Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Hypoimmune induced pluripotent stem cell-derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice.

doi: 10.1073/pnas.2022091118

Figure Lengend Snippet: Fig. 1. Differentiation of B6 and B6HIP iPSCs into iCMs and iECs. (A) B6 iPSCs were differentiated into iECs and iCMs. (B) During the differentiation, the cells lost their typical pluripotent features of iPSCs and B6 iECs adopted genetic markers of endothelial cells and B6 iCMs of cardiomyocytes (representative gels of two independent experiments). (C and D) B6 iECs expressed Cd31 and VE-cadherin (C, representative pictures of five independent experiments) and formed tubular structures in vitro (D, representative pictures of five independent experiments). (E) B6 iCMs expressed alpha-sarcomeric actinin and troponin I (representative pictures of five independent experiments). (F) B6HIP iPSCs were differentiated into iECs and iCMs. (G) During the differentiation, the cells lost their typical pluripotent features of iPSCs and B6HIP iECs adopted genetic markers of endothelial cells and B6HIP iCMs of cardiomyocytes (representative gels of two independent experiments). (H and I) B6HIP iECs expressed Cd31 and VE-cadherin (H, representative pictures of five independent experiments) and formed tubular structures in vitro (I, representative pictures of five independent experiments). (J) B6HIP iCMs expressed alpha-sarcomeric actinin and troponin I (representative pictures of five independent experiments).

Article Snippet: The iEC phenotype was confirmed by immunofluorescence (IF) for expression of Cd31 (ab28364, Abcam), and VE-cadherin (sc-6458, Santa Cruz Biotechnology) with secondary antibodies conjugated with AF488 or AF555 (Invitrogen).

Techniques: In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Hypoimmune induced pluripotent stem cell-derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice.

doi: 10.1073/pnas.2022091118

Figure Lengend Snippet: Fig. 2. Allogeneic HIP iECs facilitate ischemic limb preservation. (A) In BALB/c mice, the superficial femoral artery was ligated and partially resected to induce left lower limb ischemia. (B) Mice were left untreated or received fan-shaped injections of allo or alloHIP iECs into the surrounding tissue. (C) The study protocol included assessment of iEC survival and laser Doppler perfusion imaging and histology after 28 d. (D and E) The survival of FLuc+ iEC grafts was longitudinally followed by BLI. All allo iEC grafts were rejected over 15 d (D, 5 animals), while all alloHIP iEC grafts survived and some grafts even showed proliferation (E, 15 animals). BLI signals of individual animals are plotted, representative pictures are shown. (F) Left lower extremity perfusion was serially assessed by laser Doppler imaging and showed an improvement over time only after transplantation of alloHIP iECs (mean ± SD, 15 animals with no cell injection, 5 animals in the allo group, and 15 animals in the alloHIP group; ANOVA with Bonferroni post hoc test). (G) The sequelae of CLI after 28 d were graded according to a standardized scoring system and showed improved limb preservation with alloHIP iEC treatment (parts of whole graphs, 15 animals with no cell injection, 5 animals in the WT group, 15 animals in the HIP group; Kruskal–Wallis test). (H) Immunofluorescence staining showed no engrafted FLuc+ allo iECs in allogeneic BALB/c recipients, but engraftment of FLuc+ alloHIP iECs located along the endothelial layer of larger and smaller intramuscular vessels. Costaining showed that transplanted cells retained their VE-cadherin expression (representative pictures of five independent experiments).

Article Snippet: The iEC phenotype was confirmed by immunofluorescence (IF) for expression of Cd31 (ab28364, Abcam), and VE-cadherin (sc-6458, Santa Cruz Biotechnology) with secondary antibodies conjugated with AF488 or AF555 (Invitrogen).

Techniques: Preserving, Imaging, Transplantation Assay, Injection, Immunofluorescence, Staining, Expressing

Journal: Developmental cell

Article Title: Acute brain vascular regeneration occurs via lymphatic transdifferentiation.

doi: 10.1016/j.devcel.2021.09.005

Figure Lengend Snippet: Figure 2. The transdifferentiating iLVs switch molecular and structural identities and are restricted to stand-alone iLVs (A–C) The lyve1b+kdrl BLECs in the uninjured control (arrows), but not the transdifferentiating lyve1b+kdrl+ iLVs at 3 dpt after injury (arrowheads), were positive for anti-Prox1 (A) and fluorescence in situ hybridization (FISH)-vegfr3 (B). The statistics show the ratios of vessels positive for anti-Prox1 or FISH-vegfr3 among all the lyve1b+kdrl vessels and all the lyve1b+kdrl+ vessels. (C) (n = 6 larvae; two-way ANOVA by Dunnett’s multiple comparisons test; ***, p < 0.0001). Scale bar, 20 mm. (D and E) Positive control brain BVs in the uninjured larvae (D), the lineage-tracing system indicated that the vessels double positive for GFP and Dendra2 (E, arrow) and single positive for Dendra2 (D and E) expressed the blood-brain barrier marker glut1b, but vessels single positive for GFP (D, arrowhead) did not. Scale bar, 20 mm. (F–H) Single FIB-SEM image planes (right row) indicate cross sections of the vessels shown in the left row. Note that the mural of the DsRed+Dendra2+ vessel (G) is similar to the DsRed-Dendra2+ BV (H) but much thicker than the DsRed+Dendra2 iLV (F). Color rings mark the inner and outer surfaces of murals. Arrows indicate blood cells. Scale bars, 20 and 1 mm. (I–L) Live imaging shows BLECs (I), stand-alone iLVs (J, arrows), and track iLVs (K) at 2 and 4 dpt. Note that only the stand-alone iLVs express Dendra2 and recruit the GFP+ pericytes at 4 dpt (J, arrowheads). (L) The statistics show the ratios of transdifferentiation in the stand-alone iLVs and track iLVs at 4 dpt (n = 9 larvae; two-tailed unpaired t test; ***, p < 0.0001). Scale bar, 50 mm. (M–P) The TUNEL signals in the stand-alone iLVs (M) and track iLVs (N) at 7 dpt. The statistics show the ratios of TUNEL+ cells in stand-alone iLVs and in track iLVs (O) (n = 9 larvae; ***, p < 0.0001), and the ratios of iLVs undergoing transdifferentiation (Dendra2+GFP+) or undergoing apoptosis (GFP+TUNEL+) among all the iLVs at 7 dpt (P) (n = 30 larvae; two-tailed unpaired t test; ***, p < 0.0001). Scale bar, 50 mm. Data are represented as mean ± SD. See also Figure S2.

Article Snippet: Antibody staining, combination of FISH and antibody staining Whole-mount antibody staining and combination of FISH and antibody staining were performed as previously described (Chen et al., 2019; Liu et al., 2016; He et al., 2019, 2020) using antibodies against DsRed (1:200, Santa-Cruz), GFP or Venus (1:500, Abcam and Santa Cruz), Dendra2 (1:500, Antibody-online), Prox1 (1:1000, Abcam), and phospho-EphB4a (1:250, Signalway Antibody).

Techniques: Control, In Situ Hybridization, Positive Control, Marker, Imaging, Two Tailed Test, TUNEL Assay

Journal: Developmental cell

Article Title: Acute brain vascular regeneration occurs via lymphatic transdifferentiation.

doi: 10.1016/j.devcel.2021.09.005

Figure Lengend Snippet: Figure 4. Defective EphB4a leads to derepression of Notch in the track iLVs (A–L) Triple labeling of anti-Dendra2, anti-GFP, and FISH-dll4 (A–C)/notch1a (E–G)/hey1 (I–K). In the siblings, dll4/notch1a/hey1 were activated in the Dendra2+GFP+ stand-alone iLVs (A, E, and I) and Dendra2+GFP nascent BVs (B, F, and J), but not in the Dendra2GFP+ track iLVs (B, F, and J). By contrast, in the ephB4a mutant, the Dendra2GFP+ track iLVs at 3 dpt also exhibited dll4, notch1a, and hey1 expressions (C, G, and K). The statistics show the dll4/notch1a/ hey1 expression in the vessels of sibling and ephB4a mutant (D, H, and L) (n = 6 larvae; two-tailed unpaired t test; ***, p < 0.0001). (M–P) The Notch functional reporter tp1:Venus was activated in the Dendra2+DsRed+ stand-alone iLVs (M) and Dendra2+DsRed nascent BVs (N), but not in the Dendra2DsRed+ track iLVs (N). By contrast, in the ephB4a mutant, the Dendra2DsRed+ track iLVs at 3 dpt also exhibited Venus expression (O). The statistics show the tp1:Venus expression in the vessels of siblings and ephB4a mutants (P) (n = 6 larvae; two-tailed unpaired t test; ***, p < 0.0001). Scale bar, 20 mm. Data are represented as mean ± SD. See also Figures S6 and S7.

Article Snippet: Antibody staining, combination of FISH and antibody staining Whole-mount antibody staining and combination of FISH and antibody staining were performed as previously described (Chen et al., 2019; Liu et al., 2016; He et al., 2019, 2020) using antibodies against DsRed (1:200, Santa-Cruz), GFP or Venus (1:500, Abcam and Santa Cruz), Dendra2 (1:500, Antibody-online), Prox1 (1:1000, Abcam), and phospho-EphB4a (1:250, Signalway Antibody).

Techniques: Labeling, Mutagenesis, Expressing, Two Tailed Test, Functional Assay

Journal: Developmental cell

Article Title: Acute brain vascular regeneration occurs via lymphatic transdifferentiation.

doi: 10.1016/j.devcel.2021.09.005

Figure Lengend Snippet: Figure 5. EphB4a represses track iLV transdifferentiation through suppression of Notch (A–C) The derepressed transdifferentiation of track iLV (Dendra2+DsRed+) in the ephB4a mutant (A, arrow) was rescued by treatment with DAPT, which returned to Dendra2DsRed+ (B, arrowheads). The statistics show the ratios of transdifferentiation among track iLVs in the ephB4a mutant with or without DAPT treatment (C) (n = 10 larvae; two-tailed unpaired t test; ***, p < 0.0001). (D–G) The derepressed transdifferentiation of track iLV (Dendra2+GFP+) in the ephB4a mutant without heat shock (D, arrow) or without dnMAML-Flag (E, arrow) was rescued by the heat-shock-induced, LEC-specific overexpression of dnMAML-Flag, which exhibited Dendra2GFP+ at 4 dpt (F, arrowhead). The statistics show the ratios of transdifferentiation among track iLVs in the ephB4a mutant groups (G) (n = 10 larvae; two-tailed unpaired t test; ***, p < 0.0001). Scale bar, 20 mm. Data are represented as mean ± SD. HS, heat shock. See also Figures S6 and S7.

Article Snippet: Antibody staining, combination of FISH and antibody staining Whole-mount antibody staining and combination of FISH and antibody staining were performed as previously described (Chen et al., 2019; Liu et al., 2016; He et al., 2019, 2020) using antibodies against DsRed (1:200, Santa-Cruz), GFP or Venus (1:500, Abcam and Santa Cruz), Dendra2 (1:500, Antibody-online), Prox1 (1:1000, Abcam), and phospho-EphB4a (1:250, Signalway Antibody).

Techniques: Mutagenesis, Two Tailed Test, Over Expression