Journal: Stem cell research & therapy

Article Title: Gain of 20q11.21 in human pluripotent stem cells enhances differentiation to retinal pigment epithelium.

doi: 10.1186/s13287-025-04196-7

Figure Lengend Snippet: Fig. 2 A Representative phase images of 20q11.21 RPE monolayer and cobblestone morphology (insert). B PEDF concentration in 2 months-old 20q11.21 RPE monolayers*. C Immunofluorescence images showing positivity for RPE markers CRALBP, OTX2, PEDEF, PMEL17 and negativity for OCT4 and TGFβ in A8 20q11.21 RPE. D–E Representative phase images of 20q11.21 RPE monolayer cobblestone morphology at 10 months from derivation in passage 0 and passage 3 and 4. *The data shown in (B) are presented as mean plus SEM of three independent experiments, and statistical significance was determined using Student’s t test, two-tailed. ns = not significant

Article Snippet: Primary antibodies and dilutions: mouse anti PMEL17 (Dako, Mo634, 1:50); mouse anti CRALBP (Invitrogen, MA1-813, 1:1000); goat anti OTX2 (Santa Cruz, 30,659, 1:100); mouse anti PEDF clone 10F12.2 (Millipore, MAB1059, 1:1000); mouse anti TGFB1 (Santa Cruz, 3C11, 1:500), rabbit anti OCT-4 (Cell signalling, C30A3, 1:400).

Techniques: Concentration Assay, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: A novel microRNA-based strategy to expand the differentiation potency of stem cells

doi: 10.1101/826446

Figure Lengend Snippet: ( A ) miR-203 expression, as determined by qPCR, in five temporal different stages of normal early development: oocyte, 2-cell embryo, morula, compacted morula and blastocyst. RNA was extracted from 30 different embryos and pooled in two independent groups for analysis by qPCR. RNA expression is normalized by a housekeeping miRNA (miR-16) that maintained invariable during early embryogenesis. Data represent 6 different qPCR measures. P =0.05 (Student’s t-test) comparing 2C/morula versus compacted morula/blastocyst. ( B ) Protocol for reprogramming of miR-203 mutant MEFs into pluripotent iPSCs and subsequent differentiation into embryoid bodies. MEFs were transduced with lentiviruses expressing Oct4, Sox2, Klf4, and cMyc (OSKM) in a constitutive manner. The resulting iPSCs were then treated with doxycycline (Dox) 1 μg/ml during 5 days to induce miR-203 expression. “ mi iPSCs” refers to iPSCs in which miR-203 was transiently expressed during the indicated 5 days. Dox was removed for 15-30 days before starting the embryoid body generation protocol. Samples for RNA sequencing were taken 30 days after Dox withdrawal. ( C ) Principal Component Analysis of RNAseq data from wild-type iPSCs (n=3 clones), mi iPSCs (n=4) and wild-type ESCs (n=3). ( D ) Enrichment plots of the 282-gene 2-cell signature in mi iPSCs 10 and 25 days after Dox withdrawal. ( E ) Representative images of embryoid bodies (EBs) derived from wild-type iPSCs or ESCs, or from mi iPSC and mi ESCs at day 30 of differentiation. Scale bars, 500 μm. ( F ) Quantification of the percentage of EBs from panel ( E) presenting internal large cavities and EBs beating during the indicated time course. Data are represented as mean ± s.e.m. (n=3 independent experiments). * P <0.05; *** P <0.001 (Student’s t-test). ( G ) Representative images of EBs derived from human iPSCs transiently transfected with either control (left) or miR-203 mimics (right), at different time points during the differentiation process. Scale bars, 500 μm. ( H ) Left panel shows the quantification of EBs size derived from human iPSCs transiently transfected with either control or miR-203 mimics as in panel (G) at different time points during the differentiation process. The percentage of EBs presenting internal large cavities during the indicated time course of differentiation is shown in the right panel. Data are mean ± s.e.m. (n=3 independent experiments). *** P <0.001 (Student’s t-test).

Article Snippet: The following antibodies were used: anti-OCT4 (C30A3) (1:200; Cell Signaling), Anti-HuNu (1:200; Novus, NBP2-34342), anti-Gata4 (1:200; Santa Cruz, C-20), anti-Sox2 (1:200; R&D, AF2108), anti-hSOX17 (1:200; R&D, AF1924) (Supplementary Table 8).

Techniques: Expressing, RNA Expression, Mutagenesis, Transduction, RNA Sequencing Assay, Clone Assay, Derivative Assay, Transfection

Journal: bioRxiv

Article Title: A novel microRNA-based strategy to expand the differentiation potency of stem cells

doi: 10.1101/826446

Figure Lengend Snippet: ( A ) Teratoma volume (mm 3 ) 20-25 days after subcutaneous injection of wild-type iPSCs or mi iPSCs expressing GFP. Data are represented as mean ± s.e.m. (n=8 tumors per genotype); *** P <0.001 (Student’s t-test). Representative images are shown in Fig EV2. ( B ) Incidence and representative images of specific highly differentiated tissues in teratomas. The number of tumors included in the analysis is indicated in the panel. *** P <0.001 (Student’s t-test). Scale bars, 50 μm. ( C ) Table showing the frequency of nude mice with embryo-like structures (E-Ls) in their abdominal cavity 20-30 days after intraperitoneal (i.p.) injection of 400.000-500.000 in vivo (iv)-generated wild-type iPSCs, un-induced iPSCs, or mi iPSCs. The number of independent clones tested per condition is indicated in the panel (each animal was inoculated with a different clone). ( D ) Representative example of E-Ls generated after i.p. injection of GFP-expressing mi iPSCs. H&E, hematoxylin and eosin. The following antigens were detected by immunohistochemistry: GFP, Sox2 (ectoderm), Cd34 (mesoderm), Gata4 (endoderm), AFP and CK8 (visceral endoderm of the yolk sac) and Ter119 (nucleated erythroid cells). Scale bars, 500 μm and 100 μm for higher magnifications. ( E ) Embryo tetraploid complementation assays with un-induced iPSCs, mi iPSCs or wild-type ESCs (n=3 clones per condition). Pictures on the right show a representative example of a viable “all- mi iPSC” mouse (black) generated from mi iPSCs and litters obtained from “all- mi iPSC” adult mice, which efficiently contributed to germline transmission.

Article Snippet: The following antibodies were used: anti-OCT4 (C30A3) (1:200; Cell Signaling), Anti-HuNu (1:200; Novus, NBP2-34342), anti-Gata4 (1:200; Santa Cruz, C-20), anti-Sox2 (1:200; R&D, AF2108), anti-hSOX17 (1:200; R&D, AF1924) (Supplementary Table 8).

Techniques: Injection, Expressing, In Vivo, Generated, Clone Assay, Immunohistochemistry, Transmission Assay

Journal: bioRxiv

Article Title: Changing the Waddington landscape to control mesendoderm competence

doi: 10.1101/867010

Figure Lengend Snippet: a, Top, confocal microscopy image of immunostained hESC colony after 48 hours of BMP4 + Activin A; SOX17 (pink), OCT4 (yellow), SOX2 (blue). These cells largely adopt SOX17 + endoderm fates and are uniformly OCT4 + SOX2 - . Scale bar = 100 μm. Bottom Left, contour plot of cell density as a function of OCT4 and SOX2 levels in individual cells; Bottom Right, same plot as left displayed as scatter plot with individual cells colored based on the level of endodermal marker SOX17. b, Images of hESC colonies immunostained for OCT4, SOX2, and mesendoderm marker T after 1-6 days of Activin/NODAL inhibition followed by 42h of BMP4 + Activin A stimulation. T - OCT4 - SOX2 + ectoderm cells first appear at 2 days and are widespread by 4 days of Activin/NODAL inhibition. The spatial structure seen here largely appears after BMP4 and Activin A signal induction and is likely due to a combination of local density impacts on ectoderm-directed differentiation rate and homophilic interactions between cells. Scale bar = 300 μm. c, Top, epifluorescence microscopy image of an hESC colony immunostained for the neurectoderm marker PAX6 (purple), OCT4 (yellow), and SOX2 (blue) after 6 days of Activin/NODAL inhibition. These cells largely adopt PAX6 + OCT4 - SOX2 + neurectoderm fates. Below, contour plot of cell density as a function of OCT4 and SOX2 levels in individual cells (left), same plot as left displayed as scatter plot with individual cells colored based on the level of PAX6 (right). Scale bar = 300 μm. d, Images of hESC colonies immunostained for OCT4, SOX2, and the neurectoderm marker PAX6 after 0-6 days of Activin/NODAL inhibition. PAX6 expression is first seen after 4 days and is widespread by 5 days. Scale bar = 300 μm. e, hESCs were exposed to a pretreatment of Activin/NODAL inhibition for 1, 3, or 6 days before BMP4 + Activin A signal exposure (left). Confocal microscopy images of immunostained hESCs at the end of the experiment after 48h of BMP4 + Activin A treatment (right). Increasing duration of Activin/NODAL inhibition reduced the population’s competence to produce SOX17 + endoderm and, more broadly, OCT4 + (yellow) SOX2 - (blue) mesendoderm-derived cell types. Scale bar = 100 μm. f, Scatter plot (top) and density contour plot (bottom) of cells from E, as a function of their OCT4 and SOX2 levels. Cells in scatter plot colored by level of SOX17. g, Immunofluorescence images acquired via epifluorescence microscopy showing DAPI, phosphorylated SMAD1, and phosphorylated SMAD2 levels in hESC colonies upon stimulation with BMP4 + Activin for 45 mins after 0, 3, or 5 days of Activin/NODAL inhibition. Unstimulated control shown in top row. All stimulated cells at all time points displayed nuclear-localized pSMAD1 and pSMAD2 upon BMP4 + Activin A exposure despite the loss of mesendoderm competence that occurs during this window. Scale bar = 300 μm. h, Density contour plots of cells treated as in G, as a function of pSMAD2/3 and pSMAD1/5 levels determined by antibody staining. Dotted lines drawn for reference.

Article Snippet: The following primary antibodies were used: OCT4 (1:400, Cell Signaling C30A3); SOX2 (1:400, Thermo Fisher BTJCE); SOX17 (1:100, R&D Systems AF1924); phosphorylated SMAD1/5/9 (1:200, Cell Signaling D5B10); phosphorylated-SMAD2 (1:200, Cell Signaling E8F3R); PAX6 (1:200, DSHB AB_528427); and NANOG (1:500, R&D Systems AF1997).

Techniques: Confocal Microscopy, Marker, Inhibition, Epifluorescence Microscopy, Expressing, Derivative Assay, Immunofluorescence, Control, Staining

Journal: bioRxiv

Article Title: Changing the Waddington landscape to control mesendoderm competence

doi: 10.1101/867010

Figure Lengend Snippet: a, OCT4 and SOX2 levels as determined by immunofluorescence (IF) and by OCT4:RFP and SOX2:YFP fluorescence (FP). Scale bar = 100 μm. Blue line, best-fit linear regression. b, Immunofluoresence images of H1 OCT4:RFP SOX2:YFP double reporter cells stained for NANOG, OCT4, and SOX2. Nuclei (DAPI) are shown for reference. These cells can maintain pluripotency indefinitely. Scale bar = 300 μm. c, Immunofluorescence images of H1 OCT4:RFP SOX2:YFP double reporter cells that were (top) stained for PAX6 and SOX2 after 6 days of Activin/NODAL inhibition by A83-01 or (bottom) stained for SOX17 after 42h of BMP4 and Activin A with nuclei (DAPI) shown for reference. This line’s ability to form the germ layers is unaffected. Scale bar = 300 μm. d, Schematic of the live-cell imaging apparatus constructed for this study. Cells were grown on the bottom of a permeable membrane glued to a thin stainless-steel washer and imaged from below with an epifluorescence microscope. Focus was maintained with a combination of Zeiss Definite Focus and periodic software autofocus adjustments.

Article Snippet: The following primary antibodies were used: OCT4 (1:400, Cell Signaling C30A3); SOX2 (1:400, Thermo Fisher BTJCE); SOX17 (1:100, R&D Systems AF1924); phosphorylated SMAD1/5/9 (1:200, Cell Signaling D5B10); phosphorylated-SMAD2 (1:200, Cell Signaling E8F3R); PAX6 (1:200, DSHB AB_528427); and NANOG (1:500, R&D Systems AF1997).

Techniques: Immunofluorescence, Fluorescence, Staining, Inhibition, Live Cell Imaging, Construct, Membrane, Microscopy, Software

Journal: bioRxiv

Article Title: Changing the Waddington landscape to control mesendoderm competence

doi: 10.1101/867010

Figure Lengend Snippet: a, Schematic of the cassette transduced via lentiviral vector. Under the control of the EF1alpha promoter, we placed the cyan fluorescent protein mCerulean fused in frame to a P2A ribosomal skip sequence and the gene of interest. A Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) was included to improve transcript stability. The entire sequence was flanked by the Long Terminal Repeat regions (LTR) that the viral transposases recognize to effect integration into the genome. b, Schematic of experimental protocol. 24h after seeding in pluripotency maintenance conditions, parallel samples of hESCs were subjected to simultaneous Activin/Nodal inhibition and, for the first 36h, lentiviral transduction of the gene of interest. After 72h of Activin/Nodal inhibition, one membrane was analyzed by flow cytometry while the other was stimulated with 42h of BMP4 + Activin A before analysis by flow cytometry. c, Epifluorescent microscopy images of a cell colony after Activin/Nodal inhibition and subsequent BMP4 + Activin A stimulation. Cells that were transduced with a CFP:P2A:OCT4 expression cassette expressed CFP and preferentially adopted a OCT4:RFP + SOX2:YFP - mesendoderm fate. White regions indicate selected clusters of cells expressing CFP, indicating transduction. Scale bar = 100 μm. d, Examples of two candidates, SOX9 and TFAP2C, whose overexpression altered the distribution of OCT4:RFP/SOX2:YFP ratios of cells before the signal, p(OSR | t). OCT4:RFP and SOX2:YFP levels were normalized to an hESC sample measured in the same batch. Black dotted line, wildtype. Blue, transduced. Shaded area represents one standard deviation across biological replicates. Inset, a schematic depicting the corresponding effects on the Waddington developmental landscape. e, Scatterplot of the shift in p(mesendoderm|OSR) location versus a metric of disruption of p(OSR|t) for each TF tested. Disruption of initial differentiation dynamics was measured as the Kullback-Leibler divergence of the distribution of the log ratio of OCT4:RFP to SOX2:YFP of CFP + cells using the same distribution from CFP - cells as a reference; this KL-divergence was then reported with a positive sign if the mean OSR of the transduced cells was higher than in the non-transduced cells and a negative sign if not. Candidates that were significantly different from the CFP negative control (FDR < 0.1) along either dimension are shown in red. Candidates whose effects were not consistent across replicates are shown in gray. CFP:P2A:CFP is shown in blue. Blue dotted lines indicate CFP:P2A:CFP overexpression outcomes, shown as the mean ± one standard deviation of n=4 biological replicates, for each axis. OCT1, POU6F1, PAX3 were tested but are excluded from this plot because their overexpression caused premature downregulation of both OCT4:RFP and SOX2:YFP, indicating possible differentiation to an alternative fate. Interestingly, exogenous OCT4 overexpression does not impact the dynamics of endogenous OCT4:RFP and SOX2:YFP, as evidenced by the location of the OCT4 point near zero on the y-axis. f, Immunofluorescence images showing PAX6 (magenta) and CFP (cyan) after 6 days of neurectoderm-directed differentiation using inhibitors of both Activin/NODAL and BMP. Top row, exogenous expression of CFP:P2A:OCT4; Middle row, CFP:P2A:FOXB2; Bottom row, CFP:P2A:CFP. White arrows indicate select CFP-expressing cells. Look-up tables for the channels in each condition are scaled individually because variable membrane positioning in the well created spurious changes in overall apparent signal intensity. Scale bar = 50 μm. OCT4 overexpression prevents PAX6 induction, while FOXB2 or CFP overexpression does not disrupt normal PAX6 induction.

Article Snippet: The following primary antibodies were used: OCT4 (1:400, Cell Signaling C30A3); SOX2 (1:400, Thermo Fisher BTJCE); SOX17 (1:100, R&D Systems AF1924); phosphorylated SMAD1/5/9 (1:200, Cell Signaling D5B10); phosphorylated-SMAD2 (1:200, Cell Signaling E8F3R); PAX6 (1:200, DSHB AB_528427); and NANOG (1:500, R&D Systems AF1997).

Techniques: Plasmid Preparation, Control, Sequencing, Virus, Inhibition, Transduction, Membrane, Flow Cytometry, Microscopy, Expressing, Over Expression, Standard Deviation, Disruption, Negative Control, Immunofluorescence

Journal: bioRxiv

Article Title: Changing the Waddington landscape to control mesendoderm competence

doi: 10.1101/867010

Figure Lengend Snippet: a, Over expression of three candidates, JUNB, POU2F3, and FOXB2, shifted p(mesendoderm | OSR) to keep cells mesendoderm competent for longer along the ectodermal differentiation trajectory. Black dotted: wildtype; blue: transduced cells; shaded: std (n=3, FOXB2 n=4). Inset, schematic depicting the effect of overexpression on the Waddington landscape. b, Over expression of FEZF1, TFAP2A, OTX2, and GRHL1 (top to bottom) altered both p(OSR | t) (left column) and p(mesendoderm | OSR) (middle column). Black dotted: wildtype control; blue: transduced cells, shaded: std (n=3). Left, p(OSR | t). Center, p(mesendoderm | OSR). Right, schematic as in (a). Overexpression of candidates can tune p(OSR | t) and p(mesendoderm | OSR) independently. c, Top, FACS density plot (OCT4 vs SOX2) of wild type cells (un-transduced CFP-) showing two peaks corresponding to ectodermal lineage above diagonal (41% of cells) and mesendodermal lineage, below diagonal (59% of cells). Bottom, transduced cells (CFP+) from the same population with CFP:P2A:FOXB2 cassette show 81% of cells adopting mesendodermal fate. Overexpression of FOXB2 increases the fraction of cells that adopt a mesendodermal fate. d, Fraction of cells adopting ectodermal fate (PAX6+) under ectodermal differentiation conditions. Cells with CFP:P2A:FOXB2 or the control CFP:P2A:CFP show normal ectodermal differentiation. OCT4 over expression precludes PAX6 expression and ectodermal differentiation. Error bars: std. e, The gene regulatory network governs both the progression of the cell along the developmental trajectory and shape of barrier between fates. In turn, both the cell’s location on the developmental landscape and the location of the barrier determine the cell’s fate in response to alternative signals. Only perturbations of the location of the barrier alters competence without altering the dynamics of movement along the original trajectory in the absence of a new signal.

Article Snippet: The following primary antibodies were used: OCT4 (1:400, Cell Signaling C30A3); SOX2 (1:400, Thermo Fisher BTJCE); SOX17 (1:100, R&D Systems AF1924); phosphorylated SMAD1/5/9 (1:200, Cell Signaling D5B10); phosphorylated-SMAD2 (1:200, Cell Signaling E8F3R); PAX6 (1:200, DSHB AB_528427); and NANOG (1:500, R&D Systems AF1997).

Techniques: Over Expression, Control, Expressing