Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

Gene Therapy (2011) 18, 294–303 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt ORIGINAL ARTICLE Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway D Racila1,3, M Winter1,3, M Said1, A Tomanek-Chalkley1, S Wiechert1, RL Eckert2 and JR Bickenbach1 In this study, we describe a simple system in which human keratinocytes can be redirected to an alternative differentiation pathway. We transiently transfected freshly isolated human skin keratinocytes with the single transcription factor OCT4. Within 2 days these cells displayed expression of endogenous embryonic genes and showed reduced genomic methylation. More importantly, these cells could be specifically converted into neuronal and contractile mesenchymal cell types. Redirected differentiation was confirmed by expression of neuronal and mesenchymal cell mRNA and protein, and through a functional assay in which the newly differentiated mesenchymal cells contracted collagen gels as efficiently as authentic myofibroblasts. Thus, to generate patient-specific cells for therapeutic purposes, it may not be necessary to completely reprogram somatic cells into induced pluripotent stem cells before altering their differentiation and grafting them into new tissues. Gene Therapy (2011) 18, 294–303; doi:10.1038/gt.2010.148; published online 28 October 2010 Keywords: keratinocyte; tissue repair; epidermal cells; stem cells; reprogramming INTRODUCTION In June 2006, Yamanaka1 reported that mouse embryonic fibroblasts could be reprogrammed into embryonic stem-like cells by retroviralmediated transduction of four transcription factors, Oct4, Sox2, c-Myc and Klf4. These induced pluripotent stem (iPS) cells proved that differentiated, perhaps nondividing cells, could be reprogrammed into highly proliferative embryonic cells. Since then, a multitude of reports have surfaced showing that iPS cells can be produced from a variety of mouse and human cells.2,3 In a proof-of-principle experiment, mouse iPS cells were used in therapy for a sickle cell gene defect in mice,4 thereby demonstrating their great therapeutic potential. They have also been used to make patient-specific cell lines for human studies.5 However, the iPS method, which completely reprograms the cells, cannot be used to produce cells for human therapy. Integrating retroviruses or lentiviruses were used to deliver the reprogramming factors. These viruses may cause several problems, including cancer if the integration sites affect oncogenes or tumor suppressor genes. The number of integration sites in the transduced fibroblasts was as high as 20 per clone, resulting in tumor formation in iPS cell-derived mice.3,6 Furthermore, this complete reprogramming approach requires the delivery of multiple genes, thus, enhancing the possibility of deleterious effects. Although several groups since 2007 have now shown that iPS cells can be generated without the use of viral vectors and with fewer factors,7,8 this technology still exhibits inefficient induction and a long production time, both major limitations if iPS cells are to be used for human therapy. Skin keratinocytes offer advantages over other cell types for reprogramming. The skin is the largest organ in the body, and the epidermis provides the greatest number of easily obtainable proliferative cells. Moreover, keratinocytes are 100-fold more efficient than fibroblasts when used in the various iPS cell protocols.9 In addition, our laboratory has shown that mouse epidermal stem cells, a subset of keratinocytes, can populate tissues derived from all three germ layers when injected into the developing blastocyst.10 Thus, keratinocytes offer high reprogramming and therapeutic potential. Another advantage of keratinocytes is that they are readily obtained from human skin. A recent review emphasized that signaling in the reprogrammed cells varies greatly amongst species.11 Thus, it is critical to test reprogramming in human cells whether human tissues are to be replaced. Additionally, if patient-specific cells are needed, then rapid cell production may also be critical. Keeping these two points in mind, we explored the possibility that it might not be necessary to completely reprogram human cells to iPS cells to use them to replace damaged human tissues. In this study, we tested the potential of using freshly isolated human skin keratinocytes (HSKs) for rapid alternative tissue replacement. We transiently transfected keratinocytes with the human OCT4 transcript, exposed these cells to alternative differentiating conditions and tested the functionality of these new cell types. The results presented in this study demonstrate that a transient exposure to OCT4 is sufficient to effect changes that allow directed differentiation of the human keratinocytes into functioning mesodermally derived cell types. This method could be used to provide customized patient-specific cells for therapeutic purposes. RESULTS OCT4 protein is temporally expressed in human keratinocytes To demonstrate that a transient exposure to OCT4 is sufficient to allow a change in differentiation of the human keratinocytes, we needed to show that the OCT4 transcription factor was expressed for a 1Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA and 2Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA Correspondence: Professor JR Bickenbach, Department of Anatomy and Cell Biology, 1-251 BSB, University of Iowa Carver College of Medicine, 51 Newton Road, Iowa City, IA 42242, USA. E-mail: 3These authors contributed equally to this work. Received 10 June 2010; revised 2 August 2010; accepted 6 August 2010; published online 28 October 2010 Transient expression of OCT4 D Racila et al 295 short amount of time, and when expressed, it moved to the nucleus in which it could transactivate its target genes. We transiently introduced a plasmid carrying the full-length human OCT4 transcript into human keratinocytes and determined where and for how long the OCT4 protein was expressed. Using immunocytochemical analysis, we determined that the OCT4 protein localized to the nuclei of the transfected keratinocytes 48 h after transfection (Figure 1b). Untransfected control keratinocytes showed no expression of OCT4 (Figure 1a). To investigate the temporal expression pattern, we counted the numbers of OCT4-expressing keratinocytes over 6 days (Figure 1c). At 24 h after transfection, B24% of cells expressed OCT4. This percentage peaked at 48 h with B32% of cells expressing OCT4. With each day thereafter, the percentage of keratinocytes expressing OCT4 decreased with most lost by 6 days post transfection. Thus, pcDNA3-OCT4 transiently produced the OCT4 protein that localized to the nuclei of the keratinocytes. Transfected cells show transient expression of endogenous OCT4 targe (...truncated)