Establishment and Characterization of Novel Porcine Induced Pluripotent Stem Cells Expressing hrGFP

1Division of Physiology, Livestock Research Institute, Council of Agriculture, Executive Yuan, Tainan, 71246, Taiwan 2Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, 97004, Taiwan 3Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, 40227, Taiwan 4Department of Oral Medicine, National Cheng Kung University Hospital, Tainan, 70101, Taiwan 5Institute of Oral Medicine, College of Medicine, National Cheng Kung University, Tainan, 70101, Taiwan 6School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, 11031, Taiwan 7Institute of Biotechnology, National Cheng Kung University, Tainan, 70101, Taiwan 8Institute of Biotechnology, Southern Taiwan University, Tainan, 71005, Taiwan


Introduction
Embryonic stem (ES) cells derived from the inner cell mass of the blastocyst were first established from mouse, and they could grow indefinitely with pluripotency and differentiate into all three embryonic germ layers [1]. ES cells, however, face ethical controversies because they derived from blastocysts. Fortunately, induced pluripotent stem (iPS) cells derived from somatic cells, which believed to possess similar ability as ES cells, were established in 2006 by direct reprogramming [2]. By using this technique, we could establish pluripotent cell lines easily and circumvent ethical problems.
The pig, a common livestock species, has the potential to serve as a great research model for human biomedicine, and has been considered an optimal model for preclinical development of therapeutic approaches because the organ size, immunology, and whole animal physiology are similar to human [3][4][5]. Porcine embryonic stem (pES) cells, like human embryonic stem (hES) cells, were maintained on the feeder layer without supplement of leukemia inhibitory factor (LIF) [6]. Also, the pES cells shares similar colony morphology, and expressed the same pluripotency markers including Oct4, AP, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, but not SSEA-1 which is characterized to mouse ES cells [7,8]. Therefore the pig is a potentially useful model in human regenerative medicine.
In our previous studies, we successfully established pES cells expressing humanized recombinant green fluorescent protein (pES/ hrGFP + cells) [8]. These cells ameliorated the Parkinson's disease and spinal cord injury in the rat models by xenotransplantation [9,10], and also the periodontal furcation defects in a porcine model by allotransplantation [11]. In present study, porcine induced pluripotent stem cells expressing hrGFP (piPS/hrGFP + ) were generated from porcine ear fibroblasts (pEF) by introducing four human transcription factors (Oct4, Sox2, Klf4, and c-Myc) constructed in lentivirus vectors, and the common criteria for iPS cells were investigated. The main goal for present study was to pave the way for transplantation study, especially allotransplantation. By detecting hrGFP expression, we could easily monitor the growth, differentiation, and migration of grafted cells. In, addition, we expect that piPS/hrGFP + cells not only could be used as cell resources to study the Parkinson's disease, spinal cord injury, and periodontal furcation defects, but also have the potential for future therapeutic application on regenerative medicine by allotransplantation.

Induction and culture of green fluorescent protein expressing porcine ear fibroblasts
The pEF in this study was derived from the ear dissection of Livestock Research Institute Black Pig No. one (a topcrossing breed established from Taoyuan and Duroc pigs, No. 53501) and trypsinized to single cells by 0.25% (w/v) trypsin-0.02 mM EDTA (Invitrogen, Grand Island, NY, USA). The pEF were maintained in Dulbecco's modified eagle medium (DMEM, high glucose and no pyruvate, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 100 units/mL penicillin-100 g/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C with an atmosphere of 5% CO 2 in air. The methods for induction of hrGFP by electroporation-mediated transfection were described in previous study [8]. Briefly, the pEF cells was trypsinized to single cells and adjusted to a concentration of approximately 5×10 4 cells/mL in phosphate buffered solution (PBS). Approximately 20 μg of pAAV-hrGFP Control Plasmid (Stratagene, Santa Clara, CA, USA) were added into the cells suspension in the electroporation cuvette (Cuvettes PlusTM, Model No. 620, BTX, San Diego, CA, USA). Electroporation with condition including 2 DC pulses, 150 V/cm of field strength, and 10 msec duration time was performed by the Electro Cell Manipulator (BTX ECM 2001, San Diego, CA, USA). Approximately 40% of pEF expressed hrGFP after electroporation. When the total cell number was scaled up to 15 million, the pEF were collected for hrGFP positive sorting by flow cytometer (FACSVantage SE, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). We obtained more than 95% hrGFP expressing pEF and nominated as pEF/hrGFP + cells.
For isolation and establishment of piPS cells, the colonies exhibited dome-like morphology were picked up about 1 month post-infection. These cells were subsequently expanded and maintained in ESM on mitomycin C (Sigma-Aldrich) inactivated STO feeders (mouse embryonic fibroblasts, CRL-1503, ATCC, Manassas, VA, USA). These putative piPS/hrGFP + cells were regularly subcultured every 5 to 7 days.

Characterization of the plurioptencyt markers
The putative piPS/hrGFP + cells were fixed in 10% (v/v) neutral buffered formalin for 30 min, and permeabilized with 0.3% (v/v) Triton X-100 for 10 min after washing with PBS three times. After permeabilization, the cells were incubated with blocking solution [5% (v/v) FBS in PBS containing 0.1% (v/v) Tween-20] for 2 h at room temperature, and then incubated with primary antibody diluted with blocking solution (1:200 dilution) at 4°C overnight. After washing with PBS three times on the next morning, the cells were incubated with secondary antibody diluted with blocking solution (1:200 dilution) for 2 h at room temperature. The cells were then washed twice with PBS again, and stained with 4,6-diamidino-2-phenylindole (DAPI).

Karyotype analysis
G-banding was used for karyotyping analysis and carried out as previously described [8,13]. Briefly, The piPS/hrGFP + cells were then mitotically arrested with colcemid (KaryoMax ® Colcemid solution, Invitrogen) at a final working concentration of 0.02 µg/mL at 39°C for 30 min. Cells were harvested for hypotonic treatment for 30 min in 0.56% (w/v) KCl aqueous solution following removal from colcemid treatment on dish (in situ method). The cells were pelleted by centrifugation at 800 × g and fixed in cold Carnoy's fixative (3:1, v/v, of absolute methanol to glacial acetic acid) for 10 min. After a second wash in Carnoy's fixative, the cells were resuspended in 2 mL fixative. Slides were prepared by dropping the cell suspension onto dry microscope slides prewashed with fixative. Immediately after dropping, the slides were exposed to a flame to burn off the fixative, incubated 30 seconds to 1 minute in a trypsin (1:250) solution (0.1 g trypsin in 100 mL isotonic buffer), rinsed for a few seconds in a jar with FBS (2-3 mL FBS in 50 mL isotonic buffer), rinsed in isotonic buffer, and then stained in 5% Gurr's Giemsa staining solution (Invitrogen) for 2 min. The stained slides were rinsed, air dried and examined under a microscope at 1,000 × magnification with oil immersion. The images were then analyzed by Applied Images software (AI cytovision 2.8, 2002, Applied Images Group, Gainesville, GA, USA).

Embryoid body formation, differentiation, and embryonic germ layers determination
For the formation of embryoid body (EB), piPS/hrGFP + cells were removed from their feeders and subjected to suspending culture by hanging drops in the bacteriological Petri dish [8]. The piPS/hrGFP + cells were harvested and cultured in 20 μL of ESM on the lid of 100mm sterile bacteriological Petri. The cells were cultured at 37°C with an atmosphere of 5% CO 2 in air for 7 days. The medium was regularly changed every other day. After 7 days culture, the formed EB was transferred to gelatin-coated 48 well in the same medium for another 14 days to induce in vitro spontaneous differentiation.

Teratoma formation and in vivo tracking
All animal experiments in this study were performed in accordance with ethical guidelines and following approval of the Livestock Research Institutional Animal Care and Use Committee (IACUC).
For teratoma formation analysis, five female nonobese diabetic/ severe combined immunodeficiency (NOD-SCID) mice (Bio-LASCO, Taiwan) at 8 weeks of age were anesthetized with Zoletil/Rompun mixture (15 mg/kg). The piPS/hrGFP + cell suspension of 1×10 6 cells in 100 μL ESM were injected subcutaneously into the left doral flank. Length, width, and height of teratoma were measured every 15 days after injection during the 3-month experimental period.
For in vivo monitoring of these grafted piPS/hrGFP + cells in NOD-SCID mice, live animal fluorescence optical imaging system, the In Vivo Imaging System (IVIS 50, Xenogen Corp., Alameda, CA, USA), was used for non-invasive tracking every 15 days during the 3-month experimental period. The relative fluorescence values of treatment groups were calculated as the fluorescence intensity detected from the region of interest (ROI) on mice in each treatment group divided by that of the control groups at the same time window.

Histological analysis of teratoma
Three months after the injection, teratomas were surgically dissected from the NOD-SCID mice. Samples were fixed in 10% (v/v) neutral buffered formalin for 1 week, and embedded in paraffin after serial dehydration by alcohol. Samples were dissected at 3 μm in thickness and stained with hematoxylin and eosin by standard procedure.

Statistical analyses
All data were displayed as mean ± SEM.

Generation of piPS/hrGFP+ cells
The pEF derived from the ear fibroblasts were collected and cultured in DMEM supplemented with 10% FBS after trypsinization ( Figure  1A). Thereafter, the pEF/hrGFP + cells were obtained by transfecting pEF with pAAV-hrGFP Control Plasmid using electroporation. ( Figure  1B). The experimental schedule for piPS/hrGFP + cells isolation was summarized in Figure 1C. The pEF/GFP + cells were further infected with human Oct4, Sox2, Klf4, and c-Myc constructed in lentivirus vectors. On day 2 after infection, the infection medium was withdrawn, and the cells were maintained in ESM. The infected cells grew into round shape and aggregated approximately two weeks later ( Figure  1D), and the typical dome-like morphology of ES colony appeared about day 30 after infection ( Figure 1E). The dome-like colonies were further mechanically picked up and disaggregated into small clumps by continuous pipetting. The cell clumps were then plated onto mitomycin C inactivated STO feeder layers and subsequently flatten into typical undifferentiated pES colony morphology ( Figure 1F). These colonies induced from pEF/hrGFP + cells also successfully expressed hrGFP and were named as piPS/hrGFP + cells ( Figure 1G).   Figure 4C) and began to differentiate into cells of three embryonic germ layers. The attached cells exhibited various types of morphologies, but the morphology of cells changed frequently. The differentiation timing of each embryonic germ layer was various. Generally, neuron-like cells with obvious Nissl body first appeared on day 3 after successful attachment ( Figure 4D), and that gradually differentiated into epithelial cells ( Figure 4E). By immunocytochemical staining, the differentiated embryonic germ layers were positive for MAP2 (ectodermal maker), NFL (ectodermal maker), cytokeratin (ectodermal maker), AFP (mesodermal maker), and ANP (endodermal maker) ( Figure 4F-J). hrGFP + cells, ES cell-specific surface antigens including Oct4, AP, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 were determined. The results of immunocytochemical study showed that the piPS/hrGFP + cells were positive for those pluripotency markers (Figure 2A). The expression of endogenous pluripotency genes (pOct4, pSox2, pKlf-4, and pc-Myc) were also detected in piPS/hrGFP + and pES/hrGFP + cells. In addition, pKlf-4 and pc-Myc were prominently expressed in the pEF/ GFP + cells ( Figure 2B).

Karyotype analysis
Karyotyping of the piPS/hrGFP + cells was performed by G-banding staining for monitoring of their chromosomal normality. The results indicated that the piPS/hrGFP + cells maintained in culture for more than 90 passages possessed a normal 36 + XY male karyotype ( Figure  3).

In vitro differentiation
The piPS/hrGFP + cells formed ball-shaped EB ( Figure 4A) and retained hrGFP signal after 7 days of hanging drops culture ( Figure  4B). The EB formation rate was about 93.6 ± 4.7% (190/203, n = 10). Spontaneous differentiation of piPS/hrGFP + cells was evident when the EBs allowed to grow in gelatin-coated surface. On day 3-5 after adherent culture, the EB in ESM attached to the surface of gelatin-

Teratoma formation and in vivo tracking
For determination of the in vivo differentiation capacity, the piPS/ hrGFP + cells were injected into immunocompromised mice. One month after transplantation, all NOD-SCID mice had developed small and solid teratomas of about 0.004 cm 3 in size in dorsal flank. The teratomas grew and reached 0.55 ± 0.21 cm 3 in size three months after transplantation. The teratomas in the transplantation site were traceable by IVIS 50 through the 3-months experimental period. The relative intensity of fluorescent signal in ROI of treatment groups was 6.95 ± 1.68 folds higher than that of control groups three month after transplantation ( Figure 5A). Thereafter, the NOD-SCID mice were sacrificed for histological and immunohistochemical analysis. The dissection of teratomas revealed various tissues derived from the three embryonic germ layers, including neural tissues (ectoderm), keratin pearls (ectoderm), skeletal muscle (mesoderm), smooth muscle (mesoderm), cardiac muscle (mesoderm), cartilage (mesoderm), adipose tissues (mesoderm), and glandular structures (endoderm) (Figures 5B and 5C).
In the present study, we established the piPS/hrGFP + cells from reprogramming of hrGFP-expressing pEF cells. These novel piPS/hrGFP + cells generated in this study expressed hrGFP signal continuously and steadily for more than 90 passages. Expression of fluorescence can be detected in pEF/hrGFP + cells and pass to piPS/ hrGFP + cells, EB and teratomas ( Figure 1B, 1G, 4B, and 5A). They also possessed the typical defined characteristics of ES cells, including continuous proliferation with undifferentiated status, maintenance of a normal karyotype (Figure 3), and formation of EBs upon suspension culture ( Figure 4A and 4B). Expression of the ES cell markers including Oct-4, AP, SSEA-4, TRA-1-60, and TRA-1-81 were also detected in the undifferentiated piPS/GFP + cells, as pES/GFP + cells we described previously [8]. Expression of endogenous pluripotency genes, pOct4, pSox2, pKlf4, and pc-Myc, were detected in piPS/hrGFP + and pES/ hrGFP + cells. In addition, significant pKlf4 and pc-Myc expression but not pOct4 and pSox2 expression were detected in pEF/hrGFP + cells ( Figure 2B). This phenomenon was also shown in the previous reports of Ezashi et al. (2009) [20] and Fujishiro et al. (2012) [21]. The endogenous Klf4 expression in human fetal endothelial cells was also reported previously, and these cells allowed to be reprogrammed with Oct4 and Sox2 [26]. These results imply that piPS cells might also be able to reprogram from porcine somatic fibroblasts by using transcription factors of Oct4 and Sox2 only.
The EB formation efficiency of piPS/hrGFP + cells in this study was high (93.6 ± 4.7%) and the differentiated cells derived from three embryonic germ layers were detected after adhesive culture of EB. These results demonstrated that the in vitro differentiation capacity of piPS/hrGFP + cells generated in this study. To our knowledge, there were few reports describing the success in teratomas induction by transplanting pES cells into the nude mice [27][28][29]. Hochereau-de Reviers and Perreau (1993) [28] reported that only the embryonic disc cells derived from days 10-11 but not days 5-6 blastocysts formed teratomas when transplanted into the nude mice. Similar observation had been depicted by Piedrahita et al. (1990) [29]. They failed to induce teratomas by pES cells derived from day 7-8 embryos. The difficulty in obtaining teratomas from the porcine embryonic cells of earlier stages was also confirmed by Anderson et al. (1994) [27], who demonstrated that teratoma can only be obtained by injecting pES cells isolated from blastocysts of day 11-12. However, in the present study, piPS/hrGFP + cells formed teratomas after being transplanted into dorsal flank of NOD-SCID mice (n = 5). Other previous studies in the generation of pips cells also demonstrated the similar results [19][20][21][22][23][24]. The reason for teratomas formation of piPS cells after ectopic transplantation to SCID mice might result from different property of cells in epigenetic background via reprogramming process.
ES cells of ungulate species were rather difficult to establish from early embryos, but iPS cells provide a feasible approach for generating pluripotent stem cells. In our previous studies, transplantation of pES/hrGFP + cells-derived neuronal progenitors were successfully ameliorated the Parkinson' disease [9] and spinal cord injury [10] in the rat models. In addition, regeneration of periodontal furcation defects in a porcine model was improved by transplanted with pES/hrGFP + cells [11]. In the present study, piPS/hrGFP + cells were established and possessed very similar property as pES/hrGFP + cells we established previously [8]. In addition, the intensity of hrGFP signal in piPS/ hrGFP + cells was up to 6.95 ± 1.68 folds compared with control group. This will benefit the transplanted piPS/hrGFP + cells to easily locate, monitor and traced after transplantation. The therapeutic potential of piPS/hrGFP + cells in regenerative medicine would be further  In a nutshell, these results implicate that traceable piPS/hrGFP + cells were successfully established and opened an avenue for biomedical application in pigs.