1. Safeguarding Nonhuman Primate iPS Cells With Suicide Genes
Bonan Zhong, Korashon L Watts, Jennifer L Gori, Martin E Wohlfahrt, Joerg Enssle, Jennifer E Adair, Hans-Peter Kiem 
Molecular Therapy 
Volume 19, Issue 9, Pages (September 2011)
DOI: /mt
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Construction and functional test of suicide gene vectors. (a) Schemes of iCaspase and YCD lentiviral vectors and control eGFP vector. (b) Supplementation of 0.5 µmol/l AP20187 killed HT1080iCaspase cells but not HT1080GFP cells in an 8-day assay. (c) Bar graph: All three clones of 293TYCD cells exhibited a lower percentage of cell survival (7AAD−) with the supplementation of 5 mmol/l 5-FC (black bars) compared to 293TYCD cells without the drug (white bars). 5-FC, 5-fluorocytosine; eGFP, enhanced green fluorescent protein; PGK, phosphoglycerate kinase; SFFV, spleen focus-forming virus; YCD, yeast cytosine deaminase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Introduction of suicide genes or the control eGFP vector did not alter the pluripotency of iPS cells in vitro. (a) iPSiCaspase, iPSYCD, and iPSGFP cell colonies retain undifferentiated morphology with well-defined edges. Bars = 50 µm. (b) iPSiCaspase, iPSYCD, and iPSGFP cell colonies expressed pluripotency markers Oct4, Nanog and Tra-1-60 and were alkaline phosphatase positive. Bars = 100 µm. (c) GFP+ iPSYCD could be differentiated into CD34+ or CD45+ hematopoietic precursors via embryoid bodies formation. Bars = 100 µm. (d) GFP+ iPSYCD hematopoietic progenitor cells (HPCs) formed both erythroid-type and myeloid-type colonies in methylcellulose-based colony forming units assay on day 20. Colonies were further assayed with CD13, CD14, CD45, and CD34 by flow cytometry (n = 2). Bars = 100 µm. (e) Giemsa staining of individually cytospun colonies from iPSYCD-HPCs revealed cell morphologies consistent with various white blood cells. Bars = 50 µm. (f) GFP+ iPSYCD cells can be directly differentiated into neural precursors indicated by morphology and A2B5+ staining. BF, bright field; DAPI, 4′,6-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; iPS, induced pluripotent stem; PE, phycoerythrin; YCD, yeast cytosine deaminase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Suicide genes did not alter the self-renewal capacity of iPS cells. (a) iPSiCaspase, iPSYCD, and iPSGFP cells retained clonogenicity after single cell dissociation and GFP sorting. Lower panel shows bright field and fluorescein isothiocyanate fluorescence overlaid image of the emerged colonies 3 days after sorting, n = 3. Bars = 100 µm. (b) Total cell numbers of GFP+ iPSiCaspase, iPSYCD, and iPSGFP cells over 3 weeks in feeder-free culture, n = 3. eGFP, enhanced green fluorescent protein; iPS, induced pluripotent stem; YCD, yeast cytosine deaminase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Suicide genes efficiently and specifically controlled the cell fate of iPS cells in vitro. (a) Supplementation of 2 µmol/l AP20187 to iPSiCaspase or 5 mmol/l 5-FC to iPSYCD cells respectively induced apoptosis indicated by the shrunken morphology of iPS-derived colonies. Bars = 100 µm. (b) Quantification of unsorted, live iPSiCaspase and iPSYCD cells with or without prodrug administration on day 3 and day 9. Error bars represent one standard deviation from the mean of duplicate assessments. (c) Quantification of live (7AAD−) but apoptotic (Annexin V+) iPSiCaspase, and iPSYCD cells with and without prodrug administration on day 3 and day 9. Error bars represent one standard deviation from the mean of duplicate assessments. *P < eGFP, enhanced green fluorescent protein; iPS, induced pluripotent stem; YCD, yeast cytosine deaminase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
YCD/5-FC controlled the cell fate of teratoma-initiating iPS cells and existing teratomas efficiently and specifically. (a) iPSYCD and iPSGFP cells formed GFP+ tumors in untreated male NOD/SCID/γcnull mice 8 weeks after testis injection. Only iPSGFP cells formed tumors in treated male mice at the same time point following testis injection. Three dimensional size of tumors isolated from iPSYCD and iPSGFP cell-transplanted mice were measured ± 5-FC treatment, n = 3, **P < Bars = 500 µm and 100 µm (b) iPSGFP- and iPSYCD-tumor dissociated cells formed GFP+ tumors after injection into new mice subcutaneously 3 weeks after ± 5-FC treatment, and relative three dimension sizes of tumors were measured respectively, n = 3, **P < Bars = 100 µm. (c) iPSYCD cell-derived teratomas contained tissues from all three embryonic germ layers, and the small iPSYCD tumor observed in a single treated mouse exhibited necrosis morphology indicated by the loss of cell structure and broken nuclei (HE staining). YCD expression within tumor cells was ubiquitous, as indicated by Immunohistochemistry (IHC) staining. Yeast samples with YCD antibody staining served as positive control. iPSYCD necrotic tumor from treated mice and yeast samples with isotype antibody staining were used for negative control. Bars = 100 µm. 5-FC, 5-fluorocytosine; eGFP, enhanced green fluorescent protein; HE, hematoxylin and eosin; iPS, induced pluripotent stem; PGK, phosphoglycerate kinase; YCD, yeast cytosine deaminase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Evidence of sustained suicide gene expression in iPS cells and their derivatives. (a) Two integrations of YCD vectors were detected in iPSYCD cells by Southern blot. No YCD integrations were found in iPSGFP cells, 1ng cold probe (YCD vector) was used as positive control (b) mRNAs of YCD and iCaspase were detected in iPSYCD, iPSiCaspase cells and relative derivatives but not in iPSGFP cells. HSC, hematopoietic stem cell; iPS, induced pluripotent stem; YCD, yeast cytosine deaminase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions