EMBO/soda-nel huggingface dataset:
Genetically engineered distal airway stem cells transplantation protects mice from pulmonary infection (2019)
Figure 1

Figure 1. Constitutive expression of LL-37 protected mouse lung from bacterial infection.

A．The schematic of human CAMP transgenic mouse strain. B. LL-37(4 kD) detection by Western blotting. Prior to loading, samples were centrifuged through 10 kD ultrafiltration membranes and the equal amount of ultrafiltrate (19μg/lane) subject to immunoblotting. High-molecular-weight proteins (GAPDH) were not detected in ultrafiltrate. C. The CFU of PAO1 was measured by culturing in ultrafiltrate (upper panel) or retentate (lower panel) of mouse BALF samples from indicated mice. Initial additions of PAO1 were 1 × 103 CFU. Coculture duration, 6h. n=3. D. The bacterial CFU (per gram) in lungs of indicated mice with and without PAO1 infection (5 × 106 CFU) for 6, 24 and 48 hours. n=3. Error bars, S.E.M. E. Representative histological sections of indicated lungs with PAO1 infection (5 × 106 CFU) for 6 hours. H&E staining. Scale bar, 1000 μm (upper panel) and 50 μm (lower panel). F. Histopathological injury score of indicated mouse lungs with PAO1 infection (5 × 106 CFU) for 6 h and 24 h based on blinded expert judgement. n=3. Error bars, S.E.M. G. Gene expression level of IL-1β and IL-6 of indicated mouse lung with PAO1 infection (5 × 106 CFU) for 6 h and 24 h. n≥3. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Genetically engineered distal airway stem cells transplantation protects mice from pulmonary infection (2019)
Figure 2

Figure 2. The altered transcriptomic profiles of transgenic mouse lungs before and after PAO1 infection.

A. Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection. B. Histogram of selected differentially expressed genes of LL-37+/+ mouse lung versus wild type FVB mouse lung prior to infection. Blue bars indicated genes up-regulated in wild type FVB mouse lungs, red bars indicated genes up-regulated in LL-37+/+ mouse lungs. C-D. Enriched gene ontology classes of uninfected (C) and PAO1-infected (D) lungs. Red bar, GO class of up-regulated gene in LL-37+/+ mice. Blue bar, GO class of up-regulated gene in wild type FVB mouse lung. GO terms were ranked by the enrichment P-value. E. Protein-protein interaction network of selected genes with high expression level in PAO1-infected wild-type lung (blue) and PAO1-infected LL-37+/+ lung (red), respectively.
Genetically engineered distal airway stem cells transplantation protects mice from pulmonary infection (2019)
Figure 3

Figure 3. Engineered mDASCs possessed normal stem cell properties and enhanced antimicrobial potency.

A-C. Detection of LL-37 expression in the engineered mDASCs by immunofluorescence (A), real-time quantitative PCR (B) and Western blot (C). Scale bar, 50 μm. BF, bright field. n=10. Error bars, S.E.M. D. Anti-Krt5 (red) and anti-P63 (green) immunostaining of WT- and LL-37-mDASC colonies. Scale bar, 70 μm. E. Stem cell colony-forming efficiency of WT- and LL-37-mDASCs during 5 serial passages. n=6. F. Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers (Aqp5 and Pdpn). Left panels, bright field imaging of 3D organoids. Right panels, immunofluorescence of organoid sections. Scale bar, 20 μm. G. Co-culture of bacteria with DASC shows the antimicrobial effects in dose-dependent manner. Initial additions of PAO1 were 0.1×, 0.5× and 1 × 104 CFU, respectively. Coculture duration, 6h. n=4. Error bars, S.E.M. MOI, Multiplicity of infection. H. Co-culture of bacteria with DASC shows antimicrobial effects in time-dependent manner. Initial additions of PAO1 was 1 × 104 CFU. MOI=1. n=3. Error bars, S.E.M. I-J. Preincubation of cells with anti-LL-37 antibody, but not IgG control, significantly reduced anti-PAO1 (I) and anti-E.coli (J) effects of LL-37-mDASCs. Initial dose of bacteria was 103 CFU. Coculture duration, 18h. n=4 in I and n=3 in J. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Genetically engineered distal airway stem cells transplantation protects mice from pulmonary infection (2019)
Figure 4

Figure 4. Lung engraftment of WT- and LL-37-mDASCs after orthotopic transplantation.

A. Bright field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP-labeled WT-mDASCs (WT-Lung) or LL-37-mDASCs (LL-37-Lung) on indicated days. Scale bar, 1000 μm B. Engraftment ratio of transplanted cells in mouse lungs on indicated days. n=3. Error bars, S.E.M. *P=0.0474. C. Morphology of engrafted GFP-labeled cells in lung parenchyma by direct fluorescence. Blue color indicates nucleus DAPI staining. Scale bar, 200 μm. D. Anti-Ki67 immunofluorescence of engrafted GFP-labeled WT- and LL-37-mDASCs in lung parenchyma 21 days after transplantation. Scale bar, 50 μm.
Genetically engineered distal airway stem cells transplantation protects mice from pulmonary infection (2019)
Figure 5

Figure 5. LL-37-expressing lung had enhanced host defense ability.

A. Distribution of engrafted GFP-labeled cells in lung parenchyma by immunofluorescence 7 days after transplantation. WT-Lung: WT-mDASCs engrafted; LL-37-Lung: LL-37-mDASCs engrafted. Scale bar, 200 μm. Arrows showed the representative cells with overlapping fluorescence of GFP and LL-37. B. Intratracheal instillation of equal amount of PAO1(5 ×106 CFU per mouse) into WT-Lung (WT-mDASCs engrafted) and LL-37-Lung (LL-37-mDASCs engrafted) followed by bacterial CFU analysis in whole lung homogenates 6, 24 and 48 hours after infection. n=3. Error bars, S.E.M. C. Intratracheal instillation of equal amount of PAO1 into WT-Lung and LL-37-Lung followed by bacterial CFU analysis in BAL fluid 6, 24 and 48 hours after infection. n=3. Error bars, S.E.M. D. Down-regulated gene in LL-37-Lung enriched in Gene ontology and KEGG pathways. E. Protein-protein interaction network of selected genes with high expression level in WT-Lung (red) and LL-37-Lung (blue), respectively. F. Arterial blood gas analysis of mice with WT-Lung and LL-37-Lung following PAO1 infection. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Genetically engineered distal airway stem cells transplantation protects mice from pulmonary infection (2019)
Figure 6

Figure 6. LL-37-mDASCs transplantation protected infected mouse from pulmonary inflammation.

A. H&E staining showing histology of injured lung infected by PAO1 with WT-mDASCs or LL-37-mDASCs transplantation. Scale bar, 100 μm. B. Histopathological examination according to the lung injury scoring system based on blinded expert judgement. n=5 mice per group. Error bars, S.E.M. C. CD68 immunochemistry (brown) in infected lung with WT-mDASCs or LL-37-mDASCs transplantation. Scale bar, 50 μm. D. Quantification of brown-stained (CD68+) area by Image J software. n=5. Error bars, S.E.M. E. Gene expression level of indicated pro-inflammatory cytokines of lung infected by PAO1 with WT-mDASCs or LL-37-mDASCs transplantation. n=3. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Genetically engineered distal airway stem cells transplantation protects mice from pulmonary infection (2019)
Figure 7

Figure 7. Lung scaffold recellularized by LL-37 engineered human DASCs has enhanced anti-bacteria ability.

A. Anti-Krt5 (red) and anti-P63 (green) immunofluorescence of isolated hDASCs. Scale bar, 90 μm. B. The mRNA expression levels of LL-37 was measured by qPCR for WT- and LL-37-hDASCs. n=5. Error bars, S.E.M. C. H&E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization. Scale bar, 40 μm. D. Ex vivo biomimetic culture of LL-37-hDASCs recellularized lungs with constant media perfusion. E. Direct fluorescence image showing GFP-labeled LL-37-hDASCs before (left) and 7 days after (right) being engrafted onto the decellularized scaffold. Scale bar, 100 μm (right panel) and 1000 μm (right panel) F. Immunofluorescence of recellularized lung with indicated antibodies. Blue color indicates nucleus DAPI staining. Scale bar, 50 μm. G. The recellularized lung by LL-37-hDASCs displayed growth inhibitory effect on PAO1 and E.coli. Initial dose of bacteria was 2× 104 CFU. Culture duration, 24h. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Expanded View legends.

Table EV1. Lists of top 50 differently expressed genes between indicated experimental groups by RNA-Seq analysis on WT or LL-37+/+ lungs before or after PAO1 infection.
Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys (2022)
Figure 1

Figure 1. Fetal ovary development in cynomolgus monkeys.

(A) Scheme of female germ cell development in cynomolgus monkeys. Wpf, weeks post-fertilization; PGCs, primordial germ cells.

(B) Gross appearances of developing cy ovaries at 8−18 wpf. Scale bar = 1 mm.

(C) Hematoxylin and eosin (H&E) staining of a cy fetal ovary at 8 wpf. Arrows indicate oogonia. M, mesonephros. Scale bars = 100 μm (top), 40 μm (bottom).

(D) H&E staining of the ovarian cortex in cy fetuses at 10−18 wpf. The ovarian cortex was divided into the outer and inner cortex at the line bisecting the ovarian cortex. Black arrows, oocytes bearing a large nucleus with condensed, thread-like chromatin; white arrows, primordial follicles. Scale bar = 50 μm.

(E) Immunofluorescence (IF) analysis for oogonia (POU5F1, TFAP2C), RA responsive (ZGLP1, STRA8), meiotic (γH2AX, SYCP1), and oogenic (ZP3, TP63) markers during cy fetal ovary development. Representative images for each marker are shown. Germ cells were marked with DDX4 (magenta). Nuclear DAPI staining is shown in white. The upper-right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate the stage (wpf) of the fetus. Scale bar = 20 μm.

(F) Representative images of germ cells immunostained for SYCP3 (green), DMC1 (red), and DDX4 (magenta) at each meiotic sub-stage. Nuclear DAPI staining is shown in white. The sub-stages of meiosis were defined based on the staining patterns of SYCP3, DMC1, and DAPI as described in Materials and Methods. Scale bar = 10 μm.

(G) Percentages of germ cells at each meiotic sub-stage as defined in Fig 1F. The average percentages in the outer or inner ovarian cortex on each meiotic sub-stage were calculated from at least three fetal ovaries [8 wpf (n=4), 10/12/14/16/18 wpf (n=3)]. The color-coding is as indicated.
Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys (2022)
Figure 2

Figure 2. Derivation of cy ovarian follicles from fetal gonadal cells by xenotransplantation.

(A) Scheme of the xenotransplantation experiment. The cy rOvaries generated from in vivo ovarian cells at 8 wpf were transplanted beneath the kidney capsule of immunodeficient KSN/Slc mice (see Materials and Methods).

(B) Gross appearances (a), H&E staining (b), and IF for FOXL2/DDX4/DAPI (c) of transplanted cy rOvaries at 3/6/9/12/15 weeks post-transplantation (w-ptp). The primordial follicle-like complexes surrounded with FOXL2+ granulosa cells were observed inside the rOvaries at 12 and 15 w-ptp. Arrows indicate the rOvaries. FOXL2, granulosa cell marker; DDX4, germ cell marker. Scale bars = 1 mm (a), 40 μm (b and c).

(C) IF for oogonia (POU5F1, TFAP2C), meiotic (SYCP3, SYCP1), and oogenic (ZP3, TP63) markers in the transplanted cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of each marker (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate weeks after transplantation (w-ptp). Scale bar = 20 μm.

(D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3−15 w-ptp [3 w-ptp (n=3), 6/9/12 w-ptp (n=4), 15 w-ptp (n=6)]. The average values with SDs are shown.

(E) H&E staining of transplanted cy rOvaries at 21 w-ptp. Dashed lines indicate the transplanted cy rOvaries beneath the kidney capsule of KSN/Slc mice. K, mouse kidney. Scale bars = 200 μm (left) and 100 μm (right).
Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys (2022)
Figure 3

Figure 3. In vitro culture system for inducing cy ovarian follicles from fetal gonadal cells.

(A) Scheme of the in vitro culture system for inducing cy ovarian follicles. The cy rOvaries generated from fetal gonadal cells at 8 wpf were cultured in either an air-liquid interface or a floating condition (see Materials and Methods).

(B) Gross appearance (a), H&E staining (b, c), and IF for FOXL2/DAZL/DAPI (d) of cy rOvaries cultured in the air-liquid interface condition at 3 and 6 weeks of in vitro culture (w-ivc). FOXL2, granulosa cell marker; DAZL, germ cell marker; DAPI, nucleus. Scale bars = 200 μm (a), 100 μm (b), and 40 μm (c and d).

(C) Quantitative analysis for the size of cy rOvaries cultured in Advanced MEM or αMEM as the basal media of P-IVD medium. The mean values from more than three biological replicates are shown with SDs except for the rOvary in αMEM at 15 w-ivc [0/1/2/3/6/9/12/15 w-ivc (αMEM, n=26/23/14/23/20/9/4/1; Advanced MEM, n=14/14/14/14/9/8/7/4)]. **P < 0.01, Welch's T-test.

(D) Gross appearance (a) and H&E staining (b) images of cy rOvaries cultured with the P-IVD medium at 3/6/9/12/15 w-ivc. Arrowheads indicate primordial follicle-like structures. Scale bars = 200 μm (a) and 40 μm (b).

(E) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate the period of IVC (w-ivc). Scale bar = 20 μm.

(F) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3-15 w-ivc [3/6/9/12 w-ivc (n=3), 15 w-ivc (n=3 or 4)]. The average values with SDs are shown.

(G) Percentages of germ cells at each meiotic sub-stage as defined in Fig 1F (see also Materials and Methods). The average percentages of each meiotic sub-stage were determined from at least three cy rOvaries [3/6/9/12 w-ivc (n=3), 15 w-ivc (n=4)].
Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys (2022)
Figure 5

Figure 5. Derivation of human ovarian follicles from oogonia in vitro.

(A) Gross appearance (a), H&E staining (b), and IF (c) for FOXL2/DDX4/DAPI of the human in vivo female gonads (11W, left column) and the cultured rOvaries at 7 and 14 w-ivc. FOXL2, granulosa cell marker; DDX4, germ cell marker; DAPI, nucleus. Scale bars = 1 mm (a, left), 200 μm (a, middle/right), 50 μm (b), and 20 μm (c). g.a., gestational age.

(B and C) IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers for human in vivo female gonad (11W, B) and human in vitro cultured rOvaries at 14 w-ivc (C). Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). Scale bars = 20 μm.

(D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of human in vivo female gonads (11W) and human cultured rOvaries at 7 and 14 w-ivc. The mean values from two biological replicates are shown.

(E and F) Analyses for human germ cells by SC3-seq. (E) UHC for human germ cells in vivo and in rOvaries with all expressed genes (20,048 genes) and a heatmap of the expression levels of selected marker genes. Color bars under the dendrogram show the in vivo stage (1st bar), the period of IVC (2nd bar), and the cell type (3rd bar). The heatmap color-coding is as indicated. The color coding in bars is as indicated in Fig 5F. (F) PCA of human germ cells in vivo and in rOvaries performed by SC3-seq. The cells are plotted in the two-dimensional plane defined by PC1 and PC2 values. The color-coding is as indicated.

(G) PCA plot of all cy and human SC3-seq data merged by canonical correlation analysis (CCA). Cell types were defined after CCA followed by Louvain clustering. The color and shape coding are as indicated.
Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys (2022)
Figure 6

Figure 6. Cross-species comparison analysis of fetal female germ cell development in vivo in humans, monkeys, and mice.

(A-F) Analyses performed by 10X scRNA-seq. (A) UMAP plot of germ cells from six 10X scRNA-seq datasets (two for humans [this study and (Chitiashvili et al., 2020)], one for monkeys [this study], and three for mice [(Ge et al., 2021, Niu & Spradling, 2020, Zhao et al., 2020)]) integrated by CCA. Meiotic sub-stages were defined after CCA followed by Louvain clustering. Colors indicate the computationally assigned meiotic sub-stages defined by the expression levels of key meiotic markers conserved in all three species (see Materials and Methods). The color-coding is as indicated. M, mitotic; PL, pre-leptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene. (B) The UMAP plot highlighting cells from each dataset. The color-coding for each developmental stage is as indicated. (C) Percentages of germ cells belonging to each meiotic sub-stage. The color-coding for the germ cell stage is as indicated in Fig 6A. (D) The distribution of germ cells along the individually calculated species-specific pseudotime trajectories (Monocle 3). The color-coding for the germ cell stage is as indicated in Fig 6A. (E) Heatmap of the Pearson correlation coefficients of the average expression levels of 237 HVGs (1-1-1 orthologues) among the meiotic sub-stages in humans, monkeys, and mice (see Materials and Methods). The color-coding for the germ cell stage is as indicated. (F) Heatmap of the standardized expression levels of selected genes using 10X scRNA-seq data in all three species. Genes showing specific expression changes in humans and monkeys, but not in mice, during female germ cell development were manually selected from the list of DEGs acquired from the SC3-seq analysis for cy/human in vitro cultured rOvaries (Dataset EV6). The heatmap color-coding is as indicated. The color-coding for the germ cell sub-stage is as indicated in Fig 6E.

(G and H) IF analyses of PAX6 and CLGN with TFAP2C (oogonia marker) and DDX4 (germ cell marker) in cy in vivo fetal ovaries at 12 wpf (G) and human cultured rOvaries at 7 w-ivc (H). Nuclear DAPI staining is shown in white. Scale bars = 20 μm.

(I) IF analyses of CLGN with TP63 (oogenic marker) and DDX4 (germ cell marker) in a 13-year-old human oocyte of a primordial follicle. Nuclear DAPI staining is shown in white. Scale bar = 20 μm.
Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys (2022)
Figure 7

Figure 7. X:A ratios and XIST expression during germ-cell development in humans, monkeys, and mice.

(A) The Chr.3:A and X:A ratios (top), and Xist expression (bottom) in mouse fetal/neonatal gonadal cells analyzed by bulk RNA-seq. Dot, each datum; bar, mean. Samples at E9.5/10.5 include germ cells from male and female embryos (mix). E, embryonic day; P, postnatal day; Soma, gonadal somatic cells.

(B) The Chr.15:A and X:A ratios (top), and XIST expression (bottom) in cy female germ cells in vivo and in cultured/transplanted rOvaries analyzed by SC3-seq. The Chr.15:A/X:A ratio transitions in the embryonic and hypoblast lineages reported in (Okamoto et al., 2021) are also shown. ESC, embryonic stem cell; ICM, inner cell mass; Pre_EPI, pre-implantation epiblast; PostE_EPI, post-implantation early epiblast; PostL_EPI, post-implantation late epiblast; Gast, gastrulating cells; ePGC, early primordial germ cells. Data with small sample size (n≦3) are indicated with dots (each datum) and bars (mean).

(C) The Chr.10:A and X:A ratios (top), and XIST expression (bottom) in human female germ cells in vivo and in cultured rOvaries analyzed by SC3-seq.

(D) The autosome:A and X:A ratios (top), and XIST/Xist expression transitions (bottom) during female germ cell development in vivo in all three species analyzed by 10X scRNA-seq.

Data information: In the boxplots, the central bands represent the median values; the lower/upper hinges represent the 25th/75th percentiles, respectively; the upper limits of the whiskers represent the largest values no further than 1.5 IQR (interquartile range) from the upper hinges; the lower limits of the whiskers represent the smallest values no further than 1.5 IQR from the lower hinges; the dots represent the outliers.

LEGENDS TO EXPANDED VIEW FIGURES