TSCs. Phase-contrast images correspond to select time points. b , Periodic Acid-Schiff (PAS) staining showing strongly increased glycogen storage in CTB versus undifferentiated cells. c, Immunocytochemistry showing direct differentiation of iPSCs (JHU198i) into TE. d , e , TE cultures at day 10 (D10) with marker expression characteristic of STB cells. f , hCG secretion into the culture medium was confirmed by a urine pregnancy test in D10 TE cells derived from 3 cell lines. g , MDS plot showing gradual changes in the transcriptional profiles of WA09 ESCs differentiating into TE. h , Heatmap of the selected most highly differentially expressed genes between D0 and D10 (log fold change > 8.5, FDR-adjusted P value < 1×10 -4 ). i , Representative TEM images of WA09-TE cultures at D3 and D9 of differentiation along with ESC control (D0).

Article Snippet: All hESCs (WA07, WA09, WA14, and WA17; WiCell) and hiPSCs (JHU191i, JHU198i, and MCW032i; WiCell; WTC iPSC; Allen Institute for Cell Science) were maintained under feeder- and xeno-free conditions in Essential 8 (E8) medium (A1517001, Thermo Fisher Scientific) on microplates or T175 flasks coated with vitronectin (VTN; A14700, Thermo Fisher Scientific) at a concentration of 0.5 µg/cm 2 .

Techniques: Staining, Immunocytochemistry, Marker, Expressing, Pregnancy Test (hCG) Assay, Derivative Assay

Journal: bioRxiv

Article Title: A Comprehensive Roadmap of Human Placental Development in vitro

doi: 10.1101/2022.04.07.487558

Figure Lengend Snippet: a , Phase-contrast images of WA09-derived TSCs grown in TE3 medium. b , Passage-wise cumulative cell counts show stable proliferation of two TSC lines derived from one iPSC line (JHU198i) and one ESC line (WA09). c , Immunofluorescence showed the expression of TE/trophoblast markers in the JHU198i-TSC line at passage 28. d , Western blot analysis of TSC markers. e , RNA-seq MDS plot showed distinct clustering of hPSC, TE D10, and TSC samples, regardless of their genetic background. Early-passage (7-10) and late-passage samples were included (16-21). f , Heatmap of top differentially expressed genes (nearly 700) in TE D10 compared with hPSC (top, log2 fold change > 6, FDR-adjusted P value < 1×10 -4 ) and in TSC compared with TE D10 (bottom, log2 fold change > 3, FDR-adjusted P value < 1×10 -4 ). g , Expression of a number of markers associated with TE and trophoblast. h , Bulk RNA-seq integration with external datasets. MDS plot of transcriptional profiles of hPSC, TE (D10), and TSC and previously published iTSC , naive hPSC-derived TSC, and placental TSC . The original external sample group labels were maintained, however, the H9 ESC line is equivalent to WA09 presented here.

Article Snippet: All hESCs (WA07, WA09, WA14, and WA17; WiCell) and hiPSCs (JHU191i, JHU198i, and MCW032i; WiCell; WTC iPSC; Allen Institute for Cell Science) were maintained under feeder- and xeno-free conditions in Essential 8 (E8) medium (A1517001, Thermo Fisher Scientific) on microplates or T175 flasks coated with vitronectin (VTN; A14700, Thermo Fisher Scientific) at a concentration of 0.5 µg/cm 2 .

Techniques: Derivative Assay, Immunofluorescence, Expressing, Western Blot, RNA Sequencing Assay

Journal: bioRxiv

Article Title: A Comprehensive Roadmap of Human Placental Development in vitro

doi: 10.1101/2022.04.07.487558

Figure Lengend Snippet: a , Protocol for differentiation of TSC into STB over roughly 4 days, leading to the formation of multinucleated cells. b , Immunofluorescence of STB markers CGA and DAB2 , TSC marker TFAP2C , and gap junction protein ZO-1 ( GJP1 ) delineating individual cellular borders. c , Fusion index, which is the proportion of nuclei in multinucleated STB cells relative to all nuclei, was calculated from the fluorescent images. d , Protocol for differentiation of TSC into EVT over 14 days with passaging (“pass.”) on days 2 and 8, and typical cell morphology. e , Immunofluorescence analysis of EVT markers in WA09-TSC-derived EVT. f , Western blot analysis of EVT marker expression in hPSC, TSC, and EVT (WA09). g , Representative phase-contrast images of TSC lines derived using CEPT compared to control (Chroman 1 only). h, Ratio of the number of harvested cells per cm 2 divided by the number of seeded cells per cm 2 at each passage, in CEPT-treated and control (Chroman 1 only) TSC. i, Summary of placental cell development recapitulated by directed differentiation of hPSCs into distinct cell types expressing specific markers and showing distinct morphologies.

Article Snippet: All hESCs (WA07, WA09, WA14, and WA17; WiCell) and hiPSCs (JHU191i, JHU198i, and MCW032i; WiCell; WTC iPSC; Allen Institute for Cell Science) were maintained under feeder- and xeno-free conditions in Essential 8 (E8) medium (A1517001, Thermo Fisher Scientific) on microplates or T175 flasks coated with vitronectin (VTN; A14700, Thermo Fisher Scientific) at a concentration of 0.5 µg/cm 2 .

Techniques: Immunofluorescence, Marker, Passaging, Derivative Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: A Comprehensive Roadmap of Human Placental Development in vitro

doi: 10.1101/2022.04.07.487558

Figure Lengend Snippet: RNA-seq samples included TE derived from the three ESC lines WA09, WA14, and WA17 and D0 controls. WA09-derived endoderm (Endo), mesoderm (Meso), and ectoderm (Ecto) samples were included, as well as CC lines JEG-3 and BeWo. a , b , MDS plots of the above ESC lines (a) with and (b) without CC lines included. c , Heatmap of the 124 most highly differentially expressed genes (log-fold change > 8.5, FDR-adjusted P value < 1×10 -4 ) between TE samples and hPSC samples. d, Enrichr analysis of the 124 differentially expressed genes displayed on the heatmap. e , Enrichr analysis – ARChS4 database. f , UMAP plot of time-course scRNA-seq of WA09 hPSC differentiation into TE. g , UMAP plots colored by the expression of individual differentially expressed TE or placenta-associated genes. h , Expression of markers associated with STB identity. i , Integration of the scRNA-seq data with data derived from human peri-implantation stage embryo using the integration method in R package “Seurat”. j , The same integration method with primary placental villi tissue . VCT – villous cytotrophoblast, SCT – syncytiotrophoblast, EVT – extravillous trophoblast, FB – trophoblastic fibroblasts, VEC – vascular endothelial cells, EB – erythroblasts, HC – Hofbauer cells.

Article Snippet: All hESCs (WA07, WA09, WA14, and WA17; WiCell) and hiPSCs (JHU191i, JHU198i, and MCW032i; WiCell; WTC iPSC; Allen Institute for Cell Science) were maintained under feeder- and xeno-free conditions in Essential 8 (E8) medium (A1517001, Thermo Fisher Scientific) on microplates or T175 flasks coated with vitronectin (VTN; A14700, Thermo Fisher Scientific) at a concentration of 0.5 µg/cm 2 .

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: A Comprehensive Roadmap of Human Placental Development in vitro

doi: 10.1101/2022.04.07.487558

Figure Lengend Snippet: a , b, Single cell analysis (scRNA-seq) of TSCs (WA09) identified 4 main clusters. Trophoblast markers expressed by CTB of the placental villi and STB. c , MDS plot of miRNA expression profiles show distinct clustering of hPSC, TE D10, and TSC (JHU198i line). d , Heatmap of the expression of microRNAs of the C19MC cluster. The miRNAs marked by asterisks confirm key trophoblast criteria according to ref. 10. e , Principal component analysis of methylation profiles in iPSCs (JHU198i), TE (D10), and TSC based on MeDIP-seq. f , Absence of methylation in the ELF5 promoter region (dashed line) was found across samples. g , Low methylation levels along the long arm of chromosome 21 (q21) are consistent with trophoblast identity according to refs. 2 and 37.

Article Snippet: All hESCs (WA07, WA09, WA14, and WA17; WiCell) and hiPSCs (JHU191i, JHU198i, and MCW032i; WiCell; WTC iPSC; Allen Institute for Cell Science) were maintained under feeder- and xeno-free conditions in Essential 8 (E8) medium (A1517001, Thermo Fisher Scientific) on microplates or T175 flasks coated with vitronectin (VTN; A14700, Thermo Fisher Scientific) at a concentration of 0.5 µg/cm 2 .

Techniques: Single-cell Analysis, Expressing, Methylation, Methylated DNA Immunoprecipitation

Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: a, EB formation in the presence of DMSO, Y-27632 and CEPT. Human ESCs (WA09) cells were dissociated with Accutase and plated into 6-well ULA plates in E6 medium. Representative phase-contrast images were taken at 24 h post-plating. Scale bar, 50 μm. b, To generate single EBs, hESCs were dissociated with Accutase and plating into AggreWell plates (5,000 cells/well). Images were taken 24 h post-plating. In the presence of DMSO the vast majority cells underwent cell death and EB formation was not observed. Treatment with Y-27632 supported EB formation but significant numbers of dead cells were detected surrounding the EB. Note that CEPT enables EB formation without apparent cell death. Scale bar, 100 μm. c, Quantification of the diameter of single EBs (24 h post-plating). Data are mean ± s.d. (n = 20 EBs for Y-27632 and n = 22 for CEPT), **** P < 0.0001, two-tailed Student’s t -test. d, Single EB formation in 96-well ULA plates. Dissociated hESCs were plated into 96-well ULA plates at 2,000 cells/well. Live and dead cells were stained with calcein green AM and propidium iodide (PI) 24 h after cell seeding. Scale bars, 100 μm. e, Quantification of cell survival in single EBs at day 1 and day 7 by using the CTG 3D assay. Note the significant difference between Y-27632 and CEPT treatment at both timepoints. Data represent mean ± s.d. (n = 24 EBs for each group), **** P < 0.0001, one-way ANOVA. f, CEPT supports differentiation of single EBs into the three germ layers. Individual EBs were cultured in E6 medium to allow for spontaneous differentiation and analyzed on day 7 for the expression of PAX6, SOX17, and Brachyury using an optimized quantitative RT-PCR protocol that enabled detection of low transcript levels in single EBs (see Materials and Methods section for details). Data represent mean ± s.d. (n = 3 experiments and in each experiment 24 EBs were analyzed for each group), * P = 0.0327, two-tailed Student’s t -test. g, h, Cerebral organoids were generated by using Y-27632 and CEPT for the first 24 h. At day 30, organoids were fixed, sectioned, processed for histology (hematoxylin and eosin stain) and immunohistochemistry for FOXG1. Representative images show that CEPT treatment resulted in larger organoids and more abundant FOXG1-expressing cells. Scale bars, 400 μm.

Article Snippet: All human ESCs lines (WA01, WA07, WA09) and iPSCs (LiPSC-GR1.1 and the following lines from Coriell Institute for Medical Research: GM25256 , GM23476, GM2610 and AICS-0023 clone 20) were maintained under feeder-free condition using Essential 8 (E8) medium and vitronectin (VTN-N) (Thermo Fisher Scientific).

Techniques: Two Tailed Test, Staining, Cell Culture, Expressing, Quantitative RT-PCR, Generated, H&E Stain, Immunohistochemistry