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: Scientific Reports

Article Title: Comprehensive single-cell transcriptome analysis of autologous platelet-rich plasma therapy on human thin endometrium

doi: 10.1038/s41598-025-99468-w

Figure Lengend Snippet: Comparison of MET pathway activity between pre- and post-PRP samples. ( A ) GSVA was used to calculate the activity of the MET signalling pathway in samples from normal control and TE patients. ( B ) Epithelial cells from TE patients presented relatively high expression of VIM (red rectangle). ( C ) Epithelial cells from TE patients presented relatively low expression of KRT8 (red rectangle). ( D ) The expression of only one of the MET transcription factors, TCF4, was relatively upregulated in TE patients compared with normal patients (red rectangle). ( E ) Significant differences in MET-related gene signature scores were observed in the Str and Peri cells of post-PRP samples. ( F ) Epithelial cells from post-PRP samples presented relatively low expression of VIM (red rectangle). ( G ) Epithelial cells from post-PRP samples presented relatively similar or higher expression of KRT8 (red rectangle). ( H ) In addition to TCF4, other MET transcription factors, such as TWIST1, SMAD family members, ZEB1 and ZEB2, were activated (red rectangle). ( I ) IHC showed PRP therapy induced the expression of KRT8 and E-cad while reducing the protein level of VIM (DAB, 50 µm).

Article Snippet: After cooling to room temperature, the sections were permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 20 min, followed by blocking endogenous peroxidases with 3% (vol/vol) H 2 O 2 for 20 min. Nonspecific binding of the antibodies was blocked with 10% (vol/vol) normal donkey serum in PBS for 1 h, followed by an incubation with primary antibodies against octamer-binding transcription factor 4 (Oct-4, 1:100, Cell Signaling, USA, C30A3), STRO-1 (1:200, Merck Millipore, Ger, MAB4315), stromal cell-derived factor 1 (SDF-1, 1:200, Abcam, USA, AB25117), cytokeratin 8 (KRT8, 1:500, Hangzhou Huaan Biotechnology, China, ET1610-43), vimentin (VIM, 1:500, Hangzhou Huaan Biotechnology, ET1610-39), E-cadherin (E-cad, 1:200, Hangzhou Huaan Biotechnology, China, ET1607-75), inducible NO synthase (iNOS, 1:200, Abcam, USA, AB283655), arginase-1 (Arg1, 1:100, Cell Signaling, USA, D4E3M) and CD31 (1:200, Abcam, USA, AB281583) overnight at 4 °C, followed by an incubation with horseradish peroxidase-conjugated anti-rabbit/mouse (H + L) secondary antibodies (Dako, USA) for 40 min at room temperature.

Techniques: Comparison, Activity Assay, Control, Expressing

Journal: Molecular and Cellular Biology

Article Title: Interleukin-8 Dedifferentiates Primary Human Luminal Cells to Multipotent Stem Cells

doi: 10.1128/MCB.00508-19

Figure Lengend Snippet: Antibodies utilized for immunoblot, immunofluorescence, and immunohistochemistry analyses

Article Snippet: Antibodies are listed in . table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Antibody (clone) a Source Anti-Twist1 (10E4E6) Abcam Anti-Snail (C15D3) Cell Signaling Technology Anti-Slug (1G7) Abcam Antivimentin (RV202) Abcam Anti-ZEB1 (4C4) Abnova Corporation Anti-ALDH1/2 (H-85) Santa Cruz Biotechnology Anti-CD24 (C-20) Santa Cruz Biotechnology Anti-GAPDH (14C10) Cell Signaling Technology Anti-E-cadherin (24E10) Cell Signaling Technology Anti-N-cadherin (D4R1H) XP Cell Signaling Technology Anti-Nanog (D73G4) XP Cell Signaling Technology Anti-OCT-4 (C30A3) Cell Signaling Technology Anti-KLF-4 (D1F2) Cell Signaling Technology Anti-Sox2 (D6D9) XP Cell Signaling Technology Anti-Bmi1 (D20B7) XP Cell Signaling Technology Anti-Stat3 (124H6) Cell Signaling Technology Anti-P-Stat3 (Tyr705) (D3A7) Cell Signaling Technology Anti-EpCAM (D1B3) Cell Signaling Technology Anti-CD44 Sigma-Aldrich Anti-AUF1 (hnRNPD/AUF1) Abcam Anti-p16 BD Biosciences Anti-mouse IgG, HRP conjugate Promega Anti-rabbit IgG, HRP conjugate Promega Donkey anti-goat IgG, HRP conjugate Promega Anti-cytokeratin 14 (LL002) Abcam Anti-cytokeratin 19 (EP1580Y) Abcam Anti-cytokeratin 18 (DC 10) Abcam Anticasein (F20.14) Abcam Alexa Fluor 594–goat anti-rabbit IgG Life Technologies Alexa Fluor 488–goat anti-mouse IgG Life Technologies FITC–mouse anti-CD90 BD Biosciences Anti-CD326 (EpCAM)-APC Miltenyi Biotec PE-Cy7–mouse anti-human CD10 BD Biosciences APC-Cy7–anti-mouse/human CD44 BioLegend FITC–mouse anti-CD24 BD Biosciences PE–rat anti-CD49 BD Biosciences FITC–mouse anti-MUC-1 BD Biosciences Open in a separate window a HRP, horseradish peroxidase; FITC, fluorescein isothiocyanate; APC, allophycocyanin; PE, phycoerythrin.

Techniques: Western Blot, Immunofluorescence, Immunohistochemistry