Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: Identification and characterisation of ROR1 as a LEC marker. (A) Schematic diagram showing the three-stage lens differentiation protocol, with modification to enable ROR1-based purification of LECs. (B,C) ROR1 + cells cultured at high cell densities showed uniform polygonal morphologies that formed tightly packed monolayers (B). When cultured at low cell densities or passaged in medium containing only FGF2 (C), ROR1 + cells became large and vacuolated (arrow) with stress fibres (arrowheads; cells shown 18 days after plating; n =3). Scale bars: 100 µm. (D-G) Flow cytometry data showing expression levels of: ROR1 prior to (D) and after (E) ROR1-based purification; CRYAB after ROR1-based purification (F); and average expression levels before and after purification (G). (H) Relative mRNA transcript expression levels for PAX6, CRYAB and the lens fibre-specific gene CRYBB3 after ROR1 + cell separation (* P <0.05). (I) Pearson correlation showing high similarity (>0.96) between RNA-seq libraries generated from two independent ROR1 + cell samples. (J) Principal component analysis shows the ROR1 + RNA-seq transcriptomes are most similar to primary human LECs (circled). (K) Representative data from the ROR1 + RNA-seq libraries shows key genes required by LECs are expressed ( CRYAA , CRYAB , PAX6 , PROX1 , SOX2 , MEIS1 , MAB21L1 , BMP7 ). In contrast, genes expressed by lens fibre cells ( CRYBA1 , CRYBA2 ) or various endodermal cells ( GDF3 , VWF ), mesodermal cells ( T , GSC ), non-lens ectodermal cells ( RPE65 , NEUROD1 ) and pluripotent cells ( NANOG , POU5F1 ) are not expressed. Data shown in B,C and D-H are representative of 50 and four (respectively) independent differentiation experiments using four different hPSC lines; data are mean±s.e.m. in G,H.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Marker, Modification, Purification, Cell Culture, Flow Cytometry, Expressing, RNA Sequencing, Generated

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: Combinatorial growth factor screening identified media for ROR1 + cell expansion and differentiation. (A) Schematic diagram showing composition of the test media. FGF2 was included in the basal medium (TM32), with all combinations of the five other test pathways (eight growth factors) tested as shown [B, BMP4, BMP7; E, EGF, TGFα, H, HGF; I, insulin, IGF1; P, PDGF-AA; green square represents factor(s) present; red square represents factor(s) absent]. (B-I) Data from Hoechst-stained ROR1 + cells cultured in TM17 (B,E), TM30 (C,F) and TM32 (D,G) after seeding at low (B-D) and high (E-G) cell density, as well as average Hoechst-stained nuclei counts for all media (H,I). These data reveal that TM17 promoted expansion of ROR1 + cell cultures while maintaining expression of α- but not β-crystallins (see supplementary material Fig. S3 ). Scale bar: 20 µm. (J,K) Flow cytometry and light microscopy data show ROR1 + cells expanded, frozen, thawed and cultured for 6 days in TM17 retain high levels of CRYAB expression (J) with expected morphology (K) but without detectable expression of β-crystallins (see Table S1 and Fig. S4 ). Scale bar: 40 µm. (L,M) Light microscopy images show spontaneous production of lentoid-like structures after being expanded, frozen and thawed in TM17, then cultured in stage 2 lens differentiation medium. Cells in these cultures expressed α- and β-crystallins (see Table S2 and Fig. S3 ). Scale bars: 200 µm in L; 40 µm in M. The data shown in B-I are each representative of three independent differentiation experiments; data are mean±s.e.m. in H,I

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Staining, Cell Culture, Expressing, Flow Cytometry, Light Microscopy

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: ROR1 + cell aggregation leads to transparent and light-focusing micro-lenses. (A-Q) Light microscopy data from ROR1 + -cell aggregates and the maximal focal point below them. After 3 days of culture, the aggregates transmitted less light than the surrounding culture medium (A) and did not focus light (B). As culture progressed, the aggregates transmitted more light (C, day 7; E, day 14) and began focusing light (D, day 7; F, day 14). More-detailed characterisation of a single aggregate shows it had limited transparency (G) and focusing ability (I,K,M,O) on day 3 of culture, but by day 27 it transmitted the same amount of light as the surrounding culture medium (H) and had developed significant focusing ability (J,L,N,P). Quantification of the light transmittance and focusing ability confirms these findings (Q). Scale bar: 40 µm. The images are representative of five micro-lenses from two biological replicates; data in Q are mean±s.e.m.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Light Microscopy

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: Aggregation of ROR1 + cells induces lens fibre cell crystallin expression. (A) Real-time PCR analysis of aggregated ROR1 + cells results in decreased relative expression of PAX6 and CRYAB, and increased expression of CRYBB3 (* P <0.01; data obtained from four biological replicates and presented as mean±s.e.m.). (B-M) Immunofluorescence analysis shows that after 14 days of culture, αA-crystallin (C) was expressed uniformly throughout the bulk of the micro-lenses, whereas β-crystallin (E) and γ-crystallin (G) were not. After 24 days of culture, αA-crystallin (I), β-crystallin (K) and γ-crystallin (M) were all expressed relatively uniformly throughout the bulk of the micro-lens. The location of DAPI-stained nuclei within the day 14 (B,D,F) and day 24 (H,J,L) aggregates are shown. Scale bar: 40 µm. Each image is representative of five micro-lenses from two biological replicates.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: Evidence of progressive lens fibre cell differentiation in ROR1 + cell aggregates. Electron microscopy data from cultured aggregates. (A,B) A micro-lens cultured for 14 days shows a monolayer of LEC-like cells at the periphery of the tissue (A), and cells with small, rod-shaped nuclei (asterisk) and numerous organelles within the bulk of the tissue (B). (C) LEC-like cell with numerous organelles present at the periphery of a micro-lens after 24 days of culture. (D-G) Ultrastructural indicators of lens fibre cell differentiation within a micro-lens cultured for 42 days. (D) Ball-and-socket type membrane interdigitations (arrows) between adjacent lens fibre-like cells (inset shows a higher magnification of the region indicated with an arrow and asterisk). (E) A swollen mitochondria (arrow). (F) An enlarged nuclei with spoke-like nucleolus (inset). (G) A degraded nuclei with nuclear membrane visible as a series of vesicles (arrowheads). Scale bars: 5 µm in A-C; 2 µm in D,F,G; 0.5 µm in E. Images are representative of seven micro-lenses obtained from two biological replicates.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Cell Differentiation, Electron Microscopy, Cell Culture, Membrane

Journal: Development (Cambridge, England)

Article Title: Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro

doi: 10.1242/dev.155838

Figure Lengend Snippet: The CFTR potentiator Vx-770 inhibits light focusing in ROR1 micro-lenses. (A-D) Light microscopy data showing ROR1 + micro-lenses treated with DMSO-only (A) or 200 ng/ml Vx-770 (B) transmitted light at similar levels to the culture medium after 24 days of culture, whereas a micro-lens treated with 2000 ng/ml Vx-770 transmitted less light (C). Scale bar: 40 µm. Quantitative data are shown in D. (E-H) Light microscopy data showing ROR1 micro-lenses treated with DMSO-only (E) or 200 ng/ml Vx-770 (F) had developed focusing ability after 24 days in culture, whereas micro-lenses treated with 2000 ng/ml Vx-770 had not (G). Scale bar: 40 µm. Quantitative data are shown in H. (I-O) Light microscopy data showing micro-lenses treated after they had developed focusing ability. Micro-lenses treated with DMSO-only (I,L), 200 ng/ml Vx-770 (J,M) or 2000 ng/ml Vx-770 (K,N) all transmitted similar levels of light after 7 days of treatment (L-N) compared with before treatment (I-K). Scale bar: 40 µm. Quantitative data are shown in O. (P-V) Light microscopy data showing micro-lenses treated after having developed focusing ability. Micro-lenses treated with DMSO-only (S) and 200 ng/ml Vx-770 (T) retained focusing ability after 7 days of treatment compared with before treatment (P,Q, respectively). A micro-lens treated with 2000 ng/ml Vx-770 focused light prior to treatment (R) but did not after 7 days of treatment (U). Scale bar: 40 µm. Quantitative data are shown in V. In D,O,H,V, * P <1×10 −4 . The data shown (mean±s.e.m.) were each obtained from measurements of 15 micro-lenses from three biological replicates.

Article Snippet: The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec).

Techniques: Light Microscopy

Journal: bioRxiv

Article Title: Solid tumor-on-chip model for efficacy and safety assessment of CAR-T cell therapy

doi: 10.1101/2023.07.13.548856

Figure Lengend Snippet: a, Representative images on day 1 and 8 of the MDA-MB-231 aggregates or fibroblast spheroids after tumor-on-chip perfusion with either control T or CAR-T cells, showing different degrees of T cell infiltration within the tumor chamber. MDA-MB-231 tumor cells express GFP and are pseudo-colored in blue, fibroblasts—representing non-malignant tissue—were labeled with CellTracker TM CMFDA and pseudo-colored in cyan, whereas T and CAR-T cells were labeled in CellTracker TM Deep Red. Yellow-dashed line marks the region of interest of each tumor aggregate/fibroblasts spheroid. MvECs were present in all chips. Scalebars: 200 µm. b, Quantification of mean intensity values of the control T and CAR-T cells within each tumor aggregate’s/fibroblast spheroid’s region of interest at different time points after (CAR-)T cell perfusion. “CAR-T control” indicates the condition where CAR-T cells were perfused through chips containing ROR1 − fibroblasts spheroids; n = 7-21 MDA-MB-231 tumor aggregates/fibroblast spheroids from 3-4 chips. Data are depicted as mean with ± SEM. **p<0.01, ***p<0.001, ****p<0.0001 as assessed by Bonferroni’s multiple comparisons test. c, MDA-MB-231 tumor aggregate growth post CAR-T cell treatment in comparison to (control) T cell condition as measured by quantifying the difference of each MDA-MB-231 aggregate area at different time points compared to their initial area before (CAR-)T cell perfusion; n = 12-16 aggregates from 4 chips. Depicted are mean ± SEM. d, Quantification of mean GFP intensity fold change expressed on each MDA-MB-231 tumor aggregate on day 1 after the perfusion of either (control) T or CAR-T cells. Each dot represents one tumor aggregate and black line indicates the mean value; n = 12-14 aggregates from 4 chips. ****p<0.0001; two-tailed unpaired t test. e, The bar graph shows the quantification of control T or CAR-T cells per tumor chamber on day 1 after the tumor-on-chip was perfused at a concentration of 100,000 (CAR-)T cells/mL. Empty chip was used as control; n = 7-8 chambers from 3 chips. Data are depicted as mean with ± SEM. *p<0.05; Bonferroni’s multiple comparisons test. Scalebar: 200 µm. f, Representative images on day 1 after perfusing the CAR-T cells (red) at either 100,000 or 500,000 cells/mL concentration through the tumor-on-chip containing MDA-MB-231 aggregates (green). The focal plane was set on the aggregates for all the acquisitions. Scalebar: 200 µm. f, Flow cytometry histograms showing ROR1 expression (red) in the MDA-MB-231 cells and fibroblasts compared with the unstained control (cyan).

Article Snippet: For the analysis of the expression of extracellular markers, the following antibodies were used depending on the experiment: anti-human ROR1 APC (130-118-015; Miltenyi Biotec), anti-human CD31 FITC (130-110-806; Miltenyi Biotec), anti-human CD3 PE/Cyanine7 (317334; BioLegend), anti-human CD4 APC/Fire750 (357426; BioLegend), anti-human CD8a PerCP (300922; BioLegend), anti-human CD69 FITC (310904; BioLegend) and anti-human CD25 BV421 (302630; BioLegend).

Techniques: Control, Labeling, Comparison, Two Tailed Test, Concentration Assay, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Solid tumor-on-chip model for efficacy and safety assessment of CAR-T cell therapy

doi: 10.1101/2023.07.13.548856

Figure Lengend Snippet: a, Flow cytometry histogram plots showing ROR1 expression in the high-ROR1-expressing PDOs (red), low-ROR1-expressing PDOs (cyan) compared with the unstained control (orange). b, Representative images on day 8 of the ROR1-low and -high expressing PDOs after perfused with either (control) T or CAR-T cells. Fluorescently-labeled T cells are pseudo-colored in red. The focal plane was set on the PDOs for all the acquisitions. Scalebar: 500 µm. c, Quantification of mean intensity values of the control T (depicted as “T” in the graph) and CAR-T cells within the region of interest of each PDO on day 8. MDA-MB-231 aggregates were used as positive control; n = 14-20 PDOs from 3-4 chips. Data are depicted as mean with ± SEM. ns: not significant, *p<0.05, **p<0.01; Dunn’s multiple comparisons test. d, Representative images of the ROR1-high and ROR1-low expressing PDOs (cyan) before CAR-T cell (red) perfusion and on day 8 of the experiment. Yellow-dashed line marks the region of interest of each PDO. Scalebar: 200 µm. e, Quantification of the levels of cytokines IL-2, IL-6, IL-10, TNF-α, IFN-γ and granzyme B in the effluents of the chips after 20 h of (control) T or CAR-T cells perfusion through the tumor-on-chip containing either MDA-MB-231 aggregates (positive control for CAR-T cell treatment), ROR1-high PDOs or ROR1-low PDOs; n = 3-4 chips. Data are depicted as mean with ± SEM. Each red dot represents one chip. f, Cytokine release kinetics of the abovementioned cytokines from day 1 to 6 after CAR-T cells perfusion from day 0 to 1 through the tumor-on-chip containing either MDA-MB-231 aggregates (positive control for CAR-T cell treatment), ROR1-high PDOs or ROR1-low PDOs; n = 4 chips. Data are depicted as mean with ± SEM. Dashed line indicates the lower limit of detection. Data points from day 8 are excluded as the values are mostly below the detection limit.

Article Snippet: For the analysis of the expression of extracellular markers, the following antibodies were used depending on the experiment: anti-human ROR1 APC (130-118-015; Miltenyi Biotec), anti-human CD31 FITC (130-110-806; Miltenyi Biotec), anti-human CD3 PE/Cyanine7 (317334; BioLegend), anti-human CD4 APC/Fire750 (357426; BioLegend), anti-human CD8a PerCP (300922; BioLegend), anti-human CD69 FITC (310904; BioLegend) and anti-human CD25 BV421 (302630; BioLegend).

Techniques: Flow Cytometry, Expressing, Control, Labeling, Positive Control