Generation of Integration-Free Porcine Induced Neural Stem Cells Using Sendai virus

biomedical research. Thus, this study aimed to establish safe and efficient integration-free piNSC lines. The integration-free piNSC lines were generated by reprogramming porcine fibroblasts using the 4 Sendai virus (SeV). Here we report the successful generation of integration-free piNSC lines using the SeV, with a 7 reprogramming efficiency of 0.4%. The piNSCs can be expanded for up to 40 passages and express 8 high levels of NSC markers (PAX6, NESTIN, and SOX2). They can produce neurons and glia, 9 expressing TUJ, MAP2, TH, and GFAP. No induced pluripotent stem cells developed during 10 reprogramming, and the established piNSCs did not express OCT4. Hence, the SeV can reprogram 11 porcine fibroblast without first going through an intermediate pluripotent stage. With the SeV approach, we generated integration-free piNSCs that may be used to assess the 14 efficacy and safety of iNSC-based clinical translation in humans.


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The remarkable discovery that differentiated cells are able to be completely reprogrammed to 20 induced pluripotent stem cells (iPSCs) by viral-mediated transduction of exogenous transcription 21 factors marks a significant breakthrough in regenerative medicine [1]. The iPSCs offer an infinite 22 supply of differentiated cells for various purposes, including disease modeling in vitro, drug 23 research, toxicity testing, and autologous cell-based therapy [2]. The potential for patient-specific 1 cells to be used in autologous cell-based treatments is quite intriguing. The generation of neural 2 stem cells (NSCs) and neurons from iPSCs is one of the most clinically relevant cell types [3][4][5].  Alternatively, the forced expression of the NSC transcription factors [9][10][11] or the pluripotency 9 transcription factors, encoding Oct3/4, Sox2, Klf4, and c-Myc (OSKM) [12][13][14] converts 10 differentiated cells directly into induced neural stem cells (iNSCs) and induced neural progenitor 11 cells (iNPCs). This approach is an appealing alternative to existing iPSC technology because it 12 enables the production of patient-specific NSCs without passing through the pluripotent stage, 13 thereby decreasing the risk of tumorigenic potential [15,16]. Since the first mouse iNSCs NSCs, such as morphological, self-renewal capacity, gene and protein expression profiles, 18 epigenetic state, as well as functional multipotenc y in vitro and in vivo [9,21,22]. Additiona lly, 19 when iNSCs are transplanted into animal models for up to 6 months, they can alleviate disease 20 phenotypes without developing tumors, demonstrating their therapeutic promise for neurological 21 disorders [23]. 22 Although the iNSCs have been discovered as a feasible, effective, and autologous source for 1 medical applications, their therapeutic potential has yet to be fully explored. Porcine iNSCs 2 (piNSCs) may serve as a disease model for human regenerative medicine, as pigs have established 3 themselves as one of the most effective large animal models in biomedical research, often regarded 4 as a preferable alternative to rodent models [24][25][26]. Furthermore, preclinical evaluation of stem 5 cell transplantation using piNSCs and their differentiation cells may be utilized to determine the 6 safety and efficacy of iNSCs prior to human trials. Importantly, piNSCs are also an appealing cell 7 source for investigating pig disease in veterinary medicine. However, no commercially available 8 piNSC and their neural differentiation exist for studying pig neurological diseases, such as 9 Streptococcus suis infections and African Swine Fever. Until now, only one group has reported 10 success in generating piNPCs using nonintegrating episomal plasmids. They demonstrated that 11 piNPCs retain the capacity to grow for an extended period of time and differentiate efficiently into 12 neurons in vitro [27].

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Although previous studies have established a number of methods for directly converting somatic 15 cells to iNSCs, most of the investigations rely on integrating viral vectors (such as lentiviral or 16 retroviral approaches) [9,13,15,28,29]. These methods may result in insertional mutagenesis and 17 the persistence or reactivation of transgenes. Moreover, therapeutic translation of this technique 18 will require a thorough safety evaluation of any mutations gained during the reprogramming 19 process, as well as a fast derivation and differentiation strategy [30]. A Sendai virus (SeV) vector 20 can overcome these issues owing to a single-stranded RNA virus propagating in the cytoplasm of 21 infected cells that does neither pass a DNA phase nor integrate into the host genome, unlike the 22 other viruses. As a result, the risks of tumorigenesis can be reduced throughout the reprogramming 23 process [31]. With the SeV delivery system in kits, researchers may easily transduce the desired 1 cells with SeV carrying OSKM for reprogramming and quickly remove SeV and transgenes by 2 temperature change as temperature-sensitive. Recently, SeV-based vectors have been widely 3 utilized to generate human, and mouse integration-free iPSCs [32][33][34][35] and have been adapted to 4 generate iNSCs from human and monkey postnatal and adult fibroblasts [18]. However, the 5 generation of piNSCs using the Sev has not been explored yet. To extend our findings into clinical applications, therapeutic approaches will rely mainly on 8 personalized iNSC transplantation. We emphasize here that iNSCs require an effective 9 cryopreservation method in order to obtain good results upon transplantation. Cryopreservation 10 enables the storage and transportation of iNSCs for clinical purposes. Thus, iNSCs should be 11 cryopreserved with a high survival rate and minimal influence on cellular characteristics like 12 proliferation and differentiation potential during freezing and thawing. Ascorbic acid (Vitamin C) 13 is a water-soluble antioxidant that neutralizes the oxidative effects of radicals from the 14 cryopreservation process. To improve the survival of iNSCs during the cryopreservation 15 procedure, ascorbic acid was pretreated at various concentrations before freezing and its 16 neuroprotective effects were evaluated.

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In this investigation, we generated integration-free piNSC lines from pig fibroblasts by utilizing a 19 Sendai virus approach. The piNSCs displayed typical features of NSCs such as morphology, gene 20 expression patterns, self-renewal capacity, and differentiation potential. Moreover, pretreatment 21 of iNSCs with ascorbic acid did not affect the viability of iNSCs during the cryopreservation 22 procedure. We anticipate that piNSCs will serve as novel, easily accessible large animal models 23 for evaluating the efficacy and safety of iNSC-based clinical translation. This pig model will allow 1 us to assess the ultimate feasibility of cell-based regenerative therapy. Furthermore, our 2 integration-free piNSCs might be useful for disease modeling in pigs. As a result, this discovery 3 is beneficial for both veterinary medicine and the possibility of translation to human medicine.

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A porcine tail was received from an authorized farm in Ratchaburi Province, Thailand. Porcine 20 tail fibroblasts (PTFs) were extracted using standard procedures from the tail of a three-day-old 21 crossbred piglet, with minor modifications [36]. The PTFs were propagated in fibroblast medium 22 (FM) containing DMEM-high glucose, 10% fetal bovine serum (cat. no. SV30160,Hyclone,23 Logan, UT, USA), 1% Antibiotic-Antimycotic solution, and 1% GlutaMAX™. All cells were grown on feeder-free culture system (Matrigel-coated dishes or plates) throughout the piNSC 1 generation process. The PTFs were reprogrammed utilizing the integration-free CytoTune™-iP S 2 2.0 Sendai reprogramming kit incorporating human reprogramming factors, OSKM in FM 3 following the manufacturer's instruction, with modifications ( Fig. 1A). The PTFs (passages 3 ) 4 were seeded on 6-well plates at a density of 1x10 4 cells/cm 2 one day before viral transduction to 5 achieve approximately 60-70% confluency at the time of transduction. The PTFs were transfected 6 with SeV at a multiplicity of infection (MOI) of 5 in a FM for 24 h. The next day, the culture 7 medium containing the Sendai virus was removed and renewed with a FM. The following day, the 8 medium was switched to the iNSC medium (iNSCM) comprising DMEM/F-12 and Neurobasal 9 medium in a ratio of 1:1 supplemented with 2% B-27™ Supplement, 1% N-2 Supplement, 1% 10 antibiotic-antimycotic solution, 1% GlutaMAX™, 20 ng/mL human basic fibroblast growth factor 11 (bFGF; R&D Systems), and 10 ng/mL human epidermal growth factor (hEGF). Seven days after 12 reprogramming, the cells were dissociated with a 0.25% trypsin-EDTA solution and replaced onto 13 Matrigel-coated plate in piNSCM. From then on, the appearance of epithelium-like colonies was 14 monitored, and the medium was changed daily. Colonies with epithelium-like morphology were 15 large enough to be picked up around days 16 to 21 and transferred onto an IVF one-well dish for 16 expansion. Every 2-3 days, sub-culturing at a 1:5 ratio with Versene® Solution was conducted 17 consistently for further experiments.

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The piNSCs were passaged and plated at a density of 3x10 5 cells per well in 6-well plates, then 21 treated with ascorbic acid at various doses (0, 10, 50, and 100 uM) into NSC cultured medium for 22 12 hours before freezing to test the protective effects of ascorbic acid on cryopreservation of 23 piNSCs. A total of 5 × 10 5 cells was dissociated and placed in a cryo-tube (Nunc CryoTubes, 1 Thermo Fisher Scientific Inc., MA, USA) with 1 ml cryopreservation solution (iNSC medium with 2 10% DMSO). The vials were placed in a freezing container (Nalgene Mr. Frosty, Thermo Fisher 3 Scientific, MA, USA) for 24 h in a -80C freezer for cryopreservation. Subsequently, the vials were 4 transferred to LN 2 and stored for 1 week before thawing. Counting Kit-8 (CCK-8) assay, and the levels of nitric oxide (NO) were measured using the Griess 10 reagent (Promega) at various time points after thawing (at 1, 6, 12, 24, 36, and 48 h). A cell 11 proliferation study was performed by adding 10 ul of the CCK-8 reagent to live cells in 96-well 12 plates, and the suspension NSCs (100 ul/well) were incubated for 3 hours at 37°C with 5% CO 2 to 13 determine cell proliferation. The absorbance at 450 nm was used to determine the vitality of cells.
14 For Griess analysis, supernatant from each well was collected and transferred to a new tube.  The formation of neurospheres was investigated for iNSCs by resuspending 10,000 cells per well 1 in iNSCM in 96-well plates covered with poly (2-hydroxyethyl methacrylate). Every two days, a 2 fresh medium is added to the suspension cultures. Neurospheres were counted using a light 3 microscope seven days after the suspension and collected for further study. To induce spontaneous neuronal differentiation, piNSCs (P20) were dissociated and re-plated into 7 a 6 well dish or a 24 well plate with matrigel-coated at a density of 2 x 10 4 cells per cm 2 in the 8 neuronal differentiation medium (the piNSCM without bFGF and hEGF). The media was replaced 9 every two days for 14 days. Phase-contrast image analysis was performed every day to monitor 10 cell differentiation in each well. At days 0 (proliferating piNSCs) and 14 (neuronal differentiation) , 11 the cells were fixed with 4% paraformaldehyde for immunofluorescence analysis and were 12 manually detached for western blot analysis.

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The karyotype analysis followed a previously described procedure with minor modifications 16 [38]. Briefly, piNSC lines (P20) were cultured in a 6 cm culture dish at approximately 70% 17 confluence and treated with a 5 µg/mL colcemid solution (KaryoMAX™ Solution) for 1 h at 37°C.  The expression of the SeV genome and transgenes in both piNSCs was determined using RT-PCR.

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Cells were lysed and RNA extracted using the RNeasy Mini Kit (Genaid Biotech Ltd., New Taipei 3 City, Taiwan). The SuperScript TM III First-Strand Synthesis System was then used to reverse 4 transcribe 1 g of total RNA to cDNA. 50 ng template cDNA, 12.5L GoTaq PCR master mix 5 (Promega, WI, USA), and 0.2M of each primer were used in the PCR reaction. The PCR-amplified 6 separation was achieved on 2% agarose gels and imaged using GelRed® nucleic acid staining 7 (Biotium, Fremont, CA, USA).  as the mean ± standard error of the mean (SEM). The data was analyzed statistically using one-20 way analysis of variance (ANOVA) for comparisons of more than two groups and the Student's t-21 test for comparisons of two groups. Additionally, Tukey's test was employed as a post hoc multiple 22 comparison test for differences. All statistical analyses were carried out using the SPSS version 25 1 software (IBM, USA). Statistical significance was defined as p < 0.05. To address the question of whether PTFs can be directly converted into stably expanding 6 multipotent piNSCs, PTFs were isolated from the tail of a three-day-old crossbred piglet (Large 7 White/Landrace × Duroc). Non-integrative Sendai viral vectors carrying OSKM were transfected 8 into PTFs for 24 h (Fig. 1B). After that, cells were cultured in FM for one day to recovery and 9 then in iNSCM for another day. On day 7 after transduction, the cells were dissociated and 10 transferred onto Matrigel-coated 6 well plates in piNSCM at a density of 1 x 10 4 cells per well. On 11 day 11 after transduction, the iNSC clusters with neuroepithelial-like morphology emerged and 12 developed quickly over the next week. On days 16-21, the neuroepithelial-like morphology 13 colonies were mechanically triturated into small clusters, and re-plated onto Matrigel-coated 14 coverslips for immunostaining or onto a Matrigel-coated one-well dish for cell expansion in 15 iNSCM ( Fig. 1C, 1D). The neuroepithelial colonies expressing early NSC markers such as PAX6, 16 NESTIN, and SOX2 were continuously propagated using TrypLE Select Enzyme along the serial 17 passaging for further characterization (Fig. 1E-1G). Hence, the transfection efficiency was 0.40%, 18 as measured by the number of neuroepithelial colonies expressing PAX6, NESTIN, and SOX2 19 divided by the total number of transfected cells. We generated a total of nine iNSC lines capable 20 of proliferation in adherent monolayers (2D) (Fig. 1H) or neurospheres (3D) (Fig. 1I) as 21 suspension-grown neural cell aggregates and differentiation into neural lineages (Fig. 1J). We 22 chose only two iNSC lines based on their indefinite self-renewal potential and multipotency 23 differentiation, namely VSMUi002-B and VSMUi002-E, for further analysis. To remove the 1 temperature-sensitive SeV vectors, iNSCs were cultured at 39 °C in an incubator for 4 weeks, after 2 which the cells were collected every week for PCR detection of the remaining virus. The SeV 3 vectors were positive at passage 9 but vanished at passage 12, indicating that SeV vectors had been 4 completely eradicated. At passage 20, both piNSC lines (VSMUi002-B and VSMUi002-E) displayed a neuroepithelia l 8 morphology in adherent monolayers ( Fig. 2A). They displayed a high percentage of cells 9 expressing the NSC markers, with nearly 100% of cells staining positive for PAX6, SOX2, and 10 NESTIN, as determined by quantitative immunofluorescence analysis, indicating the formation of 11 a highly homogeneous population ( Fig. 2A, 2B). However, they did not express pluripotency-12 related genes, OCT4 ( Fig. 2A). As a result, the absence of OCT4 established that piNSC lines did VSMUi002-E than in VSMUi002-B. The PTF lacked both PAX6 and SOX2 protein expression 17 (Fig. 2C). The piNSC lines (VSMUi002-B and VSMUi002-E) had a high percentage of cells 18 expressing the proliferation marker Ki67 (75.3% ± 1.18% and 78.6% ± 1.19%, respectively) ( Fig.   19   2A, 2B). Furthermore, the population cell doubling time of the VSMUi002-B and VSMUi002-E 20 cell lines was approximately 24 h, with no significant difference (P > 0.05), and the cells had been 21 passaged over 40 times. Hence, both iNSC lines had a strong capacity for self-renewal. They 22 exhibited a typical diploid porcine karyotype (38, XY) during long term culture (Fig. 2D). Both piNSC lines were able to form neurospheres (3D) in suspension cultures with similar efficiency, 1 which were homogeneous in size and shape on day 7 (Fig. 3A, 3B). Additiona lly, 2 immunofluorescence labeling revealed that the neurospheres expressed NSC markers (PAX6 and 3 SOX2) and a proliferation marker (Ki67) (Fig. 3A). Thus, piNSCs display neural progenitor 4 features that can be obtained from PTF by Sendai virus reprogramming. To determine the ability of piNSCs to differentiate spontaneously, they were dissociated into single 8 cells and grown on a Matrigel substrate in a neural differentiation medium. After 7 days of 9 differentiation, piNSCs revealed significant morphological alterations, including a decrease in cell 10 body size (Fig. 4A), and expressed the immature neuronal marker (TUJ1). At day 14, the piNSCs 11 exhibited mature neuronal morphology, including extensive and complex neurites, which were 12 positive for the mature neuronal marker (MAP2) (Fig. 4A, 4B). The merged immunofluorescence (TH) (Fig. 4B). The piNSCs also developed into glial fibrillary acidic protein (GFAP)-positive 20 astrocytes (Fig. 4B). The staining intensities of all neuronal-positive cells (TUJ1, MAP, TH, SYN, 21 and GFAP) are the same in both iNSC lines (Fig. 4C). Moreover, qPCR analysis indicated an 22 increase in the expression of myelin basic protein (MBP), which is expressed mostly in oligodendrocytes (Fig. 4D), indicating the existence of oligodendrocytes in the differentiated 1 derivatives. To corroborate the immunofluorescence results, the endogenous TUJ1, MAP2 and 2 GFAP proteins in iNSC-derived neural differentiation were quantified by western blot analysis in 3 comparison to their parental iNSCs. The expressions of those proteins were substantially higher in 4 the neuronal differentiated from VSMUi002-E than in those from VSMUi002-B (Fig. 4B). Both 5 iNSCs lacked both MAP2 and GFAP protein expression (Fig. 4B). Interestingly, VSMUi002-E 6 also expressed TUJ1 during NSC stage. Taken together, our data indicate that piNSCs have the 7 capacity for multipotent neuronal differentiation.

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The piNSCs were treated with ascorbic acid at 0, 10, 50, and 100 uM for 24 h before freezing to 11 investigate the protective effects of ascorbic acid on cryopreservation. The cells were cultured in 12 24 well plates after being thawed for 24 hours and stained with KI67 for cell proliferation and 13 CASPASE 3 for cell apoptosis. Our findings revealed no significant differences in KI67 positive 14 cells across different concentrations of ascorbic acid (Fig. 5A, 5B). Furthermore, the absence of 15 CASPASE 3 positivity after freezing suggested that the cells did not undergo apoptosis. 16 Furthermore, after freezing, the cells were analyzed for cell proliferation using the CCK-8 test and 17 for nitric oxide (NO) levels using the Griess assay at 1, 6, 12, 24, 36, and 48 h. Our results indicated 18 that piNSCs grown in the absence of ascorbic acid proliferated more rapidly than those grown in 19 the presence of other ascorbic acid concentrations at 48 h (Fig. 5C). No significant differences in 20 NO levels across different concentrations of ascorbic acid (Fig. 5D). Our results revealed that 21 ascorbic acid had no neuroprotective impact on the cryopreservation of piNSCs. Here, we describe a safety approach for reprogramming porcine fibroblasts into iNSCs using the 3 temperature-sensitive SeV that has several benefits over prior studies: (i) the use of non-integration 4 SeV approaches avoids vector and transgenic sequences from being integrated into the iNSC  (e.g., spontaneous, undirected, and directed in vitro differentiation approaches). Our findings 23 confirmed that these piNSCs pretend to be differentiated into neurons and astroglia since 1 development into oligodendrocytes was less effective due to the short differentiation phase, 2 consistent with previous research [45].

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Moreover, we demonstrated that ascorbic acid has no neuroprotective effect on piNSC 5 cryopreservation. Contrary to another study, the NSCs generated in this culture system supplement 6 with ascorbic acid preserved their long-term growth capability, neural pluripotency, and ability to 7 differentiate into functional neurons [46].

Figure 5
The effect of ascorbic acid on cryopreservation of piNSCs. (A) Cell proliferation was determined with KI67, and cell apoptosis was determined using CASPASE3 at different ascorbic acid concentrations 24 hours after thawing. (B) the ratio of KI67-positive cells at varied ascorbic acid concentrations 24 hours after thawing. (C) The proliferation of cells was determined using the CCK-8 test at varied ascorbic acid concentrations and periods following freezing. (D) The Griess reagent was used to determine the nitrite level at varied ascorbic acid concentrations 24 hours after thawing. Scale bars represent 50 μm in (A).

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