Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency

Background Pluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive. Methods We utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR). Results The results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8. Conclusions In summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.


INTRODUCTION
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential to differentiate into all cell types of the body in vitro through embryoid body formation or in vivo through teratoma formation. Due to these characteristics, stem cells provide an option for treating a multitude of clinical problems, such as myocardium damage after heart infarction, spinal cord damage after mechanical injury, brain damage after stroke, age-related macular degeneration of the retina, liver damage, extensive skin burns, Parkinson's disease, and diabetes (Abdelalim et al., 2014;Lodi, Iannitti & Palmieri, 2011;Orlic et al., 2001;Ratajczak, Bujko & Wojakowski, 2016).
When transplanted into immune-compromised mice, undifferentiated PSCs can form teratomas, consisting of multiple tissue types derived from all three germ layers (Przyborski, 2005;Takahashi & Yamanaka, 2006). As such, there have been many efforts to differentiate pluripotent cells to cells with medical applications in an in vivo developmental environment. For example, neural stem cells (NSCs) have been differentiated in vivo through teratoma formation, and pure NSC populations exhibit properties similar to those of brain-derived NSCs (Hong et al., 2016). Similarly, fully functional and engraftable hematopoietic stem/progenitor cells (HSPCs), along with functional myeloid and lymphoid cells, have been isolated from teratomas when human iPSCs were transplanted into immunodeficient mice (Amabile et al., 2013;Suzuki et al., 2013). In addition, the teratoma assay can be applied to assess the safety of human PSC-derived cell populations that are used for therapeutic application since a small number of undifferentiated cells contaminating a given transplant material can be efficiently detected by their multi-lineage differentiation ability (Stachelscheid et al., 2013).
However, the intrinsic self-renewal and pluripotency qualities of PSCs that make them therapeutically promising are responsible for an equally fundamental tumorigenic risk (Lee et al., 2013). Studies on teratomas will contribute to a better understanding of their stepwise development processes and underlying molecular mechanisms and may provide helpful information for the development of tissue engineering technologies (Aleckovic & Simon, 2008). These facts prompted us to address the additional characteristics of teratoma growth and differentiation after PSCs injection.
In the present study, we aimed to isolate OCT4-expressing cells that escaped differentiation inside of growing teratomas and to determine whether OCT4-expressing cells still possess self-renewal and pluripotency abilities.

MATERIALS & METHODS
All animal experiments were approved by the Animal Care and Use Committees of the State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China Agricultural University (Approval number: SKLAB-2016-05-01). Briefly, mice were bred in a 12/12 h light/dark period and sacrificed by cervical vertebra dislocation.

Mouse strains
OCT4-GFP transgenic mice (Model Animal Research Center of Nanjing University) express EGFP (enhanced green fluorescence protein) cDNA under the control of the Pou5f1 promoter, which is active in pluripotent stem cells. This strain is useful for isolating pluripotent stem cells, as they specifically express green fluorescent protein. These OCT4-GFP transgenic mice were the source of the OCT4-MES and OG2 MEFs (mouse embryonic fibroblasts of 13.5-day OCT4-EGFP mice embryos) used in this study.

Derivation of MES and generation of iPSCs
To obtain OCT4-MES, uteri containing E3.5 embryos were isolated from timed pregnancies and transferred individually to the wells of a 24-well plate with irradiated mouse embryonic fibroblast (MEF) feeders. After five days of incubation, embryo outgrowths were separated from trophectoderm, individually picked, and expanded in MES medium (Dulbecco's modified eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS), L-glutamine, nonessential amino acids, β-mercaptoethanol, and 1,000 U/ml leukemia inhibitory factor).
OG2 MEFs were cultured in MEF medium (DMEM supplemented with 10% FBS, L-glutamine and nonessential amino acids); infected with retroviruses generated from pMX retroviral vectors encoding mouse Pou5f1, Sox2, Klf4 and c-Myc; and cultured on irradiated MEF feeder cells in MES medium. Subsequently, a single ESC-like colony was individually picked and expanded on feeders to establish stable lines. Both OCT4-MES and iPSCs originated from male embryos. Additional details can be found in our previous study (Pei et al., 2015).

RNA purification and cDNA preparation
Feeders were removed by plating ESCs on a gelatin-coated dish for 30 min, and unattached cells were collected by centrifugation. Total RNA was extracted from pure PSCs using Trizol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). RNA was reverse-transcribed using oligo-dT and M-MLV Reverse Transcriptase (Promega, Madison, WI, USA).

Statistical analysis
All results are presented as the mean ± standard deviation. Results were statistically analyzed using SAS (Statistics Analysis System) program. Significance of differences between samples was determined (at the significance level p < 0.05) using Kruskal-Wallis test.

Both OCT4-MES and TG iPS 1-7 are pluripotent
OCT4-EGFP mice express green fluorescent protein under the control of the pluripotencyassociated Pou5f1 promoter and are widely used to study the function of PSCs (Pei et al., 2015). These mice were used to generate mouse embryonic stem cells (MES) and iPSCs. OCT4-MES were isolated from the blastocysts of 3.5-day OCT4-EGFP mice embryos, while other mice were selected to prepare MEFs after day 13.5. The isolated MEFs were used to generate iPSCs by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. Then, TG iPS 1-7 was selected from the isolated iPSC clones. Both OCT4-MES and TG iPS 1-7 were maintained on feeder cells in the presence of leukemia inhibitory factor. They both exhibited typical MES-like morphologies (Figs. 1A and 1B). Immunofluorescent staining confirmed the expression of the three master transcription factors (OCT4, NANOG and SOX2) as well as ESC-specific surface marker SSEA-1 in OCT4-MES and TG iPS 1-7 (Figs. 1C-1J). The PCR results further demonstrated that these cells expressed pluripotency marker genes, including Pou5f1, Sox2, Nanog, Rex1, Tbx3, Nr5a2, Utf1 and Lin28a (Fig. 2). Next, in vivo teratoma formation assays were performed to further validate the pluripotency of OCT4-MES and TG iPS 1-7. Approximately 1×10 6 PSCs were suspended in 150 µl of PBS and injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to form teratomas. Three weeks after injection, OCT4-MES and TG iPS 1-7 formed teratomas in vivo, and hematoxylin and eosin (H&E) staining confirmed the formation of all three germ layers in each teratoma (Figs. 3A-3F). These results revealed that OCT4-MES and TG iPS 1-7 were pluripotent. Interestingly, we observed OCT4-positive cells growing in clusters in the teratoma masses formed by OCT4-MES and TG iPS 1-7 (Figs. 3G, 3H).

OCT4-positive cells from OCT4-MES and TG iPS 1-7 teratomas have self-renewal and pluripotency qualities
To quantify the fraction of OCT4-positive pluripotent cells in teratomas generated by OCT4-MES and TG iPS 1-7, we cut the teratomas into pieces and digested them with trypsin and then cultured the cells in MEF medium. Three days later, we found that most of these cells separated from OCT4-MES and that TG iPS 1-7-derived teratomas had the morphology of mouse embryonic cells, but a small number of cells were round and expressed GFP (Figs. 4A, 4B, 4D and 4E). After picking these cells and culturing them in MES medium, we found that they had typical MES-like morphologies, and they were AP-positive (Figs. 4C, 4F). We named these cells MES-FT and iPS-FT, which were derived from OCT4-MES and TG iPS 1-7, respectively. OCT4-expressing MES-FT and iPS-FT cells were grown in the presence of leukemia inhibitory factor, and they expressed pluripotency marker genes, including Pou5f1, Sox2, Nanog, Rex1, Tbx3, Nr5a2, Utf1 and Lin28a (Fig. 2).
The immunostaining results showed that these colonies were positive for OCT4, NANOG, SOX2 and SSEA-1 (Figs. 5A-5H). We performed in vivo teratoma formation assays to further validate the pluripotency of MES-FT and iPS-FT. MES-FT and iPS-FT formed teratomas in vivo, and the hematoxylin and eosin (H&E) staining results confirmed the formation of all three germ layers in each teratoma (Figs. 5I-5N). As in the results described above, there were also OCT4-positive pluripotent cells in the teratomas formed by MES-FT and iPS-FT (Figs. 5O, 5P).
The above results showed that OCT4-MES, TG iPS 1-7, MES-ST, iPS-ST, MES-ST and iPS-ST had pluripotency characteristics. However, MES-FT, iPS-FT, MES-ST and iPS-ST were survivors of the differentiation environment, so we wanted to know whether there were differences among these cells. Thus, we next investigated their differences.

OCT4-positive cells separated from teratomas express germ cell marker genes
To explore the gene expression patterns of OCT4-MES, TG iPS 1-7, MES-FT, iPS-FT, MES-ST and iPS-ST, cDNA was prepared from these cells without feeders for gene expression analysis. First, we detected the expression of pluripotency genes. When normalized to the values for OCT4-MES cells, the expression level of Pou5f1 was higher in MES-FT, and that of Lin28a was higher in both MES-FT and MES-ST cells, but there were no differences in the Nanog expression levels between these three cell lines (Fig. 7A). When normalized to the values for TG iPS 1-7 cells, iPS-FT and iPS-ST both highly expressed Pou5f1 and Nanog (Fig. 7B). However, there were no differences in the expression level of Lin28a (Fig. 7B). The expression of pluripotency marker genes in these cells varied slightly, but they were all within reasonable levels. Thus, these cell types were all pluripotent. Previous results have shown that PSCs that escape from differentiation inside of embryonic bodies express several markers associated with germ cell formation (Attia et al., 2014). As such, we further assayed the differences between the expression levels of important germ cell-specific genes (Dazl,Stella,Stra8,Vasa) in MES-FT, iPS-FT, MES-ST and iPS-ST (Figs. 7C, 7D). When normalized to the values for OCT4-MES, Dazl and Stella were more highly expressed in MES-FT cells, and the expression level of Stra8 was elevated nine-fold and ten-fold in MES-FT and MES-ST, respectively.
Similarly, iPS-FT and iPS-ST highly expressed Dazl, Stra8 and Vasa than TG iPS 1-7. iPS-FT also highly expressed Stella. The above results show that OCT4-positive cells separated from teratomas have elevated expression of several markers associated with germ cell formation, such as Dazl, Stella and Stra8.

DISCUSSION
ESCs and iPSCs are characterized by their ability to develop into any cell type of the adult organism. As such, they can be widely applied to the treatment of many diseases. This is especially true for iPSCs, as they do not present ethical issues. A previous report demonstrated the presence of undifferentiated human ESCs expressing the surface marker CD133 (Ritner & Bernstein, 2010). However, no additional research has been performed to investigate the characteristics of those undifferentiated cells in teratomas. Therefore, in this study, we isolated OCT4-GFP positive cells, MES-FT and iPS-FT, from teratomas generated by OCT4-MES and TG iPS 1-7, respectively. MES-FT and  Bottai et al. (2010) reported that they used 5×10 5 undifferentiated murine ESCs to cure spinal cord injury. However, some of the transplanted ESCs were found as dense aggregates in the tissue. This result supports our view that ESCs can be maintained in vivo. Another study showed that transplantation of 1 − 2 × 10 6 MES cells into SV129 mice led to tumor formation in 100% of cases, whereas transplantation of 5 × 10 5 cells produced tumors in two of six mice and transplantation of 1 × 10 5 ESCs gave rise to tumor formation in one of six transplanted mice within 100 days (Dressel et al., 2008). It can be deduced that there is likely a niche within teratomas that nurse PSCs, and the number of cells determines the niche environment. The more PSCs used for transplantation, the higher probability of tumor formation.
The  et al., 2005;Kehler et al., 2004;Tedesco et al., 2009;Toyooka et al., 2000;Wongtrakoongate et al., 2013). Stra8 is required for the chromosomal program of meiotic prophase (Soh et al., 2015). Dazl, an intrinsic meiotic competence factor, is required for Stra8-mediated initiation of meiosis in germ cells (Lin et al., 2008). Overexpression of Stra8 and Dazl genes promotes the transdifferentiation of mesenchymal stem cells and ESCs in vitro toward PGCs (Li et al., 2017;Shi et al., 2014). The elevated expression of Pou5f1, Dazl, Stella and Stra8 might indicate that the GFP positive cells separated from teratomas partially develop towards germ cells. This suggests that it is possible to isolate PGCs from teratoma differentiation models.

CONCLUSIONS
In summary, we found a small number of OCT4-expressing PSCs that escaped differentiation inside teratomas. The escaped cells kept their unique properties of selfrenewal and pluripotency and were able to form teratomas in vivo. They also expressed several markers associated with germ cell formation, such as Pou5f1, Dazl, Stella and Stra8, suggesting that these cells may partially differentiate into germ cells. Therefore, this study serves as a warning that medical workers using stem cells to treat specific diseases must pay careful attention to prevent tumor formation because OCT4-expressing cells retain pluripotency, and it is feasible to isolate germ cells from teratomas. This study of PSCs that remain undifferentiated within teratomas has provided critical information for further investigation of the applications of stem cell therapy and for obtaining germ cells from in vivo differentiation models.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This study was funded by grants from the National Key Research and Development Program (2016YFA0100202), the China National Basic Research Program (2011CBB01001,