Volume 12, Issue 1, Pages (July 2005)

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Volume 12, Issue 1, Pages 28-32 (July 2005) Cellular Manipulation of Human Embryonic Stem Cells by TAT-PDX1 Protein Transduction Young Do Kwon, Sun Kyung Oh, Hee Sun Kim, Seung-Yup Ku, Seok Hyun Kim, Young Min Choi, Shin Yong Moon Molecular Therapy Volume 12, Issue 1, Pages 28-32 (July 2005) DOI: 10.1016/j.ymthe.2005.03.010 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Transduction of TAT-FITC peptide into three hESC lines. Fluorescence confocal microscopy of (A) day 4 colonies of hESCs and (B) day 1 EBs after treatment of control free FITC and TAT-FITC at the concentration of 50 μM for 1 h. (A) Original magnification, ×100; scale bars, 200 μm. (B) Original magnification, ×200; scale bars, 100 μm. (C) Flow cytometry analysis of day 1 EBs treated with control free FITC (open region) and TAT-FITC (gray region) at the concentration of 50 μM for 1 h. The EBs were treated with trypsin and the dissociated cells were washed with PBS and analyzed. Molecular Therapy 2005 12, 28-32DOI: (10.1016/j.ymthe.2005.03.010) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Transduction of TAT-PDX1 protein into EBs. (A) Schematic representation of PDX1 and TAT-PDX1 proteins. Gray box represents the Antennapedia-like protein transduction domain in the PDX1 protein. (B) Purity of recombinant proteins. Purified proteins were run on a 10% gel and stained with Coomassie Brilliant Blue R solution. Positions of molecular weight markers are indicated. Lane 1, PDX1; lane 2, TAT-PDX1. (C) Western blotting of cell lysates of EBs treated with TAT-PDX1 or PDX1 protein at a concentration of 0.2 μM. Mouse anti-Penta·His HRP conjugate and the enhanced chemiluminescence system were used to probe the proteins. No signal was detected in the case of PDX1. (D) Stability of TAT-PDX1 and PDX1 proteins in culture medium. Day 1 EBs were treated with TAT-PDX1 or PDX1 protein at a concentration of 0.2 μM and the culture media were prepared at the indicated times. Western blotting was performed to detect the proteins. Molecular Therapy 2005 12, 28-32DOI: (10.1016/j.ymthe.2005.03.010) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Transcriptional activation of pancreatic genes by transduced TAT-PDX1 protein. (A) Real-time RT-PCR analysis of Pdx1, insulin, IAPP, and GLUT2 mRNA levels. Total RNA quantity was normalized to GAPDH mRNA level. Day 1 EBs were treated with TAT-PDX1 and control PDX1 for 1 week with daily exchange of medium. (B) Immunofluorescence staining with mouse anti-His-tag antibody and rabbit anti-human C-peptide antibody. Day 1 EBs were treated with TAT-PDX1 and PDX1 for 1 week and were attached to Matrigel-coated coverglasses. Original magnification, ×400; scale bars, 40 μm. Molecular Therapy 2005 12, 28-32DOI: (10.1016/j.ymthe.2005.03.010) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions