Figure 2.
Analysis of platelet parameters and abnormal megakaryopoiesis in RUNX1/AAVS1 competitive repopulation animals. (A) Platelet counts for animal 1 and animal 2 plotted in comparison with CRISPR control animals. The dotted lines indicate the upper and lower range of normal RM platelet counts (2 SDs from mean). (B) Sections of BM core biopsies obtained from animal 1 and 2 and a CRISPR control animal after transplantation were stained with H&E (upper panel) or via immunohistochemistry for the megakaryocyte marker CD61 (lower panel). Representative microscopic images are shown. CD61+ megakaryocytes stain bright pink. (C) Platelet aggregation results for the RUNX1/AAVS1 and CRISPR control animals. (Animal 1: 23 months–after transplantation, Animal 2: 17 months after transplantation). (D) Mpl (TPO receptor) expression on platelets for animals 1 and 2 and for CRISPR control animals by flow cytometry. Right panel shows relative MFI. (Animal 1: 20 months after transplantation, Animal 2: 14 months after transplantation). (E) Circulating TPO concentrations in PB plasma of RUNX1/AAVS1 animals 1 and 2 and CRISPR control animals by ELISA. CRISPR control animal 1 was used for statistical analyses. (Animal 1: 22 months after transplantation, Animal 2: 16 months after transplantation). (F) Targeted deep sequencing on CD34+ HSPCs, Gr, and CD41a+ megakaryocytes isolated from BM (Animal 1: 15 and 17 months after transplantation, Animal 2: 9 and 11 months after transplantation). The percentages of reads containing indels at the RUNX1 or AAVS1 target sites are shown. (G-I) BM-derived purified CD34+ cells from animals 1 and 2 and controls were differentiated into MKs in vitro for 14 days and analyzed by flow cytometry. (G) CD41a expression. (H) The percentage of each ploidy class within the in vitro differentiated CD41a+ MKs is shown. (I) Each ploidy subpopulation was sorted for targeted deep sequencing. The percentage of reads containing indels at the RUNX1 or AAVS1 target sites are shown. Bar represents 20 μm (B). ELISA, enzyme-linked immunosorbent assay; Gr, granulocyte; H&E, hematoxylin and eosin stain; MFI, mean fluorescence intensity; SD, standard deviation.

Analysis of platelet parameters and abnormal megakaryopoiesis in RUNX1/AAVS1 competitive repopulation animals. (A) Platelet counts for animal 1 and animal 2 plotted in comparison with CRISPR control animals. The dotted lines indicate the upper and lower range of normal RM platelet counts (2 SDs from mean). (B) Sections of BM core biopsies obtained from animal 1 and 2 and a CRISPR control animal after transplantation were stained with H&E (upper panel) or via immunohistochemistry for the megakaryocyte marker CD61 (lower panel). Representative microscopic images are shown. CD61+ megakaryocytes stain bright pink. (C) Platelet aggregation results for the RUNX1/AAVS1 and CRISPR control animals. (Animal 1: 23 months–after transplantation, Animal 2: 17 months after transplantation). (D) Mpl (TPO receptor) expression on platelets for animals 1 and 2 and for CRISPR control animals by flow cytometry. Right panel shows relative MFI. (Animal 1: 20 months after transplantation, Animal 2: 14 months after transplantation). (E) Circulating TPO concentrations in PB plasma of RUNX1/AAVS1 animals 1 and 2 and CRISPR control animals by ELISA. CRISPR control animal 1 was used for statistical analyses. (Animal 1: 22 months after transplantation, Animal 2: 16 months after transplantation). (F) Targeted deep sequencing on CD34+ HSPCs, Gr, and CD41a+ megakaryocytes isolated from BM (Animal 1: 15 and 17 months after transplantation, Animal 2: 9 and 11 months after transplantation). The percentages of reads containing indels at the RUNX1 or AAVS1 target sites are shown. (G-I) BM-derived purified CD34+ cells from animals 1 and 2 and controls were differentiated into MKs in vitro for 14 days and analyzed by flow cytometry. (G) CD41a expression. (H) The percentage of each ploidy class within the in vitro differentiated CD41a+ MKs is shown. (I) Each ploidy subpopulation was sorted for targeted deep sequencing. The percentage of reads containing indels at the RUNX1 or AAVS1 target sites are shown. Bar represents 20 μm (B). ELISA, enzyme-linked immunosorbent assay; Gr, granulocyte; H&E, hematoxylin and eosin stain; MFI, mean fluorescence intensity; SD, standard deviation.

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