Derivation of induced pluripotent stem cells from pig somatic cells Toshihiko Ezashia, Bhanu Prakash V. L. Telugua, Andrei P. Alexenkoa, Shrikesh Sachdevb, Sunilima Sinhaa, and R. Michael Robertsa,b,1 Divisions of aAnimal Sciences and bBiochemistry, University of Missouri, Columbia, MO 65211 Contributed by R. Michael Roberts, May 13, 2009 (sent for review April 15, 2009) For reasons that are unclear the production of embryonic stem cells For reasons that are unclear, the establishment of porcine ESC from ungulates has proved elusive. Here, we describe induced from ICM of blastocysts and the epiblast of slightly older pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts embryos has proven to be elusive. There has been a similar lack by lentiviral transduction of 4 human (h) genes, hOCT4,hSOX2, of success with other ungulate species. The earliest reports hKLF4, and hc-MYC, the combination commonly used to create iPSC announcing the derivation of ESC-like cells from ICM of pigs in mouse and human. Cells were cultured on irradiated mouse appeared in the early 1990s (19–21), but these ESC-like cells and embryonic fibroblasts (MEF) and in medium supplemented with many others since then, including ones for cattle, goat, and knockout serum replacement and FGF2. Compact colonies of alka- sheep, as well as for pig, have failed to meet the full criteria to line phosphatase-positive cells emerged after Ϸ22 days, providing define them as ESC (11–13). There are a number of reasons that an overall reprogramming efficiency of Ϸ0.1%. The cells expressed might explain the problems encountered, including choice of the porcine OCT4, NANOG, and SOX2 and had high telomerase activity, wrong stage of embryo development to establish the cultures, inappropriate culture and cell passage conditions, and contam- but also continued to express the 4 human transgenes. Unlike ination by more vigorously growing cells, such as the endoderm human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but and trophectoderm of the blastocyst from which the culture was negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix derived. Attempts to create pluripotent cells from embryonic (porcine) microarrays and real time RT-PCR supported the conclu- germ cells have also run into difficulties. As a result, this field of SCIENCES sion that reprogramming to pluripotency was complete. One cell research has languished without major breakthroughs for well line, ID6, had a normal karyotype, a cell doubling time of Ϸ17 h, over a decade. These difficulties, as well as the potential value of APPLIED BIOLOGICAL and has been maintained through >220 doublings. The ID6 line ESC from ungulate species, have been discussed at length in formed embryoid bodies, expressing genes representing all 3 germ several recent reviews (11, 13–15, 22). layers when cultured under differentiating conditions, and tera- Many recent papers (23–29) have reported the generation of tomas containing tissues of ectoderm, mesoderm, and endoderm induced pluripotent stem cells (iPSC), with properties almost origin in nude mice. We conclude that porcine somatic cells can be indistinguishable from those of ESC, by transgenic manipulation of reprogrammed to form piPSC. Such cell lines derived from individ- mouse and human somatic cells. In most of these examples, the ual animals could provide a means for testing the safety and same 4 ‘‘reprogramming genes’’ were able to establish the iPSC (25, efficacy of stem cell-derived tissue grafts when returned to the 26). In addition, the efficiency of the overall process could be same pigs at a later age. improved by the inclusion of various small molecules and growth factors (30). The major focus of this report describes the derivation iPS ͉ reprogramming ͉ OCT4 of porcine-induced pluripotent stem cells (piPSC) based on the same strategy as that used for the mouse and human, namely ectopic expression of reprogramming genes in somatic cells by the luripotent cells from the mouse were first described in 1981 (1). use of lentiviral vectors. An important justification for establishing PSuch cells, which are usually known as embryonic stem cells such a technology is that the ability to derive iPSC from a particular (ESC), are most commonly derived from either the inner cell mass pig, conduct tissue transplantation on the same animal at a later (ICM) of the blastocyst or from the early epiblast. The cells are truly time, and then follow the success of the transplant over the course pluripotent in the sense that they have the potential to differentiate of months or even years would be a particularly valuable advance. into all of the cell types found in the body and, under appropriate Finally, the ability to provide iPSC from animals with valuable traits culture conditions, to proliferate more or less indefinitely (2, 3). would provide a permanent source of cells for clonal propagation Their discovery provided a new and powerful model for studying that would likely avoid the inefficiencies and problems arising from the programs that drive differentiation and senescence. Crucially, somatic cell nuclear transfer (SCNT), where many of the cloned murine ESC also afforded a means for ‘‘knocking out’’ and ‘‘knock- offspring die or are developmentally abnormal even if they survive ing in’’ genes in mice by homologous recombination, because to term (31). mutated cells with the genetic alteration can be introduced into blastocysts to give rise to chimeric mice that, in turn, can pass on the Results mutated gene through their germline (4, 5). The derivation of Generation of piPSC. Four reprogramming genes (OCT4, SOX2, human ESC from blastocysts, more than 15 years after the first KLF4, and c-MYC), each integrated into separate lentiviral description of ESC in the mouse (6), presaged their recent deriva- tion from other species, including the dog (7), cat (8), and rat (9, 10). Author contributions: T.E., B.P.V.L.T., S. Sachdev, and R.M.R. designed research; T.E., This discovery also heralded the prospects of using ESC in regen- B.P.V.L.T., A.P.A., and S. Sinha performed research; T.E., B.P.V.L.T., and R.M.R. analyzed erative medicine. Despite their undoubted promise as sources of data; and T.E., B.P.V.L.T., and R.M.R. wrote the paper. tissue transplants, many road blocks remain to using human ESC as The authors declare no conflict of interest. a source of transplant material, especially as a means to test the Freely available online through the PNAS open access option. efficacy of therapies and the safety of the transferred cells in Data deposition: The data reported in this paper have been deposited in the Gene animals whose anatomy and physiology better resemble the human Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE15472). than the mouse (11–15). The pig is a potentially useful model in this 1To whom correspondence should be addressed. E-mail: [email protected]. regard because of similarities in organ size, immunology, and whole This article contains supporting information online at www.pnas.org/cgi/content/full/ animal physiology (16–18). 0905284106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905284106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 A BC D E FG Fig. 1. piPSC colonies derived from PFF. (A) A phase contrast image of PFF. (B) Phase contrast image of granulated piPSC similar to mouse and human iPSC begin to emerge Ϸ3 weeks after viral infection. (C) A representative piPSC colony after serial passage resembling hESC shown at low magnification, and (D) at a higher magnification. (Scale bar: A–C, 500 ␮m; D,50␮m.) (E) The piPSC colonies express alkaline phosphatase (AP), and display (F) nuclear localization of OCT4 (green) and surface SSEA1 (red). (Scale bar: 100 ␮m.) (G) Some of the piPSC colonies have a disposition for spontaneous differentiation as evi- denced by the area (arrow) on the right side of the colony. The differentiated cells exhibit cobblestone morphology with a relatively low nucleus to cyto- Fig. 2. Gene expression analysis of piPSC. (A) RT-PCR analysis for expression plasm ratio (Scale bar: 500 ␮m). of selected pluripotency genes in piPSC, PFF, and H9 hESC. The primers were chosen for their specificity toward the porcine (p) genes rather than their human orthologs, but those for pc-MYC and pKLF4 showed cross-reactivity. (B) vectors, were introduced into porcine fetal fibroblasts (PFF) and Hierarchical clustering of microarray data for 3 piPSC lines (IC1, ID4 and ID6), and 2 PFF cells (1 and 2) by using Pearson-centered single-linkage rule. All of PFF expressing enhanced green fluorescent protein (EGFP) the genes (n ϭ 8,015) that displayed a fold-change of Ն1.3 of their normalized (Fig. 1A and Fig. S1). Twenty-two days after transduction, expression between piPSC and PFF and a P value of Յ0.05 were used in the compact colonies comprised of cells, which were positive for analysis. The values next to the branches represent Pearson distances. (C) Fold alkaline phosphatase (AP), stage-specific embryonic antigen differences (Log2) in expression of select genes between the piPSC and PFF. Black (SSEA)1, and OCT4, were noted (Fig. 1 B–F). Individual bars on the right-hand side of the axis represent those genes that were up- colonies were mechanically dissociated by using a pulled Pasteur regulated in piPSC compared with PFF, whereas the gray bars on the left side of pipette. A colony’s component cells were transferred to fresh the axis were down-regulated genes. *, P value Յ 0.05 and **, P value Յ 0.01. medium in 24-well plates coated with irradiated MEF. After Ϸ4 days, well-developed secondary colonies (Fig.