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Figure 3 | Stem Cell Research & Therapy

Figure 3

From: Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cells

Figure 3

Characterization of transgene-free induced pluripotent stem cells. (A) Pluripotency analysis of transgene-excised clones. del-ARiPSCs stain positive for pluripotency-associated markers Oct4 and SSEA-4. Scale bar: 40 μm. (B) PluriTest analysis of HES I3, fl-ARiPSC, del-ARiPSC cells and parental fibroblasts to assess pluripotency induction. Cells are distributed based on pluripotency and novelty scores, as indicated by color density background. Red, pluripotency; blue, nonpluripotency. (C) Heat-map representation of pluripotency and fibroblast-specific markers. Expression of pluripotency-associated markers Oct4, Sox2 and Rex1 and fibroblast specific markers Thy1 and Col5a2 in induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) used for this study. (D) Venn diagram showing differentially expressed genes. Comparison of differentially expressed genes amongst HES I3, fl-ARiPSC and del-ARiPSC cells. (E) Histological analysis of teratoma analysis of excised clone. Teratoma analysis after injection of the excised clone into SCID mice showed formation of all three germ layers: ectoderm (primitive neuroepithelium), mesoderm (cartilage, muscle) and endoderm (glands). (F) Karyotype analysis of transgene-excised clone. Cytogenetic analysis on G-banded metaphase cells from excised clone (p13) and all 20 cells demonstrated a normal male karyotype. HES I3, human ESC I3 line; fl-ARiPS, fl-ARiPS iPSC clone 1; del-ARiPS, transgene-free daughter clones of ARIPS iPSC clone 1.

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