Low-dose Polyethylenimine-Mediated Transfection to Generate Human Induced Pluripotent Stem Cell: an optimized protocol

Human dermal broblasts (HDF) can be reprogrammed through different strategies to generate human induced pluripotent stem cells (hiPSCs). However, most of these strategies require high-cost materials and specic equipment not readily accessible in most laboratories. Hence, liposomal and virus-based techniques can replace with polyethyleneimine (PEI)-mediated transfection to overcome these challenges. However, few researchers have addressed the PEI's ability to transfect HDFs. This study used PEI reagent to transfer oriP/EBNA1-based vector into HDFs to produce hiPSC lines. We rst described conditions allowing the ecient transfection of HDFs with low cytotoxicity and without specic types of equipment and optimized several parameters relevant to the transfection procedure. We then monitored the effect of different N/P ratios on transfection eciency and cytotoxicity using ow cytometry and uorescent microscopy. By the results, we found that transfection eciency was greatly affected by plasmid DNA (pDNA) concentration, PEI concentration, order of combining reagents, serum presence in polyplexes, and the duration of serum starvations. Moreover, using the optimized condition, we found that the N/P ratio of 3 achieved the highest percentage of HDFs positive for pGFP (~ 40%) with minimal cell toxicity. We nally generated hiPSCs using the optimized protocol and oriP/EBNA1-based vectors. We conrmed hiPSC formation by characterizing tests: Alkaline Phosphatase (AP) staining, immunocytochemistry (ICC) assay, real-time PCR analysis, in vitro differentiation into three germ layers, and karyotyping test. In conclusion, our results indicated that 25kD branched PEI could eciently transfect HDFs toward generating hiPSCs via a simple, cost-effective, and optimized condition. a normal karyotype of 46 demonstrated using chromosomal G-band analysis. These results suggest that hiPSC lines were successfully generated using our optimized PEI25K protocol.


Introduction
Induced pluripotent stem cells (iPSCs) have opened great perspectives for disease modeling, high throughput drug discovery, and cell therapy in a patient-speci c manner [1]. The genetic reprogramming of somatic cells toward a stem-cell-like state overcomes both the limitations of adult stem cells and the ethical issues encircling ESC use. In this approach, reprogramming of somatic cells occurs through forced and ectopic expression of de ned pluripotency transcription factors named OCT4, SOX2, NANOG, KLF4, and cMYC [2]. Although iPSCs provide an assurance platform for generating disease models and drug screening, the clinical application of the iPSCs has been limited due to the lack of safe and e cient pluripotency gene delivery systems [3]. Several strategies can be used to induce pluripotency in somatic cells, including utilizing viral vectors, non-viral vectors, small molecules, RNAs, and proteins. Each method of generating iPSCs has its advantages and disadvantages. For instance, viruses are well-known delivery carriers with high e cient gene transfer and long-term expression potential. However, integrative properties of viral vectors arise concerns about mutations and safety [4]. Hence, using viral vectors is not recommended in clinical therapies [5]. Several scientists attempted to develop novel strategies to establish non-integrative methods. These methods are classi ed into two categories; non-integrating viral vectors like Adeno and Sendai viruses-based vectors and non-viral vectors such as various nano-carrier systems. In the last two decades, there has been a growing interest in nanoparticle delivery systems for generating iPSCs [6] due to their great potential to enhance stem cell and biology research [7,8]. Among these vectors, cationic lipids are commonly administered in the iPSC technique. However, the cost of such commercial reagents such as Lipofectamine can be a limiting factor for labs facing nancial problems. In addition, several other successful nanoparticulated delivery vehicles have been introduced in the literature for generating iPSCs including polyketal nanoparticles, cationic bolaamphiphile, Poly-b-amino esters, and Calcium phosphate nanocomposite [9][10][11] (See [8] for a comprehensive review) Although the research on a greater understanding of such nanoparticles has being conducted with promising results, the optimized protocol and the properties enabling these nanoparticles to overcome several barriers to intracellular DNA delivery still need further investigations [12,13]. For example, in spite of the successful reprogramming ability of poly-b-amino esters, integrated transgenes were found in clones of hiPSCs and further investigations are still required to evaluate the feasibility of poly-b-amino ester-plasmid as a non-viral reagent [8]. Moreover, evidence have shown more potency of PEI for delivering genes of interest into cells in comparison to to one of the most common nanoparticulated delivery vehicles; calcium phosphate [14].
Despite common usage of different non-viral reagents for generating iPSCs from a wide range of somatic cells, cationic polymers such as polyethylenimine (PEI) which is one of the golden standard reagents for nucleic acid delivery applications [15,16], have been less investigated in iPSC technology. Cationic polymers are attracting considerable interest due to their easy generation, the capability of large scales transfection, the versatility of DNA-polymer conjugation, long storage stability, and economic cost. PEI belongs to one of the most studied and effective families of manufactured polycations, which are available with different sizes (KDa) and structures (linear or branched) depending on the particular synthesis procedure [17]. The PEI's ability to form complexes with nucleic acids and carry them into the nucleus has been widely documented in vivo and in vitro conditions [18]. PEI and its derivatives have a high concentration of positively charged nitrogen atoms giving them a considerable buffering capacity due to better condensation of large negatively charged molecules such as DNA [19]. The signi cant buffering capacity of PEI led to the proton sponge hypothesis which describes that unprotonated amines of PEI can absorb protons facilitating the subsequent release of polyplexes into the cytoplasm [20]. In addition, Wang et al. showed that the total amount of pDNA within the cytosol was similar among cultured cells after transfection, showing PEI's prominent ability to penetrate cell membranes [21]. Seo et al. also evaluated the e ciency of PLAG/PEI/DNA nanoparticles in transfection and virus production in comparison with liposome-DNA complexes [22]. The authors showed superior transfection e ciency and viral yield in PLAG/PEI/DNA group when compared with liposome-DNA complexes, particularly in multiple gene transfection. These properties make PEI a good alternative for the delivery of desired genes and nucleic acids [23,24].
However, despite the known considerable strength of PEI in the cell transfection process, not much attention has been given to the application of PEI in iPSC generation, which is a novel and gold strategy in personalized as well as regenerative medicine. In a comparable attempt, Drozd et al. investigated the generation of hiPSCs from cells of broblastic and epithelial origin by means of episomal reprogramming vectors via four reagents; Lipofectamine, PEI, FuGENE6, and FuGENE HD [25]. At the preliminary stages, they observed the highest expression levels achieved from PEI and FuGENE6 reagents [25]. Although PEI gave the highest transfection e ciency, the authors decided to use FuGENE6 for further experiments in their study due to lesser cytotoxic effects. Therefore, PEI dose-dependent toxicity on cells and tissues has remained unclear and controversial [26].
The cytotoxicity and e ciency of PEI have been linked to PEI's physicochemical properties such as molecular weight, branching ratio, and particle size [27,28]. Most techniques have focused on re ning PEI size, branch density, and deacylation to reduce PEI-mediated cell toxicity. But these methods are laborintensive and time-consuming [29,30]. Besides such methods, the way the polyplex is prepared can also signi cantly impact transfection e ciency and cytotoxicity [31][32][33][34]. So considering several parameters such as DNA amount, PEI amount, the ratios of moles of the amine groups of cationic polymers to those of the phosphate ones of DNA (N/P ratio) assists further optimizing transfection e ciency and cytotoxicity of polyplexes. To better clarify the properties of PEI/ plasmid DNA (pDNA) complexes for generating hiPSCs from Human Dermal Fibroblast (HDF), we intended to optimize several conditions regarding PEI/pDNA complexes. Herein, we investigated the in uence of different parameters relevant to the formation of PEI-pDNA complex, such as pDNA concentration, PEI concentration, order of combining reagents (i.e., PEI, pDNA), serum content of polyplexes, and serum starvation of cells before transfection. Then, after optimizing the polyplex preparation, we studied the in uence of N/P ratio on HDF transfection to nd the most e cient protocol for generating hiPSC lines in which 25KDa branched PEI and oriP/EBNA1-based vectors was used. Therefore, the main goal of the present study was to develop an e cient protocol for the PEI-mediated transfection of HDF toward hiPSCs comparable in ease to other non-viral commercial transfection reagents. We believe that our ndings provide experimental evidence that could be usefully employed in other experiments in the area of safe and cost-effective iPSC generation.

2-1-Determining the best condition for transfection of HDFs
Based on our previous experiments, PEI25KD at N/P ratio of 12 could e ciently transfect standard cell lines such as Human Embryonic Kidney (HEK) cells. Thus in the current study, different experiments conditions were adjusted based on PEI/pGFP ratio of 12 at the rst stand. Fluorescent microscopy, ow cytometry, and MTT assay were performed to nd the most e cient condition with few cytotoxicity effects. The following parameters were set and lead to nd an optimized condition for PEI-mediated transfection (See Fig. 1 & Table 1):

Plasmid concentration:
The uorescence microscopy and ow cytometry method showed that the PEI/pDNA complex with 1µg pDNA e ciently transfected HDFs. In contrast, using 2µg pDNA decreased the transfection e ciency. Moreover, the HDF samples treated by PEI/pDNA complex containing 2µg DNA showed less than 70% cell viability.

PEI concentration:
We denoted that re-diluting PEI in distilled water before complex formation to reach 40 µg/ml concentration considerably increased transfection e ciency and cell viability compared to concentrated PEI (1mg/ml).
3. The sequence of adding reagents: Our results showed that the order of adding reagents affects the e ciency of transfection. Adding PEI to pDNA increased the transfection e ciency while the vice versa order decreased the rate of transfection. However, the cell viability percentage did not differ signi cantly between samples treated by PEI/pDNA vs. pDNA/PEI complexes.

10% FBS treatment during PEI/pDNA complexation:
MTT assay showed that FBS signi cantly reduced the cytotoxicity of PEI/pDNA complexes. However, uorescent microscopy and ow cytometry assay determined that the presence of the FBS in polyplexes environment during complex formation dramatically decreased the transfection e ciency.

HDFs serum starvation before transfection:
Based on the uorescent microscopy and ow cytometry results, 1 and 2h serum starvation before transfection showed similar transfection e ciency. Nevertheless, 2h FBS-free culture before transfection signi cantly decreased the cell viability measured by MTT assay 48h post-transfection. At the next step, the optimized condition was chosen to measure the transfection e ciency among different N/P ratios of PEI/pGFP complexes (i.e., 3, 4, 6, 8, 10, and 12). GFP-positive cells were determined using ow cytometry and uorescent microscopy. Our results showed that the transfected cells with N/P ratio of 3 displayed the highest percentage of the cell expressing GFP (an average of ~ 40%) ( Fig. 2B & 2C). When we increased the ratio of PEI/pGFP, the amount of GFP-positive HDF signi cantly decreased in comparison to N/P ratio of 3 (p= 0.04). However, the reduction in transfection e ciency followed by increasing the N/P ratio has not occurred linearly ( Fig. 2B & 2C). Among N/P ratios of 4, 6, 8, 10, and 12, N/P ratio of 4 achieved higher transfection e ciency (29.1%).
Moreover, the uorescent microscopy results were consistent with the ow cytometer ndings. Fluorescent images represented that the N/P ratio of 3 caused more GFP-expressing cells when compared to N/P ratio of 12 ( Fig. 2A).

2-3-Cytotoxicity of PEI complexes
MTT assay showed that increasing N/P ratio signi cantly decreased the cell viability index (p=0.001) (Fig.  2D). The cell viability of all HDF groups are represented in

2-4-Generation and characterization of hiPSCs
Complexes with PEI/pDNA ratio of 3 containing 1µg pDNA, diluted PEI (40 µg/ml), and no FBS complexing by adding PEI to pDNA, which assures a balance of considerable transfection e ciency and acceptable toxicity ( Fig. 3), was chosen to further transfect HDFs to investigate the potency of this protocol to generate hiPSCs through transfection with oriP/EBNA1 vector. On day 14, post-transfection colonies emerged. Colonies exhibited hESC-like morphology with refractive edges and three-dimensional growth (Fig. 3A). An alkaline phosphatase assay was performed to distinguish the fully reprogrammed hiPSC colonies. AP staining illustrated that 1-3% of rounded colonies expressed alkaline phosphatase enzyme. The colonies with hESC colonies morphology could be better noticeable from non-iPS colonies upon passaging and were individually and manually picked for expansion and analysis (Fig. 3A). Under sterile conditions and using a 1 ml syringe, hiPSC colonies were broken into pieces on days 30-35 and transferred onto a 1% Geltrex-coated 12-well plate containing 0.5 ml PluriSTEM. Plates were incubated at 37 °C, 5% CO2 incubator. Colonies were attached to the culture plate 48 hours after colony transferring, and from then on, the medium was replaced every day. When the colonies covered 80-90% of the surface area of the culture plate, they were considered ready for multiple passaging to obtain hiPSC-lines ready for characterization.
Immuno uorescence staining was administrated for the expanded colonies after 8-10 passages.
Immuno uorescence microscopic images displayed the expression of pluripotency markers, including KLF4, OCT4, SOX2, and cMYC ( Fig. 3B). Furthermore, real-time PCR analyses showed that all chosen clones expressed the endogenous reprogramming and pluripotency factors of nanog, sox2 and oct4. Relative expression levels (REI) for these pluripotency genes were markedly increased in the iPSCs compared to the parental HDFs ( Fig. 3C and Supplementary data S1 and S2 online). The genetic stability of the hiPSCs, were also con rmed by karyotyping (Fig. 3D). The established hiPSC line exhibited a normal karyotype of 46 XY as con rmed by chromosomal G-band analysis at passages 8-10. Moreover, the differentiation potential of generated hiPSCs were examined using embryonic body formation and spontaneous differentiation (Fig. 3E). The expression of three germs layer markers, including msx1 (mesoderm), gata4 (Endoderm), and sox1 (ectoderm) were checked by real-time PCR analysis. All differentiated markers were upregulated in hiPSC-derived differentiated cells in comparison to progenitor hiPSCs (Fig. 3F).

Discussion
The iPSC technology opens up an unlimited source of variable human cell types for regenerative medicine.
Administrating economical transfection methods such as cationic polymers provide a simpli ed platform for large-scale production of iPSCs. This method can be used in all iPSC institutes, especially in the labs facing nancial issues. In the current investigation, we represented a cost-effective and optimized protocol for transfecting HDFs through a gold standard reagent (i.e PEI) and oriP/EBNA1-based vectors to produce hiPSCs. Previous evidences have con rmed the capability of PEI to transfect several types of cells such as HEK293 [2], mesenchymal [39], neural [40], and embryonic [41] stem cells. However, with the aim of generating hiPSCs, our investigation is of the pioneers setting and standardizing several parameters relevant to HDF transfection process using PEI25K.
Our results revealed that using 2 µg pDNA implies a lower transfection rate and higher cell death in comparison to 1 µg pDNA, which may be related to the aspect ratio of PEI/pDNA. Higher concentrations of pDNA require a higher amount of PEI for modi cation and permeabilization of endosomal membrane, which causes a higher cytotoxic effect of polyplexes on cell viability [42,43]. In addition, regardless of PEI dosage, an excess of pDNA itself can also contribute to cellular toxicity [44,45]. There are pieces of evidence showing that regardless of the transfection method, the concentration of nucleic acid is also critical for enhancing e ciency and minimizing toxicity [44,46]. For example, Juckem et al. suggested through empirical testing that an optimal starting point is one microgram per well of a 12-well plate resulting in maximum protein expression and minimal toxicity [44]. However, previous studies reported different e cient amounts of pDNA in transfection procedures, partly due to the different sensitivity of diverse cell types to the dosage and concentration of exogenous nucleic acid [44]. In addition, we found that a lower concentration of PEI (dissolved in a low ionic solvent such as distilled water) could prominently increase both transfection e ciency and cell viability compared to concentrated PEI. Besides the lesser toxic effect of PEI at lower concentrations [47], low-concentrated branched PEI have different formation and hydration properties, facilitating transport and diffusion of the polyplexes through cell membrane [48]. Moreover, we hypothesized that diluted PEI might provide more free branches than a concentrated state, advancing DNA binding a nity and improving cellular uptake [49]. In addition, diluted PEI might form a thicker layer around pDNA than concentrated PEI, which enhances the protonation state of PEI/pDNA polyplex structure and provides an electrostatic and physical barrier to DNases with cationic surfaces [24].
Our results also represented that adding PEI to pDNA can increase transfection e ciency relative to the converse order related in part to the electrostatic interactions between the positive charges of cationic polymers and negative charges of pDNA [50]. Therefore, even subtle changes in the sequence of adding reagents may in uence the physicochemical properties and reactivity of reagents and subsequently change the transfection e ciency of the polyplexes [51][52][53].
Furthermore, we found that the presence of serum in polyplexes signi cantly decreased transfection e ciency due to the interfering role of serum in dissociating and disrupting the structure of PEI/pDNA complexes [18]. However, the MTT test showed that the existence of FBS prominently increased the cell viability, which may be attributed to the potential of serum proteins in absorbing free PEIs [18]. Previous studies also con rmed that the presence of FBS in polycations-based transfection reduces both cytotoxicity and transfection e ciency [54]. Therefore, in the current study, using FBS during polyplex formation was not favored by transfection e ciency and is not recommended in HDF cell type.
Apart from the impact of FBS in the structure of polyplexes, it is evident that FBS in cell culture media before transfection can affect the e ciency of PEI/pDNA delivery to the cells. Serum starvation induces cell cycle synchronization (G0/G1 proliferation delay), which eventually increases transfection e ciency [55,56]. Moreover, the competitive interaction of serum proteins and polyplexes with cell surface reduce the e ciency of transfection. Herein, we examined the impact of 1h vs. 2h serum starvation on HDFs' transfection e ciency and cell viability. We discovered that both groups showed the same transfection e ciency. Yet 2h serum starvation decreased cell viability in comparison to 1h starvation.
Nevertheless, the optimized serum starvation period differs in other studies depending on the studied cell types [57].
Given together, our optimized protocol consists of 1µg pDNA, diluted PEI (40 µg/ml), PEI to pDNA addition sequence, serum-free polyplexes, and 1h serum starvation of HDFs. Besides, since N/P ratio plays an essential role in in uencing the degree of complexation, particle diameter, transfection e ciency, and cytotoxicity of carriers [34], in the next step, we investigated the effect of N/P ratio on transfection e ciency and cell viability using the optimized protocol. We observed that the PEI/pDNA ratio of 3:1 led to the highest transfection e ciency and cell viability in comparison to other higher N/P ratios representing the acceptable strength of low-dose PEI in successfully carrying a large-sized plasmid (~ 20kb) into HDFs.
We hypothesized two assumptions regarding this nding. Our rst assumption is related to pDNA binding ability of PEI and particle aggregation. The pDNA dosage for all N/P ratios has been xed (1µg) while the amount of PEI has been raised for increasing N/P ratio. We assumed that due to higher amounts of PEI and more positive charge in respect to the negative charge of pDNA, the pDNA binding ability of PEI has been increased at higher N/P ratios. Thus, despite the potential of higher buffering capacity at higher N/P ratios, extra condensing of pDNA has been occurred, preventing the easy release of pDNA into cytoplasm of cells [58]. Moreover, at higher N/P ratios, polyplexes might have more challenging cellular uptake due to greater sizes, and more particle aggregation [58,59]. In line with our nding, Cheraghi et al. also assessed the effect of different PEI/ DNA ( re y luciferase) N/P ratios of 3, 6, 12, 18, 24 on transfection e ciency of MCF7 and BT 474 Cells. They observed a decrease (approximately more than 5 fold) in transfection e ciency from N/P ratio of 12 to 18 [58]. They also interpreted the occurrence of this difference to be as a result of higher particle aggregation, and consequently more challenging cellular uptake at higher N/P ratios. In addition, pDNA size may also have a role in either the mechanism of pDNA release from cationic reagent or intracellular migration of pDNA from the cytoplasm to nucleus[60]. In our study plasmid size was approximately high (~ 20kb), adding further challenges regarding particle aggregation at higher N/P ratios[61].
Our second assumption is related to the observed relative higher cytotoxic effect of PEI at higher N/P ratios in the current study. We assumed that even though the transfection might occur for a substantial number of cells at N/P ratios greater than 3 (e.g. 4, 6, 8, 10, and 12), but due to higher toxic effects of polyplexes at higher N/P ratios, the number of dead cells had been also relatively increased. Therefore, when we washed the transfected cells for ow cytometry analysis, dead cells (even transfected ones) were removed and washed from the plate and subsequently did not enter the ow cytometry analysis.
The maximum e cacy at a low-dose N/P ratio found in this study differs from previous studies, which used higher polyplex N/P ratios to improve the transfection e ciency even at increasing N/P ratio higher than 32, zeta potential (surface charge of polyplexes) increases, and subsequently, transfection e ciency will enhance. However, to achieve an acceptable transfection e ciency, they also observed an unavoidable decrease in cell viability by increasing N/P ratio.
We concluded that there could be two possible explanations regarding our nding of acceptable etransfection e ciency in a low N/P ratio. First, our optimization led to signi cant transfection even at a low N/P ratio of 3. In line with our results, evidence shows that the complex formation protocol can strongly in uence transfection e ciency and cytotoxicity [33]. In other words, reagent/DNA ratio and complex formation conditions is of paramount importance for obtaining maximum e cacy with the least side effects in cell transfection process [63]. Second, in the current study, we used low ionic solvent to dissolve PEI. Evidence have shown that PEI/pDNA complexes in high ionic conditions (e.g. HEPES buffered 150 mm saline (HBS)) exhibited a strong tendency for aggregation at low N/P ratios, which can be avoided by increasing the N/P ratio to 6 or higher [33]. While polyplexes in low ionic solvents represented an opposite behavior [33]. Therefore, by administrating the optimized conditions and N/P ratio which we presented in the current study, we circumvented issues with either transfection success or high cytotoxicity.
Finally, PEI polyplexes containing oriP/EBNA1 vector were used to generate iPSCs from HDFs through the optimized condition and N/P ratio. In this study, the e ciency of colony formation was about 2 colonies per 150000 input cells. Although e ciency was low, it is still su cient to establish iPSC lines from HDFs

Conclusion
In this study, we represented that PEI25K can transfect HDFs to generate hiPSCs with minimal cytotoxicity.
Owing to high molecular weight (> 25 KDa), PEI exhibits toxicity issues which are in part due to the absorption of anionic serum proteins onto polyplex surface between cationic polymers and anionic plasma proteins of serum [69]. However, we assumed that a low-dose ratio and an optimized polyplex preparation condition helped minimize polyplexes' toxicity effects on our cell type studied (i.e., HDFs). Our protocol is safer, less costly, and less complicated than conventional viral and lipid-based non-viral methods. Nevertheless, it is worthy to note that some variables other than those aforementioned here may also in uence the way polyplexes behave and consequently transfection e ciency and cytotoxicity. We recommend future studies to optimize different parameters relevant to the transfection process with the aim of hiPSCs generation. In sum, using our optimized PEI mediated transfection method is highly recommended, especially in large-scale transfection programs, as an alternative to more expensive commercial reagents for generating hiPSCs. In addition, we could propose that by setting the vital parameters, using PEI on iPSC generation will be a very important primary step toward the extension of the eld. At the later phases, we can further improve and optimize PEI e cacy through PEI derivatives for iPSC generation.

Materials And Methods
This study was approved by Ethics Committee of cellular and molecular research center, Iran University of Medical Sciences (IUMS). All methods were carried out in accordance with Iranian guidelines and regulations regarding the generation of hiPSCs following a protocol approved by Royan Institute.

4-1-Human Dermal Fibroblast culture
Primary HDFs were purchased from Royan Institute, Stem Cell Biology and Technology Department [35,36]. The cells were isolated from a skin biopsy of a healthy 37-year-old Iranian male who donated a skin biopsy in reprogramming studies and iPSC lines generation. HDFs were maintained in a medium containing Dulbecco's modi ed Eagle's medium (DMEM; Gibco), 10% fetal bovine serum (FBS, Gibco), 100 units/ml penicillin, and 100 µg/ml streptomycin (Pen/Strep) (Gibco). They were at passage four at the time of transfection. Informed consent was obtained from the donor.

4-2-PEI/pGFP (Green Fluorescent Protein) complexes
One mg branched PEI (molecular weight of 25KDa) was purchased from Polysciences Inc. and dissolved in 1ml sterile water and ltered with a 0.25 μm syringe lter. GFP plasmid (pGFP) (Addgene #64904) was used to detect the most e cient PEI-mediated gene delivery [37]. Then, immuno uorescence imaging and ow cytometry were performed to detect transfected cells. At the rst step, we used N.P ratio of 12 to test different conditions and to nd the best-optimized protocol relevant to complex formation. For this purpose, pDNA concentration, PEI concentration, order of combining reagents, FBS content of complexes, and duration of serum starvation prior to transfection were adjusted (See Table 3 for more details on the different conditions of complex formation). Based on the optimized protocol of complex formation, we assessed the impact of different N/P ratios on transfection e ciency and cell toxicity. We prepared different N/P ratios of PEI/pGFP complexes, including 3, 4, 6, 8, 10, and 12. For this purpose, we re-diluted 1, 1.44, 2.16, 2.88, 3.6, and 4.3µl PEI25K (1mg/ml) in distilled water to the nal volume of 25µl. 1µg/ml of pGFP was also diluted in distilled water to the nal volume of 25µl. These polyplex ratios were chosen based on the established protocol in our laboratory. Then, the contents of each PEI tube were added to 25µl of pGFP, and tubes were mixed by gentle pipetting or 10 seconds vortexing. The complexes were incubated at room temperature for 30 minutes. Finally, the synthesized complexes were added to HDFs in 250µl free FBS medium dropwise. After 4h incubation, we replaced the media with fresh HDFs medium.

4-5-hiPSCs Generation
The transfection was performed based on the optimized protocol described earlier. Brie y, the 4 th passage of HDFs were seeded on Geltrex (Life Technologies) coated 24-well plates the day before transfection. When HDFs reached 70-80% con uence, they were transfected by oriP/EBNA1-based vector genes presented in Fig. 4  The normal chromosomal constitution was veri ed by conventional karyotyping of generated iPSCs.
After washing with PBS, cells were incubated with 0.075 M KCl for 20 minutes at 37 °C. Cell xation was performed by administrating methanol/glacial acetic acid solution (3:1). Conventional cytogenetic analysis was performed on hiPSCs using conventional QFQ-banding at 450 bands resolution according to the International System for Human Cytogenetic Nomenclature. A minimum of 10 metaphase spreads were analyzed for each sample and karyotyped using a chromosome imaging analyzer software (Chromowin software, Tesi Imaging).

4-6-4 Embryonic body (EB) formation
The hiPSCs were harvested using Dispase (1 mg/ml, StemCell technologies) when they reached 80-90% con uence. Then the cells were re-suspended in differentiation medium (high glucose DMEM, 2 Nm Lglutamine, 0.1 Mm nonessential amino acid, 0.1 Mm b-mercaptoethanol, 20% FBS) and incubated at 37˚ C with 5% CO2. The hiPSCs were cultured in suspension, and the medium was refreshed every 2 days. After 1 week, EBs had formed. The following day, EBs were transferred to 1% Geltrex-coated 6-well plates and cultured in the high glucose DMEM media for 7 days. Finally, the cells were harvested, and gene expression was evaluated by real-time PCR technique. Table 4 represents the primer sequence used for real-time PCR analysis in this study.

4-7-Real-time PCR analysis
Real-time PCR was performed to evaluate the expression level of pluripotency genes (oct4, sox2, nanog) to con rm generated iPSCs lines. Moreover, gata4, msx1, and sox1 genes were checked in hiPSC deriveddifferentiated cells to ensure trilineage differentiation (endoderm, mesoderm, and ectoderm, respectively) (  Table 4. The relative expression level of βactin was used for normalization. pGFP, diluted PEI (40 µg/ml), PEI-added-to-pGFP complexes, and PEI/pGFP complexes without FBS are more in number than 2µg pGFP, concentrated PEI (1mg/ml), pGFP-added-to-PEI complexes, and PEI/pGFP complexes with 10% FBS respectively. (B) Quanti cation of transfected HDF cells with different conditions through ow cytometry analysis. All analyses were performed on 3 independent HDF transfections. The xaxis denotes uorescence, and the y-axis shows the number of cells detected at a de ned uorescence by the ow cytometer. Untransfected HDFs were used as a reference, and the gating was set such 1.2% of cells were untransfected. 1µg pDNA (11.7%), diluted PEI (15.53%), PEI-added-to-pGFP complexes (13.1%), and PEI/pGFP complexes without FBS (16.92%) resulted in more percentage of cells positive for pGFP in comparison to 2µg pDNA (3.72%), concentrated PEI (3.98%), pGFP-added-to-PEI complexes (8.58%), and PEI/pGFP complexes with 10% FBS (3.62%) respectively. (C) Error bars represent the standard error of the mean (SEM). This graph displays the percentage of GFP+ cells. The x-axis represents the different conditions relevant to complex formation, and the y-axis represents the percentage of transfection as determined by ow cytometry (asterisk denotes differences at p\0.05). (D) Effect of pDNA concentration, PEI concentration, the sequence of adding reagents, FBS content of polyplexes, and serum starvation before transfection on cell viability measured by MTT assay. The data are expressed as a percentage of the absorbance of untreated cells and presented as the mean of three independent replicates ± SEM.
Asterisk denotes a signi cant difference (p<0.05). of the mean (SEM). This graph shows the percentage of GFP+ cells determined in (3B). Asterisks denote signi cance level at p<0.05. The x-axis represents the N/P ratios compared in this manuscript, and the yaxis represents the percentage of transfection as determined by ow cytometry. (D) Cytotoxicity of different N/P ratios. The effect of different complexes with the same optimized condition but different N/P ratios of 3,4,6,8,10 and 12 on cell viability were measured by MTT metabolism. Results are expressed as a percentage of the absorbance of untreated cells and presented as the mean of three independent replicates ±SEM. The mean percentage of cell viability at N/P ratios of 3,4,6,8,10, and 12 were 91%, 86% 79%, 76%, 72% and 69% respectively. detect pluripotent markers, including KLF4, OCT4, SOX2, and cMYC. The nuclei of all cells were stained blue with DAPI (Scale bars 100µm). (C) Expression levels of transgenes oct4, sox2, and nanog were assessed at passages 8-10 by real-time PCR and plotted relative to βactin. HDFs were used as negative controls. The error bar indicated standard deviation (SD). (D) Karyotype analysis of HDF-induced pluripotent stem cells. Karyotype analysis revealed a normal karyotype of 46 chromosomes (E) hiPSCs were grown in suspension, and EBs were detected after 8 days. EBs were cultured in Geltrex-coated 6-well plates for an additional 7 days, and cells were checked for their adherent status (Scale bar 200µm) (F) realtime PCR analysis was performed for differentiation markers of the three germ layers in EBs. βactin was utilized as an internal control.