No Detection of Potential Cancer Risk for Free-Viral Reprogrammed Stem Cell-Derived Dopaminergic Neurons from Adult Mice Fibroblasts

Abbreviations: AD: Alzheimer’s Disease; BDNF: Brain Derived Neurotrophic Factor; Chat: Choline Acetyltransferase; c-Myc: cellular Myelocytomatosis Oncogene; DA: Dopamine; Dcx: Doublecortin; iPSC: Induced Pluripotent Stem Cell; GAPDH: Glyceraldehyde 3-Phosphate Dehydrogenase; Klf4: Kruppel-like Factor 4; Oct4: Octamer-binding Transcription Factor 4; PD: Parkinson Disease; RFU: Relative Fluorescence Units (real time qPCR); SLC6A2: Norepinephrine; SLC6A4: Serotonin; Sox2: SRY (sex determining region Y)-box 2; TH: Tyrosine Hydroxylase; Vector 20866: Vector pCAG2LMKOSimO; UTRs: Untranslated Regions


Introduction
Stem cell replacement therapy has potential clinical applications by inducing neuroplasticity in neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease (AD) and spinal cord injury, and possibly neuropsychiatric illnesses [1,2]. Re-programming stem cells from somatic cells of individual patients with specific diseases reveals innovative profits for the personalized medicine as there are less immune response rejections to such cell replacement.
One critical limitation of the use of induced pluripotent stem cells (iPSCs) derived from re-programmed somatic cells for longterm cell culturing is the fact that the viral-based (usually retroviral) transfection methods applied in the host cells pose a considerable risk of tumor formation. This is attributable to the random and permanent integration of viral genomic DNA into the host cells' genome. Although Wernig et al. [3] successfully prevented toxin-induced Parkinsonism After careful review of current publications, we considered that the non-viral system that was created by Kaji et al. [4] had the most advantages: high re-programming efficiency; one vector (for most easy opening) packed with four transcription factor genes -cMyc, Klf4, Oct4 (Octamer-binding transcription factor 4), and Sox2 (SRY sex determining region Y-box 2), the most important of re-programming genes; and the subsequent excision of these re-programming factors; as well as one fusion of vector marker mOrange with the re-programming genes in the same reading-frame for common gene expressions. Hence, we concluded this system showed promise to be a good model for us to evaluate potential cancer risks in free-viral reprogrammed stem cellderived dopaminergic neurons from adult mice fibroblasts in longterm cell culture circumstances.

Plasmid structure
One free-viral vector pCAG2LMKOSimO (vector 20866, Addgene) was used for re-programming somatic cells into iPSCs for our experiments [4]. This vector was constructed with free-viral within one single vector frame c-Myc-Klf4-Oct4-Sox2-(MKOS)-IRES-mOrange, flanked by loxP sites with a 2A-peptide-linked reprogramming cassette. Their expressions were transcribed by driving universally synthetic CAG enhancer/promoter [16,17]. This vector holds more exclusive advantages as described above.

Primary cell cultures
The fibroblast tail-tips from two-month adult (CD1) mice were segregated. The protocols were permitted by the Carleton University Animal Care Committee, in accordance with The Animals for Research Act of Ontario and the Guidelines of the Canadian Council on Animal Care (CCAC). The matrix of 100 × 60 mm plates or 6-well plates was prepared by coating culture vessels with laminin (final coating concentrations from 1 μg/cm 2 of surface area, Invitrogen 23017-015) and Poly-D-Lysine (final coating concentrations from 1 μg/cm 2 of surface area, Millipore A-003-E). Fibroblasts were composed with Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/ F12, Invitrogen, 10565-018). Tissues were chipped into small pieces on a plate with 4 mL DMEM/F12, and then were enzymatically digested using 2 mg/mL of filter-sterilized papain (Worthington, LS003119) at 37°C for 45 minutes. Consequently, tissues were removed and spun down, and the supernatant re-suspended in 5 mL of knockout DMEM (Invitrogen, 10829-018). Tubes containing cells were additionally centrifuged for 4 minutes at 200×g, after the supernatants were removed and cell pellets re-suspended in 41.5 mL DMEM/F-12 (Invitrogen, 10565-018). A total of 50 mL medium of DMEM/F-12 was prepared by adding 7.5 mL knockout™ serum replacement (Invitrogen, 10828-028), 0.5 mL MEM Non-Essential Amino Acids Solution (NEAA) (Invitrogen, 11140-050), 0.5 mL L-glutamine (Invitrogen, 25030-081), 91 μl (final 0.1 mM) β-mercaptoethanol (Invitrogen, 21985-023), and 50 µl of 10 µg/mL (final 10 ng/mL) bFGF (Fibroblast growth factors, Invitrogen 13256-029). Cells were raised at 37°C and 5% CO 2 in an ambient humidified atmosphere; and were freshly nourished every third day with the proper medium.

Initiation of pluripotency by non-viral reprogramming genes
Cells at 90% confluence were transfected with/without reprogramming genes containing vector 20866 by applying lipofectamine® 2000 Transfection Reagent (Invitrogen, 11668-027). Cells were continually fed with fresh media to maintain re-programming and the induction of pluripotency. The cluster-appearances of stem-cell-similar colonies expressing the mOrange-positive marker were observed starting from Days 5-6 after transfection. The iPSCs were confirmed by expressions of SSEA-1 (stem cell stage-specific antigens, Millipore SCR002) and GF141 (Stem Cell Factor Protein, Millipore GF141). The mOrange-positive iPSCs at Day 20 were further distinguished by live staining with GF141, and then were transferred to cultures containing a Neural Induction Medium on freshly gelatin-coated plates (Attachment Factor is sterile 1X solution containing 0.1% gelatin, Invitrogen, S-006-100).

Immunocytochemistry
In order to assess stem cell markers, Embryonic Stem (ES) Marker Sample Kits (Millipore, SCR002 and GF141) were employed. The kits are respectively comprised of monoclonal antibodies for the detection of cell-surface Stage-Specific Embryonic Antigens-1 (SSEA-1), and the expression of Stem Cell Factor (SCF) protein antigens (GF141). Mouse Nestin Antibody (R & D system, MAB2736) was applied as a neural progenitor mark for the verification of neuronal progenitor cells. For the tyrosine hydroxylase (TH)-positive dopaminergic neurons marker, TH antibody (ImmunoStar, 22941) was utilized. Briefly, the primary antibodies were diluted at a 1:25 ratio in a blocking solution (1X PBS/4% normal goat serum/0.3% Triton™ X-100), and then incubated for 1 hour at room temperature following 3 washes with 1X Rinse Buffer. Furthermore, the samples were incubated with fluorescein isothiocyanate (FITC)-labeled secondary antibodies for 1 hour at room temperature. Fluorescence images were shot using an Olympus 1x2-UCB series microscope with Media Cybernetics image analysis software. Living cell culture samples were evaluated using a Zeiss Axiovert 40 CFL microscope with Lumenera Infinity software.

Quantitative real-time PCR
Total RNA from culture cells was segregated using a GeneJET RNA Purification kit (Thermo scientific, K0732), and then quantitative Real-time PCR (qRT-PCR) was measured by applying a LightCycler ® 480 RNA Master Hydrolysis Probes kit (Roche, 04991885001). All TaqMan-gene-specific primers (Supplementary Table 1) were designed by using Integrated DNA Technologies' web application PrimeTime qPCR Assays. The values of gene expressions were normalized to total levels of a control housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All tests were also normalized for PCR sample loading and reaction. Relative fluorescence unit (RFU) is a unit of measurement used in analysis of fluorescence detection in real time PCR. This data is represented in one of three independent experiments for ct (threshold cycle; at 35 cycles) plots in the Figures.

Genomic DNA analysis of tyrosine hydroxylase
Under the forced direction described above, the final product was dopaminergic neurons throughout the entire progressive staging cell culturing. Since it is difficult to measure dopamine using current technology due to its instability, tyrosine hydroxylase (TH, ratelimiting enzyme for dopamine synthesis) represents TH-positive dopaminergic neurons. Consequently, TH-positive dopaminergic neurons were investigated in order to evaluate the potential risk of four re-programming genes for the host cell's genomic integration or mutagenic incidences. Thus, cells were continually cultured long-term for 6 months following their conversion into mature dopamine neurons.
Specifically, re-programmed stem cells-derived dopamine neuronal cells and parallel original tail-tip cells, which were cultured in the absence of the re-programming factors as one control for cell culture conditions, were sequenced for investigation, along with additional two tissue sample controls that were comprised of untreated in vivo brain tissue (midbrain) and tail tissue. A total of four groups were individually analyzed at the same time. We examined the TH genomic DNA sequences with full 13-coding regions of cDNA (ID numbers Exons 1-13) and their two-ended partial UTR regions. The design of specific PCR primers (Supplementary Table 1

Statistical analyses
All calculations were produced using StatView version 6.0, a statistical software package from the SAS Institute Inc. Significant findings were followed up on using the Bonferonni analyses.

Virus-free reprogramming genes converted mice mature fibroblasts into iPSCs
In order to optimize current re-programming methods established by Kaji et al. [4], the tail-tip fibroblast cells from 2-month-old adult mice (Figure 1a) were transfected with the free-viral vector 20866 containing the following four re-programming transcription factors: Oct4, Sox2, Klf4, and c-Myc. The iPSC identity during Days 17-20 was identified by the expression of the fluorescent vector mOrange marker (Figure 1b and 1c) and further verified by stem cell markers -GF141 (Figure 1d), and SSEA-1 (Figure 1e). The colonies were further selected and flourished on freshly gelatin-coated plates, which offered effective reprogramming. The non-programmed cells were cultured in parallel in the same conditions (Figure 1f).

Neural progenitor cells derived from iPSCs
Upon successfully generating pluripotency cells by using freeviral re-programming system, our auxiliary interests were to produce neuronal phenotype cells. By applying the specific Neural Induction Medium, iPSCs were transformed into neural progenitor cells from Days 20 to 35 (Figure 2a, with nestin staining as a neural progenitor mark; and Figure 2b), suggesting significant differences with the control group in the morphology (Figure 2c).

Generation of mature dopaminergic neurons from neural progenitor cells
Following the transformation of the neural progenitor cells, cells were further steered to differentiate into midbrain-like mature dopaminergic neurons (Figure 3a) by using the Dopamine Neuronal Differentiation Medium from Day 36 to 50. The cells developed to form mature neural networks (Figure 3b). The presence of the neuronal marker tyrosine hydroxylase (TH) was detected for verification ( Figure  3c). However, no significant modification was observed in the original non-reprogrammed adult fibroblasts in the parallel cultures ( Figure  3d).

Long-term cell culture for preparation of cancer risk assessments
Long-term observation of these cell lines until 6 months after the initial fibroblast cell re-programming revealed that the cultured dopaminergic neurons still had a pronounced ability to form mature neuronal networks. In contrast, at the 6-month mark, tail-tip fibroblast cells grown in control cultures (i.e., without cell re-programming and differentiating media) maintained their original shape and failed to form any visible cellular networks (Figure 4a). The TH genomic DNA of total Exons 1-13 were further abstracted to produce PCR products on gel from these long-term culturing cells (Figures 4a and 4b) for cancer risk assessments (Figure 4c).

Overall assessment for potential cancer risk
We assessed overall potential cancer risk by the expression of four re-programming genes cMyc, Oct4, Sox2, and Klf4 (all 4 genes for both exogenous and endogenous gene expressions), plus two stem cell bio-markers SSEA1 and Nanog (all two genes for endogenous gene expressions) for 6-month cultured mass iPSC-derived neurons. These were compared with 3 control groups: original non-reprogrammed mice tail-tip cells from parallel cell cultures, original mice midbrain tissues, and original tail tissues. The results suggested that six gene expressions of the 6-month cultured dopaminergic neurons did not differ from that of the three control groups in mRNA level by real time qPCR ( Figure 5).

Screening and identifying final product at end of cell culture
During the first 50 days under the forcibly directed conditions of the cell cultures, the re-programmed stem cells developed in five different stages: tail-tip fibroblasts, iPSCs, neural progenitor cells, dopamine neural progenitors, to dopaminergic neurons. The dopaminergic neurons were the final product. After 6 months, we further screened out and distinguished gene expressions for the most important neurotransmission factors: TH, doublecortin (Dcx), choline acetyltransferase (Chat), serotonin (SLC6A4), and norepinephrine (SLC6A2) from the samples of the cultured iPSC-derived neurons. We measured mRNA expressions of these genes by RT-qPCR-TaqMan with specific primers. Our results detected that there was only a boost in TH gene expression; the other genes did not present any expressions ( Figure 6).

Tyrosine hydroxylase genomic DNA analysis
Based on the overall assessment for potential cancer risk and elevated TH as the final product, we further scrutinized the TH    c Each pair on PCR gel in same size bp for total 10-pair genomic DNA TH from a cells on left side, and from b cells on right side above Figure 4: Long-term cell culture. After 6 months, the neuronal cells were displaying further signs of mature network formation from the re-programmed stem cellderived dopaminergic neurons (Figure 4b). Importantly, control tail cells that were not subject to the re-programming procedures kept their original shape in the same time period (Figure 4a). Genomic DNA TH was abstracted (Figure 4c) from both cell lines (Figure 4a and 4b) for further cancer risk assessments. A total of ten pairs with the same size (bp) of PCR products (from Figure 4a and 4b) were selected to run on gel representing complete Exons 1-13 (ID numbers) of TH genomic DNA (Figure 4c). In each of the same size pairs on gel, the cells band (PCR products) on the left side were from the original control non-programmed tail cells (Figure 4a), while the cells band on right side came from the re-programmed stem cell-derived dopaminergic neurons (Figure 4b). Here, the sizes (bp) of PCR product bands on the gel showed that of genomic DNA TH in Fig. 4-c. The relevant sizes with Exons 1-13 and cDNA, which matched with related genomic DNA, were also suggested in Figure 4c in order to highlight the relationships among them. Brain tissues Figure 5: Overall evaluation of cancer risk. By using real time qPCR with TaqMan-gene-specific primers for four re-programming genes (cMyc, Oct4, Sox2, Klf4) plus two stem cell bio-markers SSEA1 and Nanog, the overall potential cancer risk was assessed by comparing 6-month bulk cultured re-programmed stem cellderived dopaminergic neurons with other 3 control groups: original non-reprogrammed mouse tail-tip cells from parallel cultures, original mice midbrain tissues, and original mice tail tissues. There were no significant differences in the gene expressions of all biomarkers among the four groups. The values of the gene expressions were normalized to the level of control housekeeping gene GAPDH.
genomic DNA through gene sequencing. We examined whether or not the re-programming genes occurred with incidents of genomic DNA integration into host cells and/or mutagenesis of host cells.
Our results (Figure 7 and Supplementary data) demonstrated that all cells and tissues had identical genomic DNA sequences, with no any evidence of integration or mutagenesis for TH genomic DNA after reprogramming.

Discussion
At present, the re-programming of genes for transformation of  somatic cells into stem cells are predominantly connected with viral vectors, which are a safety concern for potential cancer risks due to viral genomic DNA integration and mutagenesis for host cells. This has seriously limited potential clinical applications. Recently, researchers have made great advances to create non-viral vector models to replace viral models. However, the risks are not completely eliminated, which has attracted our attention for compulsory evaluation. In addition, the mechanism of non-viral re-programming is still unclear. In order to reach these conclusions, we chose and applied an existing excellent non-virus-vector system with four re-programming genes (vector 20866) for our studies.
This system efficiently re-programmed adult mouse fibroblasts (tailtips) to produce iPSC, progressively differentiated neuron progenitor cells, and TH-positive dopaminergic neuron cells, which was the final product under the forcibly directed conditions of the cell cultures. Our experiments showed that only TH gene expression was detected, while the other important neurotransmitters (Dcx, Chat, serotonin, and norepinephrine) did not present any expressions. The results did not surprise us, since the conversion of tail-tip fibroblasts into neuronal cells was being cultured under forced conditions for producing only unique neuron cells and further dopamine cells [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34].
Significantly, there have been no published reports demonstrating the exact mechanisms of "gene re-programming" for non-viral systems. The possibilities are thought to include: 1) the insertion of foreign genes; 2) the modification of existing genomic DNA; and 3) the activation or silencing of existing genes. We observed a tissueand-cell-specific pattern of TH expression, which could be viewed as lending support to an epigenetic mechanism of reprogramming. Tail-tips and re-programmed stem cell-derived dopaminergic neurons showed the exact same genomic sequences of TH, however, only the re-programmed stem cell-derived dopaminergic neurons presented gene expression. These findings suggest that the mechanism of reprogramming may make silence TH expression in the tails to present boosted gene expression in the re-programmed dopaminergic neurons. Although we did not know the detailed pathways for gene silencing, the re-programmed stem cell-derived dopaminergic neurons may not involve changes in the actual genetic code at the genomic DNA level. However, this could potentially involve epigenetic factors, wherein methylation or acetylation of DNA sequences could be influencing TH expression following re-programming. Explicitly, the mechanism of re-programming might involve epigenetic changes that occur via a pattern of synthesized mRNA and protein, which acts to promote gene silencing in adult mice fibroblast tail-tips, which may then be converted into the activated expression of TH-positive dopaminergic neurons.
Of note, two directions may become apparent for the complete procedure: 1) the re-programming genes such as cMyc, Klf4, Oct4 and Sox2 may be present from gene expression to silence, and their functional role is only for "starting" cell life as embryotic cells, but not for developing into "mature" and "specific" types of cells such as neuron, heart, and lung, etc. 2) Gene TH-positive dopaminergic neurons may advance into phenotype from silence to activity, which are fully developed into "mature" and "specific" cell types under the forcedly direction conditions such as specific mediums. The contradictory directions cause development for re-programmed somatic cells to develop into specific types of cells, while re-programming genes become into silence. The entire developing stages handle progression from genotype to phenotype.
Given the association of cancer with mutagenic events, our findings support the argument that our free-viral pluripotency-inducing system may provide a lower risk of genetic pathology than the commonly used alternate viral procedures. Thus, the ultimate significance is that our results may provide numerous potential downstream clinical applications. However, this is speculative and it is still unclear as to the nature of the gene-transcription factor interactions occurring during (and after) re-programming. Free-viral methods lessen the potential risk of detrimental mutagenic events and the re-programming mechanism could involve the activation of previous silent genes from tail-tip cells to dopaminergic neurons. In order to confirm that this method carries less risk, the indication of tumorigenesis with varied approaches such as immunocompromised mice and soft agar assays should be further to provide further supporting evidence.
In conclusion, we determined that the non-viral system was an efficient and safe system by which to re-programmed somatic cells into iPSC and subsequent developing neuron cells. This system may become an innovative method for future therapeutic applications, such as neuroplasticity and neuroprotection.