Improvement of transfection with reprogramming factors in urinederived
cells

Human-induced pluripotent stem cells (iPSCs) are an accessible source of adult-derived, patient-specific pluripotent stem cells for use in basic research, drug discovery, disease modeling, and stem cell therapy. Improving the accessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application. Therefore, our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells (RECs) using different extracellular matrices and transfection reagents. In this study, we began by culturing urine-derived cells from healthy donors to establish a primary culture of renal epithelial cells, followed by their characterization. Subsequently, we generated iPS-like cells by transfecting renal epithelial cells (RECs) with vectors expressing Oct4, Sox2, L-Myc, Lin-28, and Klf4, and we compared the efficacy of different extracellular matrices and transfection reagents. The resultant iPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markers Oct3/4, Nanog, Lin28, and Klf4. We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like cells under the conditions tested. Moreover, the three matrices are similar in their efficiency of obtaining iPS-like cells. This report provides an experimental protocol for obtaining and generating iPS-like cells from urine samples for further cell therapy research on different human diseases.


Introduction
Since it has been shown that the human somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) by expressing specific transcription factors, it has opened unprecedented opportunities in basic research, drug discovery, disease modeling, and stem cell therapy. The first report on iPSCs was published in 2006; iPSCs were generated by exposing murine fibroblasts to retroviruses expressing four reprogramming factors: Oct3/4, Sox2, c-Myc, and Klf4 (Takahashi and Yamanaka, 2006). Then, the first generation of human iPSCs was constructed by the transduction of adult human fibroblasts with the same reprogramming factors (Takahashi et al., 2007). Along with these reports, another research group generated human iPSCs by the transduction of human fetal fibroblasts with the Oct4, Sox2, Lin28, and Nanog reprogramming factors (Yu et al., 2007). The hazard of retrovirus genome integration into the host genome raises the possibility of insertional mutagenesis and oncogene activation (Anson, 2004). Therefore, the production of virus-free iPSCs is a critical safety concern for their potential clinical application in cell and gene therapy. Okita and colleagues described an efficient method to generate virus-free iPSCs derived from mouse embryonic fibroblasts by transient plasmid expression (Okita et al., 2008). Patient-specific iPSCs have been widely used to study the mechanisms of various diseases and test new pharmacological therapies in a variety of human diseases (Crandall and Lalande, 2013). These iPSCs can be generated from various cellular sources ranging from skin fibroblasts to blood cells; however, endometrial and urine-derived cells hold tremendous clinical interest because they can be obtained through noninvasive methods to minimize the collateral risks of biopsy in patients with diseases such as diabetes or hemophilia (Ding et al., 2015). Due to variation between iPSC clones obtained from different cell sources, feeder-free systems, and culture conditions, it remains important to establish a simple protocol for obtaining iPSCs regardless of the cellular source or patient's background (Nagasaka et al., 2017;Chin et al., 2009).
This study aimed to report a simple protocol for obtaining iPS-like cells from urine-derived renal epithelial cells from healthy donors using nonviral vectors. Here, we present a comparison of commercial extracellular matrices and transfection reagents in urine-derived cells. Our results showed that the transfection reagent is a significant factor to consider in the efficacy of obtaining iPS-like cells; meanwhile, there was a similar efficacy among the three matrices tested. These results can be further used to support protocol selection for the generation of iPS-like cells from patients with different medical conditions.

Materials and Methods
Urine sample collection Urine samples were obtained from three healthy male donors. This work was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee from the School of Medicine, Universidad Autónoma de Nuevo León (Monterrey, México) under Reg. HT17-00002. All volunteers who participated in this study signed written informed consent before donating urine samples.
Donors were asked to clean their urethral area with flushable, antibacterial, premoistened wipes, and discard the first urine stream into the toilet before collecting their urine in sterile containers. All samples (ranging between 100 and 200 mL each) were processed within 60 min post-collection.
Primary renal epithelial cell culture Urine samples were transferred to 50 mL-tubes inside a culture hood and centrifuged at 300 × g for 10 min at room temperature. The supernatant was carefully discarded by pipetting inside a culture hood, leaving approximately 0.2 mL of urine in the tube, and the cell pellets were washed three times with 10 mL of 1X PBS containing 100 U/mL penicillin/streptomycin (Gibco, Thermo Fisher Scientific, Lafayette, CO, USA, #15240062) at 300 × g for 10 min at room temperature. The cell pellet was resuspended in 1 mL of Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) (Gibco, Thermo Fisher Scientific, Lafayette, CO, USA, #11320033) supplemented with the Renal Epithelial Cell Growth kit (RECG) (ATCC, Manassas, VA, USA #PCS-400-040) and 100 U/mL penicillin/streptomycin. From this point onwards, this medium mixture is referred to as the primary medium. The cell suspension was seeded into 12-well plates coated with 0.1% L-gelatin (Sigma-Aldrich, St. Louis, MO, USA, #G1393) in 1X PBS. Every day during the first three days post-isolation, 1 mL of the primary medium was added. Beginning the next day, half of the medium was replaced with fresh renal epithelial cell basal medium (RECBM) (ATCC, Manassas, VA, USA, #PCS-400-030) supplemented with the RECG kit and 100 U/mL penicillin/ streptomycin daily before visible cells/colonies appeared and after the establishment of a primary culture of renal epithelial cells (RECs). The first full change of medium was made after the first colonies were observed to maintain factor secretion from the urine-derived cells and avoid unnecessary stress.
stem cell reagent (STEMCELL Technologies, Cambridge, MA, USA, #05872) and seeded into a 48-well plate to continue clonal expansion. The iPS-like colonies were expanded for two months prior to further assays.

iPS-like cells characterization
iPS-like expanded colonies were seeded into a 24-well plate coated with Matrigel, Geltrex or vitronectin and incubated until 80% confluence was reached. Then, the cells were fixed with methanol/acetone, washed with 1X PBS, and blocked with 3% normal goat serum for 30 min at 4°C. Cells were washed and incubated with the following primary antibodies overnight at 4°C: anti-Nanog ( Cellular nuclei were stained with DAPI for 10 min and washed with 1X PBS. The slides were mounted with VectaShield ® and analyzed by fluorescence microscopy (Leica, DM1000), followed by photographic documentation with the Qcapture Pro 7 program (QImaging). Representative microscope fields are shown.

Analysis of gene pluripotency expression in iPS-like cells
For the analysis of gene expression, RNA isolation from clones obtained of iPS-like cells was performed with the GeneJet RNA Purification kit ® (Thermo Fisher Scientific, Lafayette, CO, USA, #K0731) according to the manufacturer's instructions. One microgram of the RNA transcripts was used for cDNA synthesis using the Maxima First Strand cDNA Synthesis kit ® for RT-PCR (Thermo Fisher Scientific, Lafayette, CO, USA, #K1641). Once the cDNA was obtained, the RT-qPCR was performed using the Maxima SYBR Green/ ROX qPCR Master Mix ® (Thermo Fisher Scientific, Lafayette, CO, USA, #K0221) with the following oligonucleotides: Oct3/ 4, Sox2, Nanog, Lin28, Nodal, Rex1, and GAPDH. GAPDH was used to normalize the gene expression (Tab. 1). qPCR reactions were performed using a StepOne Real-Time PCR System (Applied Biosystems). Calculations were made using the 2 −ΔΔCt threshold cycle method and normalized to the expression of the endogenous gene GAPDH.

Statistical analysis
The results were analyzed by one-way ANOVA and Tukey's multiple comparisons test and plotted using Prism software v.6 (GraphPad, Inc., San Diego, CA, USA). p < 0.05 indicates statistical significance.

Results
Establishment of primary renal epithelial cell culture from the urine samples of healthy donors The isolation and culture of renal epithelial cells generate colonies with different morphologies named types 1 and 2.
Type 1 cell colonies have irregular shapes and consist of spindle-like cells. In contrast, type 2 cell colonies are smooth (Dörrenhaus et al., 2000). To obtain renal epithelial cells, we isolated urine-derived cells from the samples of healthy donors. Within the first day after isolation, the cell morphology was similar to that of squamous cells and predominated by small, round cells. After 6 to 9 days, small colonies with irregular and smooth edges were observed, which was consistent with renal epithelial cell morphology. At 12 to 15 days after their isolation, cells were harvested after no more than four passages to continue the expansion of the renal cells to further characterize and reprogram the cells (Figs. 1a and 1b).
Reports have indicated that renal cells express epithelial markers as cadherin, cytokeratin, zonula occludens-protein 1 (ZO-I), and others (Zhou et al., 2012). Immunofluorescence confirmed that primary cultures of RECs express the epithelial-specific markers: cytokeratin, E-cadherin, β-catenin, ZO-I, and proximal tubular epithelial markers: neutral endopeptidase (CD10) and aminopeptidase N (CD13) (Fig. 2). The expression of epithelial-specific marker proteins indicated the successful establishment of a primary renal epithelial culture from the urine samples of healthy donors. We observed an isolation rate of 75% among all the donor samples containing cells that proliferated to colonies.
Renal epithelial cell reprogramming using nonviral vectors and different extracellular matrices Somatic cells can be reprogrammed to iPSCs by overexpressing certain defined transcription factors. iPSCs can be generated by using nonintegrative vectors, such as adenovirus or plasmids, with the latter preferable for clinical applications (Stadtfeld et al., 2008;Okita et al., 2013). We transfected RECs at passage 3 with nonviral vectors expressing the reprogramming factors Oct4, Sox2, Lin28, L-Myc, and Klf4 to induce cell dedifferentiation into pluripotent stem cells. Morphological changes are indicative of cell reprogramming. After 12 days of transfection, we observed small colonies made up of polyhedral cells with large nuclei and scarce cytoplasm, which are similar to the morphology criteria of ESC (embryonic stem cell)-like colonies (Nagasaka et al., 2017). By day 20, compact colonies were observed, selected, picked up under the microscope, and expanded for further characterization (Figs. 3a and 3b).
Three different extracellular matrices and two transfection reagents were tested. Cells seeded on Matrigel, Geltrex or vitronectin generated more iPS-like colonies when transfected with Lipofectamine stem cell reagent than with FuGENE reagent. Statistical analysis revealed no significant difference between the extracellular matrices; moreover, upon transfection reagent analysis, we detected that Lipofectamine stem cell reagent is significantly more effective than FuGENE in obtaining iPS-like colonies from urine-derived renal epithelial cells in these conditions (Fig. 3c).

iPS-like cells derived from RECs express pluripotency markers
Next, we aimed to determine whether the initial iPS-like colonies could maintain their pluripotent state after clonal expansion. Immunofluorescence confirmed that the iPS-like cells with no more than three passages expressed the following pluripotency-specific proteins after their expansion: Oct4, Klf4, Lin28, and Nanog (Fig. 4). Moreover, to confirm the pluripotency of the iPS-like cells obtained, we conducted a qPCR assay to assess the expression of ESC markers. The results confirmed that the iPS-like cells expressed pluripotency-specific genes: oct3/4, sox2, lin28, nanog, nodal, and rex1 (Fig. 5). Of the genes analyzed, nanog, nodal, and rex1 were not used to reprogram the RECs. Due to the expression of pluripotency-associated transcription markers, we can conclude that iPS-like cells were successfully generated from urine-derived renal epithelial cells.

Discussion
The human kidney contains an extensive network of tubules; those cells from the renal tubular system and urinary tract are detach and excreted daily in the urine (Rahmoune et al., 2005).
Urine-derived cells are a source of somatic cells obtained without invasive intervention, which is the principal advantage of urine-derived cells over other somatic cells sources, such as mesenchymal stem cells, fibroblasts, or blood cells. Therefore, urine-derived cells have a prospective clinical future to generate iPSCs-specific of patients (Manaph et al., 2018).
In this study, we performed the isolation of urine-derived cells from healthy adult donors, the establishment of primary cultures of renal epithelial cells, and their further reprogramming into iPS-like cells using nonviral vectors expressing Oct4, Sox2, L-Myc, Lin-28, and Klf4. In addition, we compared the abilities of different commercial extracellular matrices and transfection reagents to support the efficient acquisition of iPS-like colonies.
There have been several reports of urine-derived cells isolated from patients with different disease conditions (Chen et al., 2013;Park et al., 2015;Afzal and Strande, 2015). Our study is based on a previously reported culture method (Zhou et al., 2011). We used DMEM/F-12 supplemented with RECG factors as a primary medium and renal epithelial cell basal medium (ATCC) containing RECG factors (ATCC) as urine-derived cell culture medium; meanwhile, Zhou and coauthors used DMEM/F-12 and renal epithelial cell basal medium (Lonza) supplemented with SingleQuot factors ® (Lonza). We observed colonies with irregular and smooth edges that appeared after 3-6 days of culture, which is consistent with the observations of Zhou et al. (2011). RECs cultures were established from over 75% of the isolates from healthy donor samples that contained viable cells; this percentage is higher than the isolation rates of 37% and 52% reported from healthy donor samples (Dörrenhaus et al., 2000;Belik et al., 2008).
Next, we seeded renal epithelial cells in Matrigel, Geltrex or vitronectin and dedifferentiated the RECs into iPS-like cells using nonviral reprogramming factors. iPS-like cells obtained
from the culture with Matrigel, Geltrex, or vitronectin extracellular matrices had an hESC (human embryonic stem cell)-like morphology with central and prominent nuclei, scarce cytoplasm, and small and polyhedral colonies. Importantly, the iPS-cell colonies expressed the pluripotency markers Oct4, Klf4, Nanog, and Lin28. Additionally, we confirmed by qPCR the expression of pluripotency genes oct3/4, sox2, nanog, lin28, nodal, and rex1. Of the genes analyzed, nanog, nodal, and rex1 were not used to reprogram the RECs. Nevertheless, further assays are required for the exhaustive characterization of the iPS-like cells generated with each experimental condition. Over the years, progress has been made in defining the molecular arrangement of components in the basement membrane due to variation between iPSCs lines obtained with different feeder-free systems and culture conditions (Amit et al., 2004;Sun et al., 2009;Nagasaka et al., 2017). Interactions between cells and ECMs have important roles in the regulation of cell functions, and their composition change with different cell types and phenotypes (Frantz et al., 2010). Cai et al. (2015) show that the surfaces coated with ECMs from bone marrow mesenchymal stem cells (MSCs), dermal fibroblasts, and osteoblasts promoted cell adhesion more strongly than surfaces coated with ECMs from osteosarcoma cells. Additionally, the ECMs promoted the proliferation of MSCs while they inhibited the proliferation of osteosarcoma cells. Therefore, the coating cell source is important for investigating the effect of FIGURE 2. Expression of renal epithelial cell-specific markers. RECs were obtained from urine samples from healthy donors. Microphotographs show positive signals (red channel) for cytokeratin, E-cadherin, β-catenin, ZO-1, which are specific markers for epithelial cells; CD10 and CD13, which are specific markers for renal epithelial cells, detected by immunofluorescence. Nuclei were stained with DAPI. Ab IgG refers to a non-specific IgG antibody as a control. Scale bar = 50 µm. specific or a combination of a few proteins from ECMs. In another study, Rojas and coauthors found that a fibrinogen matrix improved cardiac iPSC retention in an experimental model of ischemic heart failure (Rojas et al., 2015).
Matrigel/Geltrex, one of the most widely used extracellular matrices for the feeder-free growth of undifferentiated hESCs, is extracted from Engelbreth-Holm-Swarm mouse tumor and consists of a mixture of laminin, collagen IV, heparan sulfate proteoglycan, and nidogen-1 (Kleinman et al., 1982;Stojkovic et al., 2005). Individual basement membrane components have been examined in other reports. Cells seeded on surfaces coated with laminin, collagen IV, and fibronectin results on compact colonies of hESCs, although cultures maintained on fibronectin or collagen IV did not contain as many colonies as those maintained on Matrigel or laminin (Xu et al., 2001), which suggest the importance of all basement membrane components for the establishment of undifferentiated hESC colonies.
Additionally, the expression and function of iPSCs integrin extracellular matrix receptors have been investigated on iPSCs cultures with feeder layers, Matrigel, or vitronectin to understand the interaction between iPSCs and extracellular matrix components. Cultures maintained on Matrigel require β1 integrins for adhesion, while cultures maintained on vitronectin require αvβ5 for adhesion. In contrast, blockade of β1 integrins did not affect adhesion to vitronectin, and the inhibition of αvβ5 did not affect adherence to Matrigel. However, integrins β1 and αvβ5 were shown to mediate iPSCs proliferation on vitronectin, whereas only β1 was required for iPSCs proliferation on Matrigel (Rowland et al., 2010). Furthermore, we have shown that more iPSC colonies were obtained with Matrigel than with vitronectin. This observation could be due to the high content of laminin and collagen IV in Matrigel, which interacts with iPSCs through β1 integrins such as α2β1, α3β1, α7β1, and α11β1 to promote cell adherence, while only αvβ5 integrins are responsible for adherence to vitronectin (Braam et al., 2008;Miyazaki et al., 2008).
The selection of the optimal transfection method is essential for the efficient establishment of iPSC colonies. The transfection capability of Lipofectamine 2000 and FuGENE was reported in mouse embryonic stem cells (Tamm et al., 2016). Additionally, Lipofectamine 3000 was used to report a simple protocol for transfection of MSCs, a cell type known to be difficult to transfect (de Carvalho et al., 2018). In this study, we compared the capability of Lipofectamine stem cell reagent with FuGENE reagent to establish a simple method to obtain iPS-like cells from RECs, which are difficult to transfect. We observed more iPS-like colonies using Lipofectamine Stem Cell reagent than FuGENE reagent regardless of whether Matrigel,   Molinari and Sayer, 2019). In this report, we tested a non-viral method for generating integration-free iPS-like cells under different feederfree culture conditions and transfection reagents using urinederived cells, which are obtained without invasive intervention.
Compared to currently used non-viral methods that use sophisticated transfection methods to obtain iPSCs from urine-derived cells and other somatic cells obtained through invasive procedures on different diseases (Lee et al., 2017;Li et al., 2016), the conditions tested here represent an improvement in iPSCs technology for experimental research Characterization of iPS-like cells by immunofluorescence. Microphotographs show positive signals (red channel) for the pluripotency markers Oct4, Klf4, Nanog, and Lin28. Nuclei were stained with DAPI. Ab IgG refers to a nonspecific IgG antibody as a control. Scale bar = 50 µm.
FIGURE 5. Gene expression of pluripotency markers. iPS-like cells were analyzed by real-time PCR. The graph shows the expression of pluripotency genes oct3/4, sox2, nanog, lin28, nodal, and rex1. Reference control was RECs. GADPH was used for normalization. Mean ± SD are shown. due to the accessibility to obtain iPS-like cells from urinederived cells using different transfection reagents and extracellular matrices. We report results using different conditions to obtain iPS-like cells, which can be very useful when deciding which method to use in experimental research.

Conclusions
A human urine-derived renal epithelial cell primary culture from healthy donors was performed. Additionally, iPS-like cells were obtained from a primary culture of RECs by a virus-free and feeder-free system. We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like colonies from urinederived cells under the conditions reported. Moreover, the three matrices are comparable in their efficiency to obtain iPS-like cells. This report provides an experimental protocol to obtain and generate iPS-like cells from urine samples for further cell therapy research on different human diseases.

Author Contributions
All authors made substantial contributions to this study. ORR, MLA, JPT, and EPG participated in data collection and manuscript preparation, ORR, MLA, JPT, AVO, ASD, OSC, and RMOL, participated in the analysis and interpretation of data, LVC, HRR, AGG, OSC, participated in manuscript preparation and revision, MLA and RMOL designed the study and participated in manuscript preparation. All authors read and approved the final version of the manuscript.

Acknowledgement:
The authors thank Bianka Dianey Camacho Zamora for her help in PCR real-time advisory. ORR thanks CONACyT for its support through a scholarship under register number 296354.
Funding Statement: This research was funded by the Programa de Apoyo a la Investigación Científica y Tecnológica (PAICyT; grant no. SA813-19) from the Universidad Autónoma de Nuevo León and the Consejo Nacional de Ciencia y Tecnología (CONACyT; grant no. CB-2015/255725).