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