Xenofree generation of oral keratinocytes from induced pluripotent stem cells derived from adult human fibroblasts for oral mucosal regeneration

Xenofree generation of oral keratinocytes from induced pluripotent stem cells derived from adult human fibroblasts for oral mucosal regeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Xenofree generation of oral keratinocytes from induced pluripotent stem cells derived from adult human fibroblasts for oral mucosal regeneration Ridhima Das, Hassan R.W. Ali, Tarig Osman, Mohammed A. Yassin, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7842122/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigated the potential of xenofree generation of oral keratinocytes from induced pluripotent stem cells (iPSCs) derived from adult oral and skin fibroblasts for oral mucosal regeneration. Using an extracellular matrix-based protocol, iPSCs were differentiated into cells with a phenotype and molecular profile of oral epithelium, proven through morphological and molecular analyses. Differentiation to epithelial squamous lineage was achieved in serum-containing and xenofree conditions, although the former showed higher efficiency. The differentiated cells successfully formed pluristratified squamous epithelial tissues in 3D organotypic cultures, mimicking oral mucosa. In vivo tests using an immunodeficient mouse model validated the viability of multilayered squamous epithelial-like tissues, with distinct oral-specific markers identified in tissues derived from gingival fibroblast iPSCs. These findings demonstrate the feasibility of using iPSCs to create functional oral mucosal sheets, highlighting their potential for clinical applications in regenerative therapies, though further optimization is necessary to enhance differentiation efficiency under xenofree conditions. Biological sciences/Biotechnology Biological sciences/Cell biology Biological sciences/Stem cells induced pluripotent stem cells xenofree oral mucosa keratinocytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Intraoral surgical wounds occurring post trauma or after tumour excision are either left open or undergo primary closure if small, while they require skin grafts, flaps or delayed closure in case of large defects ( 1 ). The gold standard for treatment of large defects is the use of skin grafts to prevent infection of the wound bed and promote healing, and this requires usually a second surgical procedure leading to increased post-surgical morbidity. Oral mucosa grafts are best suited, but due to limited availability they are not used for treatment of large defects ( 2 ). Split-thickness skin grafts are commonly used, but due to presence of various adnexa and the different texture of skin, these are not ideal for the treatment of large oral mucosal defects ( 3 , 4 ). Oral mucosa engineering holds great promise as an alternative to conventional grafting methods, offering a potential solution to overcome these limitations. As a relatively recent field within tissue engineering, it combines cells, materials, and bioactive factors to generate a three-dimensional (3D) model of oral mucosa. It aims to reproduce the real anatomical structure and function of oral mucosa ( 5 ). Stem cells are undifferentiated pluripotent/multipotent cells that can self-renew and differentiate into specialized cells such as epithelial cells, blood cells, adipocytes, etc ( 6 ). Based on their origin, stem cells can be classified as embryonic stem cells (ESC), adult stem cells, and induced pluripotent stem cells (iPSC) ( 7 ). ESCs are a potentially powerful tool for use in tissue engineering, but the use of human ESCs is still not approved ethically. The best source for stem cells is from the patient itself, since this would not involve ethical or immunological concerns. However, the adult stem cells, particularly the epithelial ones, are still very challenging to propagate and amplify in culture for oral epithelial regeneration. The iPSCs generated from adult somatic cells are thus promising candidates for use in cell based therapies ( 8 ). Use of pluripotent stem cells in regenerative medicine has advanced considerably since Takahashi and Yamanaka developed the iPSCs from somatic cells in 2006, by forced reprogramming of human adult fibroblasts using the transcriptional factors Oct3/4, Sox2, c-Myc, and Klf4 ( 9 , 10 ). The iPSCs can self-renew indefinitely without raising any ethical concerns associated with the use of ESC. In the past decade, studies have been focused on numerous aspects of the iPSCs such as optimization methods, mechanisms of reprogramming, differentiation, as well as their clinical applications ( 11 ). Xenofree expansion of the cells avoids the immunological complications which may be associated with the use of animal derived products like foetal bovine serum (FBS). Studies in the last few years have suggested that the use of human platelet lysate (PL) can support in vitro clinical grade propagation of human cells, especially for stem cells/progenitor cells. PL contains factors and nutrients which promote cell growth and are essential for wound healing and regeneration of damaged tissues ( 12 – 15 ). PL has been proven as a suitable alternative to FBS for propagating human cells in culture for an array of applications in tissue engineering and cell therapy ( 16 , 17 ). To date, several groups, including ours, have reported procedures to differentiate mouse ESCs ( 18 ) and human iPSCs derived from skin adult fibroblasts into epidermal keratinocytes ( 19 – 21 ). The iPSCs reprogrammed from normal oral fibroblasts either in FBS containing or xeno-free conditions, have not been used so far for differentiation into oral keratinocytes and for regeneration of either in vitro or in vivo oral mucosa. In this study, we aimed to investigate if oral/gingival and skin adult fibroblast-derived iPSCs (G-iPSCs and S-iPSCs, respectively) can be induced into oral keratinocytes as a source of cells for oral mucosal regeneration. To achieve this, we tested various conditions and modifications of published protocols known to be efficient in inducing skin keratinocytes from skin fibroblast-derived iPSCs or in inducing epithelial progenitors from mouse iPSCs ( 22 – 24 ). MATERIALS & METHODS All experiments were performed in accordance with relevant guidelines and regulations. Cell culture Primary cell isolation and culture Dermal and gingival tissue specimens were obtained from two healthy female volunteers (Donor 1: aged 40–50 years; Donor 2: aged 50–60 years) following acquisition of informed consent in accordance with ethical protocols, as previously described ( 17 , 25 ). Two distinct anatomical sites were sampled from each donor: dermal biopsies were harvested from the anterior forearm region, and gingival specimens were excised from the attached gingiva overlying the maxillary first molar. Primary human normal oral fibroblasts (NOF) and normal skin fibroblasts (NSF) were isolated from the attached gingiva and dorsal forearm skin, respectively as previously described ( 26 ). Mycoplasma testing was regularly performed once every two weeks using MycoAlertTM mycoplasma detection kit (Lonza) as per manufacturer’s instructions. Normal oral keratinocytes (NOK) were also isolated from the attached gingiva following previously described protocol established in our laboratory ( 26 ) and were routinely grown on plastic surfaces (Nunc) with no feeding layers, in keratinocyte serum free medium (KSFM) supplemented with 1 ng/ml recombinant human epidermal growth factor (rhEGF, Gibco), 25 µg/ml bovine pituitary extract (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 µg/ml streptomycin (Gibco), 0.25 µg/ml amphotericin B (Gibco). Fibroblasts were grown in Dulbecco′s Modified Eagle′s Medium - high glucose (DMEM) (D6429, Sigma) supplemented with 10% FBS (Fischer scientific), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B (Gibco). NOF and NSF were also grown in xenofree conditions using Dulbecco′s Modified Eagle′s Medium - high glucose (DMEM) (D6429, Sigma) supplemented with 5% PL (BergenLys, unfiltered, 4-month storage), 1% heparin, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B (Gibco). NOF and NSF were reprogrammed to gingival and skin iPSCs (G-iPSCs and S-iPSCs respectively) using Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) as described previously ( 17 , 27 ). They were routinely cultured on plates coated with Geltrex (Gibco) in Stem-flex media (Gibco) with 1% AB/AM (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B ( 17 ). Differentiation and characterization of human iPSCs The iPSCs were differentiated into keratinocytes following slightly modified previously published protocols for differentiation of NSF into skin keratinocytes or mouse iPS into epithelial lineages ( 22 – 24 ). Two of the protocols used were growth factors (GF) based and contained 25 ng/ml bone morphogenic protein 4 (BMP4) and 1uM retinoic acid (RA). RA promotes the differentiation of iPSCs into the ectodermal lineage while BMP4 blocks neural lineage commitment ( 28 ). One protocol (Protocol I) involved, in addition, the use of 20 ng/ml rhEGF and 20 ng/ml basic fibroblast growth factor (bFGF), while the other one (Protocol II) involved the use of N2B27 medium. The N2B27 medium combines DMEM/F12 and Neurobasal medium (Gibco) in a 1:1 ratio, supplemented with 0.1 mM nonessential amino acids, 1 mM glutamine, 55 µM 2-mercaptoethanol (2-ME), N2 supplement (100x) (Life Technologies), B27 supplement (50x) (Life Technologies), 50 µg/ml ascorbic acid, 0.05% bovine serum albumin (BSA), 50 U/mL penicillin-streptomycin, 100 ng/ml bFGF (Life Technologies), and 10 µM Y27632 Rock inhibitor (Sigma-Aldrich) ( 22 ). Protocol I lasted for nine days, and protocol II lasted for twenty-four days. The extracellular matrix (ECM) based protocol (Protocol III) used the ECM produced by mitomycin C-metabolically inactivated fibroblasts. This protocol involved also the use of 25 ng/ml BMP4 in the media in addition to ECM and it lasted fourteen days. Protocol III was modified from ( 23 ) to minimize the use of xenogeneic derived biological materials as follows: instead of ECM produced by mitomycin-treated mouse PA6 cells as feeder layer, the oral and skin fibroblast-derived iPSCs were grown in this study on a feeder layer produced by mitomycin C - treated human normal primary oral and skin fibroblasts ( 25 ). The morphology of the iPSCs was observed daily from the first day to the last day of the differentiation protocol using a light microscope. Images were taken every second day when the media was changed. Differentiated cells obtained from all the three protocols were characterized by light microscopy and immunofluorescence and based on the obtained results, Protocol III ( 23 ) was further used to produce keratinocytes in both xenofree and xenogeneic conditions. Collection and use of normal human oral mucosa and normal human skin samples from healthy adult donors were conducted as previously described ( 17 , 25 ), under the project “Modulation of Stem Cell Plasticity for Regenerative Medicine Applications”, approved by the Regional Committee for Medical and Health Research Ethics, Western Norway (REK Vest; Approval No. 80005; Approval Date: 17 January 2020). All donors were healthy adults and provided written informed consent for participation and the use of their de-identified samples in research. Molecular assays Immunofluorescence Differentiated iPSCs were cultured on cover slips and fixed with 4% paraformaldehyde (PFA) at room temperature (RT). The cells were then permeabilized with 0.1% Triton X, blocked using 5% BSA in phosphate buffered saline (PBS), and the immunostaining was done using anti-pancytokeratin antibody (DAKO, clone AE1/AE3, 1:100). Alexa Fluor 488 anti-mouse IgG (BioLegend) was used to detect the site of reaction according to the manufacturer`s instructions. The coverslips were then mounted on glass slides with Vectashield containing DAPI (Vector Labs) and the images were captured using a florescence microscope (Axio Imager Z2, Carl Zeiss, Oberkochen, DE). NOK and undifferentiated G-iPSCs were used as positive and negative controls, respectively, as previously described ( 25 ). All experiments were repeated at least 3 times in duplicates. Gene expression analysis The differentiation of iPSCs was assessed also at mRNA level by investigating the expression of several selected pluripotency and epithelial phenotype-related genes, as previously described ( 25 ). Total RNA was extracted using RNeasy Mini Kit (Qiagen) as per manufacturer’s instructions. Measurement of total RNA concentration was done using Nanodrop spectrophotometer (Nanodrop Technologies, USA). Three hundred nanograms of total RNA was converted to cDNA using the High-capacity cDNA kit as per manufacturer’s instructions (Applied Biosystems, Carlsbad, US). TaqMan ® gene expression assays (Applied Biosystems ® ) were used to detect mRNA levels of pluripotency markers (POU5F1-Oct3/4, Nanog, Sox2,) and epithelial markers (cytokeratins 5, 13, 18 and 19 - KR 5, 13, 18, 19). Real-time quantitative polymerase reaction (RT-qPCR) amplification was performed using AB 7500 PCR system for 40 cycles. qPCR was performed on a Step One Plus system, using TaqMan gene expression assays (Applied Biosystems, CA, USA). Data was analysed using the ∆∆Ct method. The expression of the genes was normalized to that of the housekeeping gene, GAPDH. The expression of the pluripotency markers in differentiated iPSCs was presented as fold changes relative to their expression in undifferentiated matched iPSCs. The expression of the epithelial markers in differentiated iPSCs was presented as fold changes relative to NOK. An overview of the primers used for the gene expression analysis is presented in Table 1 . All experiments were repeated at least 3 times in duplicates. Table 1 List of TaqMan probes used in the study Gene Symbol Gene Name TaqMan assay ID GAPDH Glyceraldehyde-3-phosphate dehydrogenase Hs02786624_g1 SOX2 SRY-box 2 Hs04234836_s1 NANOG Nanog homeobox Hs02387400_g1 POU5F1 POU class 5 homeobox 1 Hs04260367_gH KRT5 Keratin 5 Hs00361185_m1 KRT13 Keratin 13 Hs02558881_s1 KRT18 Keratin 18 Hs02827483_g1 KRT19 Keratin 19 Hs00761767_s1 Flow cytometry The differentiated cells were characterized for expression of pluripotency markers: TRA-60 (Stemcell Technologies, clone TRA-1-60R, PE-conjugated, #60064PE, 1:20), SSEA4 (R&D, clone FAB1435A, APC-conjugated, 1:100), Sox2 (R&D, IC2018G, Alexa Fluor® 488-conjugated, 1:100), Oct-3/4 (Santacruz, clone C-10, Alexa Fluor® 488-conjugated, sc-5279), and for the epithelial marker E-cadherin (R&D, clone FAB18381A, APC-conjugated, 1:20). A number of 10⁵ cells were fixed in 4% PFA for 15 min, and permeabilized via 0.1% Triton X, for 15 min. Unspecific binding of antibodies was prevented with blocking buffer (composition 5% BSA in PBS) for 10 min at 4°C. Conjugated monoclonal antibodies were then added to the pellet on ice and the cells were incubated in the dark for 30 min at 4°C before washing. Stained samples were analysed and compared to the corresponding unstained samples (negative control) as well as isotope controls ( 25 ). The final quantification was performed with a BD Accuri flow cytometer (BD Biosciences®) and the data was analysed using FlowJo (FlowJo®, LLC, Ashland, OR USA). NOK and undifferentiated matched G-iPSCs and S-iPSCs were used as positive controls for epithelial and pluripotency markers, respectively, as described in ( 25 ). All experiments were repeated at least 3 times in duplicates. Immunoblotting Cell lysates for blotting were prepared in RIPA lysis buffer (Sigma-Aldrich, St. Louis, US) containing protease and phosphatase inhibitors (Halt ™ Protease Inhibitor Cocktails, Thermo Scientific). Protein concentration was measured using Pierce bicinchoninic acid (BCA) kit (Thermo Scientific). A total of 25 µg protein was loaded on 4–12% pre-casted polyacrylamide gels (GenScript), resolved and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). The membranes were blocked with 5% BSA or 5% non-fat dry milk (Biorad) for 1 hr at RT, and incubated with anti-pancytokeratin, (DAKO, clones AE1/AE3, 1.200) and β-actin (Cell Signalling, 1:3000) overnight at 4º C. The following day the membranes were washed with tris-buffered saline Tween 20 (TBS-T) and then incubated with secondary antibodies anti mouse IgG, HRP-linked (Cell Signalling, 1:3000) anti rabbit IgG, HRP-linked (Cell Signalling, 1:3000) for 1 hour at RT. The reaction was visualized using super signal west femto chemiluminescent substrate (Thermo Scientific, Waltham, US) in SynGene G: Box scanner (Syngene, Frederick, US). The protocol is as describe din ( 25 ). All experiments were repeated 3 times. 3D organotypic cultures 3D organotypic cultures were generated by growing both oral and skin fibroblast-derived iPSCs that were differentiated following Protocol III in both FBS and PL on top of oral fibroblast-populated collagen type I (BD Biosciences) biomatrices, using a well-established protocol in our laboratory ( 25 , 26 ). Simple collagen gels were prepared on ice by mixing 7 vol. (3.40 mg/ml) of rat tail collagen type I (Collaborative Biomedical), 2 vol. reconstitution buffer (261 mM NaHCO3, 150 mM NaOH, 200 mM HEPES) pH 8.15, 1 vol. DMEM 10× (Sigma) and 1 vol. FBS (Gibco) or PL. All 3D organotypic cultures were grown in serum free FAD medium (DMEM: Ham’s F-12 / 3:1) supplemented with 1 µM hydrocortisone, 10 ng/ml rhEGF, 10 ng/ml KGF, 0.8 µM insulin, 0.25 mM transferrin, 0.25 mM L-ascorbic acid, 15–30 µM linoleic acid, 15 µM bovine serum albumin, 2 mM L-glutamine (all from Sigma)( 25 ). In some cultures, 10ng/ml GM-CSF was also added ( 29 ). Cultures were grown for three days submerged and for further for seven days at the air-liquid interface on lens paper on metal grids. On the 11th day, the 3D organotypic cultures were harvested, fixed in formalin, embedded in paraffin, cut and hematoxylin-eosin stained for histological analysis, as described in ( 25 ). All experiments were repeated 6 times in duplicates or triplicates. Immunohistochemistry To stain the tissues obtained in 3D organotypic cultures, we followed our previously described protocol ( 25 ); 4 µm sections were cut, deparaffinized and rehydrated by immersion in xylene and diminishing concentrations of alcohol. Sections were then incubated for 1 hour at RT with one of the following monoclonal mouse anti-human primary antibodies: anti-pancytokeratin (DAKO, AE1/AE3, 1:100), CK13, CK10 (CRUK, 1:10), and anti-vimentin (DAKO, 1:2000). Envision+ ® anti-mouse (DAKO) was used to detect the site of reaction according to the manufacturer`s instructions. The reaction was visualized using 3,3′-diaminobenzidine tetrahydrochloride (DAB). Sections were then counterstained with hematoxylin (DAKO), dehydrated and cover slipped ( 25 ). Immunohistochemistry was repeated at least 2 times in duplicates for 4 out of the 6 repetitions of the 3D organotypic assays. 3D printing of poly ɛ-caprolactone (PCL) chambers for xenotransplantation of mucosal sheets A 3D CAD model was designed (dimensions in Fig. 1 A, B) with Magics software (EnvisionTEC, Germany), then sliced into layers at a slice thickness of 80% of the inner needle diameter (ID). In accordance with the manufacturer's guidelines (3D Bioplotter® RP, EnvisionTEC), a slice thickness of 0.32 mm was applied to a stainless- steel needle 0.4 mm in diameter (ID). Before adding 2.5 g of PCL (MW 45 kDa, melting temperature = 60°C, Sigma-Aldrich) granules to the cartridge, it was preheated to a preheating temperature of 110°C. The PCL was then kept constant at this temperature during the entire printing time. Chambers were extruded at the predefined designs, as described in ( 25 ). In vivo mice models A total of 16 NSG (NOD/SCID ILgamma2 deficient) mice were used for this study as previously described ( 25 ). These mice (aged between 15–17 weeks, weighing 29-34g) are immunocompromised and lack B and T lymphocytes, as well as natural killer cells. Prior to surgical implantation of PCL chambers and/or subcutaneous cell injection, NSG mice were anesthetized with 3–4% isoflurane for induction and 1–2% for maintenance via nose cone, delivered in oxygen. Anesthesia depth was verified by pedal withdrawal reflex. Pre-operative analgesia with buprenorphine (0.05 mg/kg, subcutaneously) was administered. The differentiated iPSCs were both injected subcutaneously within the pre- implanted PCL chambers (6 mice, Fig. 6 A), as well as implanted as a mucosal sheet after being pre-assembled in vitro in 3D OT models seven days prior to xenotransplantation and then glued to the lower edge of the PCL chambers with tissue glue Histoacryl® (10mice, Fig. 6 B), as previously described ( 25 ). After 14 days post-implantation, animals were euthanized using carbon dioxide (CO₂) inhalation in a gradually rising concentration (20–30% chamber volume/min), followed by cervical dislocation to ensure death. Tissues were harvested immediately thereafter for downstream analysis. All animal experiments are reported in compliance with the ARRIVE 2.0 (Animal Research: Reporting of In Vivo Experiments) guidelines. In situ hybridization (ISH) To identify the transplanted human cells, ISH for the human specific repetitive Alu sequence was used as previously described ( 17 , 25 ). ISH was performed by RNAscope 2.5 High-Definition Brown Assay according to the manufacturer’s instructions (all from Advanced Cell Diagnostics, Newark, CA, United States). Briefly, the tissue sections were baked at 60°C for 1 h followed by deparaffinization in 100% xylene and twice for 5 min each and then two times in 100% ethanol. The slides were then treated with an endogenous peroxidase-blocking reagent, for 15 min by boiling in target retrieval buffer and then treated with protease buffer for 30 min at 40°C. The slides were then incubated with the target Alu probe for 2 h at 40°C, followed by signal amplification. For colorimetric detection, 3,3′- Diaminobenzidine (DAB) was used for 5 min at RT followed by counterstaining with hematoxylin for 5 min. A peptidylprolyl isomerase B (PPIB) Positive Control Probe was used to validate the assay, as previously described ( 25 ). Statistical analysis The statistical analyses were done using GraphPad Prism 9 (GraphPad Software). Data are presented as mean values (+/- standard deviation). Statistical significance was determined by using one-way ANOVA analysis with Tukey’s test for multiple comparisons. A p-value of < 0.05 was considered statistically significant. This work was included in the doctoral thesis of RD, University of Bergen, 2022 ( 25 ). RESULTS Differentiation of G-iPSCs and S-iPSCs into keratinocytes using growth factors (GF) or extracellular matrix (ECM) enriched protocols Both GF enriched (Protocols I and II) and ECM enriched (Protocol III) protocols induced changes in the morphology of G-iPSCs and S-iPSCs, as observed by light microscopy (Fig. 1 , A-H). Both G-iPSC and S-iPSC gradually changed their morphology from tight colonies of small, rounded cells with very little cytoplasm, typical for undifferentiated iPSCs (Fig. 2 J) towards larger, flattened, polygonal shape cell morphology, typical for normal oral keratinocytes (NOK, Fig. 2 I). When they became confluent, the typical cobble-stone morphology of keratinocytes in monolayer was observed in all conditions, including the condition in which FBS was replaced by PL in the ECM enriched protocol (Fig. 1 G, H). Immunofluorescence staining using an antibody against pancytokeratin showed various expression in iPSCs differentiated by all the protocols tested. Nearly all iPSC differentiated through ECM enriched protocol (Protocol III) in FBS containing conditions showed a strong and uniform expression of pancytokeratin (Fig. 2 C), comparable to the expression in NOK (Fig. 2 G). The iPSCs differentiated through ECM enriched protocol (Protocol III) in xenofree conditions expressed also pancytokeratin, but to a lesser extent (Fig. 2 E, F). Further tests were performed using iPSC differentiated based on ECM-enriched protocol (Protocol III) only. Characterization of differentiated cells obtained from iPSCs in FBS and xenofree conditions RT-qPCR analysis showed that differentiated iPSCs (both G-iPSCs and S-iPSCs) still expressed the pluripotency markers Oct3/4, Nanog and Sox2. Expression of Oct3/4 and Nanog in both G-iPSC and S-iPSC was lower in xenofree conditions (PL) than in FBS-containing conditions (Fig. 3 A, B), and the difference was statistically significant for Oct3/4 only. However, expression of Sox2 was higher, although not significantly, in PL conditions (Fig. 3 C). All differentiated iPSCs expressed the epithelial markers cytokeratins (KR) 5, 13, 18, and 19. The iPSCs differentiated in FBS-containing conditions showed higher expression of these markers when compared to differentiated iPSCs in PL, although the differences were not found to be statistically significant (Fig. 3 D-G). Flow cytometric analysis showed also decreased expression of pluripotent stem cell markers TRA-60, SSEA4, Sox2 and Oct3/4 of differentiated G-iPSCs and S-iPSCs in both FBS and PL conditions (Fig. 4 A-E), although there were no statistically significant differences between iPSCs generated from fibroblasts of different origin (gingival versus skin) or between iPSCs generated in xenofree or FBS containing conditions. E-cadherin expression was higher (although not statistically significant) in differentiated iPSCs in FBS as compared to the differentiated iPSCs in PL conditions (Fig. 4 E). The western blot for pancytokeratin of the differentiated iPSCs both in presence of FBS or PL showed that the differentiated iPSCs in all conditions expressed pancytokeratin to a certain degree, but the differentiated iPSCs in FBS showed a stronger expression (Fig. 4 F). In vitro generation of oral mucosal sheets Based on the characterization of differentiated iPSCs in monolayers, tests of regeneration of oral mucosal sheets were further performed using only iPSCs differentiated through ECM enriched protocol (Fig. 5 ). The iPSCs differentiated in FBS-containing conditions formed a cohesive 5–6 multi-layered epithelial-like tissue on top of collagen gels (both those derived from skin and gingival fibroblasts), with a basal compartment with cells perpendicular on the collagen interface and a more superficial compartment with more flattened cells (Fig. 6 A, F). The iPSCs differentiated in PL also formed a multilayer epithelial-like tissue on top of collagen gels, which in some areas was even thicker (8–10 cell layers), but the tissue formed showed less distinct basal and superficial cell layers (Fig. 5 K, P). Immunophenotyping of the tissues developed from differentiated iPSCs in FBS-containing conditions showed strong expression of pancytokeratin, indicating differentiation towards stratified squamous epithelium by iPSCs differentiated in FBS (for both gingival and skin-derived iPSCs). The tissues formed on top of the collagen gels by differentiated iPSCs in PL-containing conditions showed a more patchy expression of pancytokeratin, indicating a limited differentiation towards stratified squamous epithelium when iPSCs were differentiated in xenofree conditions (for both gingival and skin-derived iPSCs). Furthermore, differentiated G-iPSCs gave rise to an epithelium that was negative for CK10 (Fig. 6 C, H,M,R,W), but positive for CK13 in the suprabasal layers both in FBS and PL conditions (Fig. 6 D,N), suggesting differentiation of the epithelium formed in the 3D organotypics on top of NOF-containing collagen gels towards an oral phenotype. The expression was, however, weaker than in the 3D models reconstructed with NOK (Fig. 6 X). As expected, vimentin was expressed by the normal oral fibroblasts (NOF) in the collagen matrix but also by few cells in the basal compartment of the tissues regenerated using differentiated iPSCs (Fig. 6 E,J,O,T), similar to the epithelial sheets reconstructed from NOK (Fig. 6 Y). Based on our previous findings which showed GM-SCF to be an important differentiation factor ( 29 ), GM-SCF was also added to some of the 3D organotypic cultures. No significant differences were observed in differentiation of the epithelium-like tissue formed by differentiated iPSCs with or without GM-CSF (data not shown). However, in presence of GM-CSF a thicker epithelium-like tissue was formed on top of the collagen gels, suggesting a higher proliferation of the epithelial-like tissue generated by differentiated iPSCs in presence of GM-CSF, especially when grown in PL conditions. Viability of in vivo xenotransplanted 3D OTs in a mice model as a proof of principle for clinical use of the oral mucosal sheets derived from differentiated iPSCs As a proof of principle, we first used an animal model based on previous literature ( 30 ) in which differentiated iPSCs admixed with fibroblasts were injected into the inner space of the PCL chambers, as explained in Fig. 6 A. The results of that experiment showed inconsistent formation of a single epithelial layer only even in the control mice injected with admixture of NOK and NOF (confirmed with pancytokeratin staining and hAlu ISH – data not shown). The model has been adapted and improved to xenotransplant differentiated iPSCs already assembled in 3D OT cultures before xenotransplantation, as more detailed explained in Materials and methods (Fig. 6 B). For this model, we first constructed 3D OTs using differentiated iPSCs on top of NOF-populated collagen gels and xenotransplanted them in NGS mice using custom made poly ɛ-caprolactone (PCL) chambers (Fig. 6 B). As control of the xenotransplantation technique, we constructed 3D OT models using NOK and NOF isolated from normal adult human oral mucosa and xenotransplanted them after 7 days of in vitro culture on the back of NGS mice, similar to the other iPSC-derived 3D OT cultures (Fig. 6 ). When cultured in FBS-containing conditions, both gingival- and skin-derived iPSCs generated multilayered epithelial structures lining the inner aspect of the PCL chambers (Fig. 7 ). Immunostaining confirmed epithelial commitment, with pancytokeratin robustly detected across the tissues. Notably, the gingival iPSC-derived epithelium lacked CK10 but displayed CK13, a profile characteristic of oral-type differentiation (Fig. 7 ). In contrast, under xenofree (PL) conditions, the epithelial layers derived from both cell sources appeared less compact, with a predominance of basaloid-like cells throughout the thickness, suggesting a reduced degree of stratified squamous maturation compared to FBS-grown counterparts. However, the epithelial-like tissues generated from iPSCs differentiated in xenofree conditions showed expression of pancytokeratin and CK13, although weaker than in FBS-containing conditions. When compared to the epithelium generated by xenotransplanted NOK-containing 3D OTs, the epithelium generated by differentiated iPSCs was less differentiated and expressed weaker CK13, while pancytokeratin expression was comparable for the FBS-containing conditions. As anticipated, vimentin staining was detected in the fibroblasts embedded within the collagen matrix of the xenotransplanted constructs, and sporadically in basal cells of the iPSC-derived epithelium, a pattern also observed in NOK-based controls. The human origin of the regenerated tissues was verified through hALU by in situ hybridization (Fig. 7 ). Overall, the in vivo findings were consistent with the epithelial differentiation and tissue organization seen in the in vitro 3D organotypic models. DISCUSSION The introduction of iPSC technology by Takahashi and Yamanaka marked a turning point in regenerative medicine by enabling the reprogramming of somatic fibroblasts into pluripotent cells without major genomic alterations ( 9 ). This advance addressed key barriers related to donor cell supply, immune compatibility, and ethical concerns ( 31 ). In the past years tremendous progress has been made in understanding the potential of iPSCs for clinical use ( 32 ). iPSC have been differentiated into a number of different cell types, such as cardiomyocytes ( 33 ) and skin keratinocytes ( 22 ). Building on protocols established for skin iPSCs, we adapted differentiation strategies to produce keratinocytes from both skin- and gingiva-derived iPSCs, which subsequently formed oral mucosal sheets. Importantly, these cells could be generated under xenofree conditions and further organized into oral-type epithelia with mesenchymal support. While previous reports have described the conversion of skin fibroblast–derived iPSCs into keratinocytes ( 22 , 23 ), our findings extend this concept by demonstrating, for the first time to our knowledge, that gingival fibroblast–derived iPSCs can also be induced into oral keratinocytes using modified culture protocols. Up to now, iPSCs have been successfully used in treatment of burns and other skin disorders for regenerative therapy in preclinical studies and it is foreseen to have a tremendous impact in the field of dermatology based on the 3D in vitro and in vivo animal studies performed during the past decade ( 34 – 37 ). However, use of iPSCs for generation of oral mucosa has been lagging. The present study is one of the first where skin and gingival fibroblast derived iPSCs were differentiated into oral keratinocytes and employed to produce 3D mucosal sheets in vitro . Cytokeratins represent key intermediate filament proteins that mark epithelial cell differentiation ( 38 ). CK13 is typically distributed across suprabasal layers of non-keratinized oral mucosa, while CK10 is characteristic of epidermis and keratinized oral regions ( 39 ). In our 3D cultures, gingiva-derived iPSCs differentiated in both FBS and PL conditions displayed CK13 in suprabasal compartments, supporting an oral-type epithelial profile. Interestingly, this pattern was more consistent and pronounced in constructs derived from gingival iPSCs than those from skin iPSCs. Although confirmation with additional lineage-specific markers is needed, these observations suggest that gingival iPSCs may retain an epigenetic memory of their tissue of origin, making them more predisposed to differentiate toward an oral epithelial phenotype. Compliance with good manufacturing practice (GMP) standards is essential when developing cellular products for therapeutic application. In this context, human platelet lysate (PL) has gained recognition as a clinically acceptable substitute for fetal bovine serum (FBS) ( 40 ), with several clinical studies already demonstrating its feasibility in both autologous and allogeneic transplantation settings ( 41 , 42 ). In our experiments, FBS appeared to promote more efficient differentiation of iPSCs into epithelial lineages. Nonetheless, xenofree conditions supplemented with PL were also capable of supporting keratinocyte induction in monolayers as well as epithelial sheet formation in 3D constructs. To enable reliable clinical translation, further optimization is required to improve the robustness of PL-based protocols and to ensure the generation of more uniform keratinocyte populations capable of forming continuous, transplantable oral mucosal sheets. To establish proof-of-principle, we developed and refined a murine transplantation system using custom-designed PCL chambers, which allowed stable engraftment of 3D organotypic constructs. These models offered flexibility in controlling extracellular matrix composition and mechanical properties, providing a supportive environment to examine epithelial–mesenchymal interactions ( 43 ). Compared to direct injection of mixed cell suspensions, chamber-based transplantation proved more reliable for maintaining stratified epithelial tissues derived from both iPSCs and NOK. The outcomes of these in vivo experiments aligned closely with our in vitro 3D culture findings, reinforcing the potential of iPSC-derived keratinocytes for reconstructing oral mucosa. However, additional refinement will be necessary to enhance reproducibility and efficiency before clinical translation can be realistically pursued. (“Part of this work was included in the doctoral thesis of RD, University of Bergen, 2022”). CONCLUSION We show here an optimized ECM based cell culture protocol for inducing efficient differentiation of skin and gingival fibroblast-derived iPSC into oral keratinocytes and successful 3D in vitro reconstruction of a tissue with a similar phenotype to oral mucosa. Such 3D tissue-engineered oral mucosal sheets might be used for the repair of clinical oral mucosal defects in the future. Further steps should be taken to optimize the differentiation of adult fibroblast-derived iPSC into oral keratinocytes in xenofree conditions for robust reconstruction of oral mucosal tissues in vivo . Declarations FUNDING This work was funded by Helse Vest (Grants No. 912260/2019 and F-13105/2024), The Research Council of Norway through its Center of Excellence funding scheme, (Grant No. 22325). AUTHOR CONTRIBUTIONS Ridhima Das: Contributed to conception, design, data acquisition, and interpretation, drafted and critically revised the manuscript. Hassan R.W. Ali, Arild Kvalheim: Contributed to generation of fibroblasts and iPSCs. Tarig Osman, Mohammed A. Yassin, Kamal Mustafa, Harsh Dongre, Siren Fromreide, Helge Ræder, Anne Christine Johannessen, Mihaela- Roxana Cimpan, Salwa Suliman, Daniela-Elena Costea: Contributed to conception, design, and critically revised the manuscript. Mihaela Roxana Cimpan, Anne Chr. Johannessen, Salwa Suliman, Daniela-Elena Costea: Supervised the work. All content was critically reviewed and approved by the authors. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Collection and use of normal human oral mucosa and normal human skin samples from healthy adult donors were conducted under the project “Modulation of Stem Cell Plasticity for Regenerative Medicine Applications”, approved by the Regional Committee for Medical and Health Research Ethics, Western Norway (REK Vest; Approval No. 80005; Approval Date: 17 January 2020). All donors were healthy adults and provided written informed consent for participation and the use of their de-identified samples in research. All experiments were performed in accordance with relevant guidelines and regulations. All animal experiments involving NSG mice were approved by the Norwegian Food Safety Authority (NFSA) under project titles “Epi-Trans—Skin and Mucosa Regeneration Using Induced Pluripotent Stem Cells” (FOTS ID: 22627; Approval Date: 3 September 2020) and “Human Tissue Models for Precision Therapy in Head and Neck Cancer” (FOTS ID: 200640; Approval Date: 1 February 2021). All procedures complied with institutional and national ethical guidelines. DECLARATION OF INTERESTS The authors declare no competing interests. DECLARATION ON USE OF AI The authors declare that they have not used AI-generated content in the preparation of the original manuscript. Availability of data and materials Additional data can be made available by the authors upon request. References Purna, S. K. & Babu, M. Collagen based dressings–a review. Burns 26 (1), 54–62 (2000). Rastogi, S., Modi, M. & Sathian, B. The efficacy of collagen membrane as a biodegradable wound dressing material for surgical defects of oral mucosa: a prospective study. J. Oral Maxillofac. Surg. 67 (8), 1600–1606 (2009). Izumi, K. & Feinberg, S. E. Skin and oral mucosal substitutes. Oral Maxillofac. Surg. Clin. N. Am. 14 (1), 61–71 (2002). Ahmad, F. & Lata, J. Collagen matrix membrane as a biological dressing in defects of the oral mucosa. Natl. J. Maxillofacial Surg. ; 12 (2). (2021). Kinikoglu, B., Damour, O. & Hasirci, V. Tissue engineering of oral mucosa: a shared concept with skin. J. Artif. Organs . 18 (1), 8–19 (2015). Weissman, I. L. Stem Cells Cell. ; 100 (1):157–168. (2000). Paz, A. G., Maghaireh, H. & Mangano, F. G. Stem Cells in Dentistry: Types of Intra- and Extraoral Tissue-Derived Stem Cells and Clinical Applications. Stem Cells Int. 2018 , 1–14 (2018). Berthiaume, F. & Hsia, H. C. Regenerative Approaches for Chronic Wounds. Annu. Rev. Biomed. Eng. ; 24 (1). (2022). Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 (4), 663–676 (2006). Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448 (7151), 313–317 (2007). Karagiannis, P. & Eto, K. Ten years of induced pluripotency: from basic mechanisms to therapeutic applications. Development 143 (12), 2039–2043 (2016). Piersma, S. R. et al. Proteomics of the TRAP-induced platelet releasate. J. Proteom. 72 (1), 91–109 (2009). Macaulay, I. C. Platelet genomics and proteomics in human health and disease. J. Clin. Invest. 115 (12), 3370–3377 (2005). Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H. & Tomic-Canic, M. PERSPECTIVE ARTICLE: Growth factors and cytokines in wound healing. Wound Repair. Regeneration . 16 (5), 585–601 (2008). Burnouf, T. et al. Blood-derived biomaterials and platelet growth factors in regenerative medicine. Blood Rev. 27 (2), 77–89 (2013). Burnouf, T., Strunk, D., Koh, M. B. C. & Schallmoser, K. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation? Biomaterials 76 , 371–387 (2016). Ali, H. R. W. et al. Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues. Stem Cell. Res. Ther. 14 (1), 199 (2023). Das, R. et al. Embryonic Stem Cells Can Generate Oral Epithelia under Matrix Instruction. Int. J. Mol. Sci. ; 24 (9). (2023). Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28 (1), 31–40 (2000). Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat. Protoc. 8 (1), 162–175 (2013). Metallo, C. M., Ji, L., de Pablo, J. J. & Palecek, S. P. Directed differentiation of human embryonic stem cells to epidermal progenitors. Methods Mol. Biol. 585 , 83–92 (2010). Kogut, I., Roop, D. R. & Bilousova, G. Differentiation of human induced pluripotent stem cells into a keratinocyte lineage. Methods Mol. Biol. 1195 , 1–12 (2014). Yoshida, S. et al. Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells. PLoS One . 6 (12), e28856 (2011). Zhao, A., Yang, Y., Pan, X., Pan, Y. & Cai, S. Generation of keratinocyte stem-like cells from human fibroblasts via a direct reprogramming approach. Biotechnol. Prog . 36 (3), e2961 (2020). Das, R. Novel Methods and Sources for Regeneration of Oral Mucosa (University of Bergen, 2022). Costea, D. E., Loro, L. L., Dimba, E. A., Vintermyr, O. K. & Johannessen, A. C. Crucial effects of fibroblasts and keratinocyte growth factor on morphogenesis of reconstituted human oral epithelium. J. Invest. Dermatol. 121 (6), 1479–1486 (2003). Bjørlykke, Y. et al. Reprogrammed Cells Display Distinct Proteomic Signatures Associated with Colony Morphology Variability. Stem Cells Int. 2019 , 1–16 (2019). Bilousova, G. & Roop, D. R. Generation of Functional Multipotent Keratinocytes from Mouse Induced Pluripotent Stem Cells. In: (eds Has, C. & Sitaru, C.) Molecular Dermatology: Methods and Protocols. Totowa, NJ: Humana; 337–350. (2013). Das, R. et al. Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium. Eur. J. Oral Sci. 130 (3), e12867 (2022). Kataoka, K. et al. Participation of Adult Mouse Bone Marrow Cells in Reconstitution of Skin. Am. J. Pathol. 163 (4), 1227–1231 (2003). Volarevic, V. et al. Ethical and Safety Issues of Stem Cell-Based Therapy. Int. J. Med. Sci. 15 (1), 36–45 (2018). Paes, B. C. M. F. et al. Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation. Cell Biol. Toxicol. 33 (3), 233–250 (2016). Sepac, A. et al. Comparison of Cardiomyogenic Potential among Human ESC and iPSC Lines. Cell Transplant. 21 (11), 2523–2530 (2012). Choudhury, S., Surendran, N. & Das, A. Recent advances in the induced pluripotent stem cell-based skin regeneration. Wound Repair. Regen . 29 (5), 697–710 (2021). Itoh, M., Kiuru, M., Cairo, M. S. & Christiano, A. M. Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells. Proc. Natl. Acad. Sci. U S A . 108 (21), 8797–8802 (2011). Soares, E. & Zhou, H. Pluripotent Stem Cell Differentiation Toward Functional Basal Stratified Epithelial Cells. Methods Mol. Biol. 2454 , 297–304 (2022). Yan, Y. et al. Effect of iPSCs-derived keratinocytes on healing of full-thickness skin wounds in mice. Exp. Cell. Res. 385 (1), 111627 (2019). Sawaf, M. H., Ouhayoun, J. P., Shabana, A. H. & Forest, N. [Cytokeratins, markers of epithelial cell differentiation: expression in normal epithelia]. Pathol. Biol. (Paris) . 40 (6), 655–665 (1992). Oubayoun, J-P., Gosselin, F., Forest, N., Winter, S. & Franke, W. W. Cytokeratin patterns of human oral epithelia: Differences in cytokeratin synthesis in gingival epithelium and the adjacent alveolar mucosa. Differentiation 30 (2), 123–129 (1985). Schallmoser, K. et al. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 47 (8), 1436–1446 (2007). Espinosa, A. et al. Implementation of a standardised method for the production of allogeneic serum eye drops from regular blood donors in a Norwegian University Hospital: Some methodological aspects and clinical considerations. Transfus. Apheres. Sci. 53 (1), 88–91 (2015). Al-Ajlouni, J. et al. Safety and Efficacy of Autologous Intra-articular Platelet Lysates in Early and Intermediate Knee Osteoarthrosis in Humans. Clin. J. Sport Med. 25 (6), 524–528 (2015). Milligan, D. A., Tyler, E. J. & Bishop, C. L. Tissue engineering to better understand senescence: Organotypics come of age. Mech. Ageing Dev. ; 190 . (2020). Additional Declarations No competing interests reported. Supplementary Files Fullwetsternblotts.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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17:49:43","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143106,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/059c4479f626b915e615e160.png"},{"id":96851743,"identity":"64c36db4-ca58-4fed-b9fb-baf79834518b","added_by":"auto","created_at":"2025-11-26 17:49:43","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113765,"visible":true,"origin":"","legend":"","description":"","filename":"7946be7bdcab447da6b33260af99cb441structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/9788b358cfb7758bff7913dd.xml"},{"id":96851747,"identity":"20996cb0-1ca2-4327-a77d-56044686c598","added_by":"auto","created_at":"2025-11-26 17:49:44","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125576,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/f6187b6590b5672609883d26.html"},{"id":96851722,"identity":"eafb7899-12c5-4ee4-b1d0-d464bd5ca51a","added_by":"auto","created_at":"2025-11-26 17:49:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":950919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative light microscopy images of differentiated iPSCs and respective controls using different GF-based (Protocols I and II) and ECM-based (Protocol III) protocols.\u003c/strong\u003e A-F images show differentiated gingival and skin fibroblast-derived iPSCs (G-iPSCs and S-iPSCs) in FBS using the three protocols. G, H are the differentiated iPSC cells in PL using Protocol III. I and J are the light microscopy images of controls: normal oral keratinocytes and undifferentiated G-iPSCs, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/896084441e143c5f5dc9ce11.png"},{"id":96851723,"identity":"a11966a4-c018-4345-9db0-21a2534e0552","added_by":"auto","created_at":"2025-11-26 17:49:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":672185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative immunofluorescent (IF) images of differentiated iPSCs and respective controls using different GF-based (Protocols I and II) and ECM-based (Protocol III) protocols after staining with anti-pancytokeratin antibody.\u003c/strong\u003e A and B are IF of differentiated gingival fibroblast derived iPSCs (G-iPSCs) using Protocols I and II, respectively. C, E differentiated G-iPSCs using Protocol III in FBS and PL, respectively. D, F differentiated S-iPSCs using Protocol III in FBS and PL, respectively. G and H are IF for the positive (normal oral keratinocytes) and negative (undifferentiated G-iPSCs) controls, respectively.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/7164c6433ef9e1b13bf360a3.png"},{"id":96851725,"identity":"976155f9-ba5b-47d7-b2aa-18d8812b0d45","added_by":"auto","created_at":"2025-11-26 17:49:43","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":141973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRT-qPCR analysis of differentiated gingival and skin fibroblast derived iPSCs in FBS and PL by using the ECM-enriched protocol for pluripotency markers Oct3/4, Nanog, Sox2 and epithelial markers KR5, KR13, KR18 and KR19.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/d2a19e869a72171c21d64c28.jpeg"},{"id":96851726,"identity":"1c2589fe-168d-44fb-997a-8e36c14f41e1","added_by":"auto","created_at":"2025-11-26 17:49:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":359373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of differentiated gingival and skin fibroblast derived iPSCs in FBS and PL using the ECM-enriched protocol by flow cytometry for pluripotency markers TRA-60, SSEA-4, Sox2, Oct3/4 (A-D) and epithelial marker Ecadherin (E) and western blot using pancytokeratin (F).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/06762ec8b1f8427d4a21b667.png"},{"id":96851740,"identity":"70d9c676-ede3-4cc2-95b9-c17964a91fbe","added_by":"auto","created_at":"2025-11-26 17:49:43","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":350910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative microscopy images of 3D organotypic cultures generated from differentiated gingival and skin fibroblast derived iPSCs in FBS and PL by ECM-enriched protocol when seeded on top of gingival fibroblast populated collagen matrices for 11 days.\u003c/strong\u003e A,F,K,P and U are histological images (haematoxylin-eosin staining) and B-E, G-J, L-O, Q-T, V-Y are images of 3D organotypic cultures stained by immunohistochemistry with antibodies against pancytokeratin, CK10, CK13 and vimentin.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/a4b3ab52c68912a3a2d5126c.jpeg"},{"id":96919796,"identity":"5a7bb392-42dc-416a-b4da-608aa86f643a","added_by":"auto","created_at":"2025-11-27 14:14:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1595508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration and real-life images of key experimental procedures involving \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etests\u003c/strong\u003e. (A) After incision and removal of the skin, the PCL chambers were glued using Histoacryl® on the connective tissue covering the muscle layer on the back of the mice and a mixture of cells was injected in the inner pocket formed by PCL chambers, or (B, C, D, E) they glued first to the outer edge of the 3D OTs and then to the back of the mice (F). After the chambers were glued to the mice, the pockets were sutured and secured with metal clams (G). Macroscopic image of mucosal sheets harvested from mice and placed in cassettes for processing.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/b31ce5a78216460b3abc0b48.png"},{"id":96851738,"identity":"cd86e08e-dc6e-4196-94a9-77b2e6afa809","added_by":"auto","created_at":"2025-11-26 17:49:43","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":239856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological images (haematoxylin-eosin) and representative immunostaining of 3D OT cultures of cells differentiated from gingival and skin fibroblast-derived iPSC (G-iPSCs and S-iPSCs) in FBS and PL seeded on top of human oral fibroblast populated collagen matrices for 7 days and then implanted in NGS mice using PCL chambers for 14 more days. \u003c/strong\u003e1,7,13,19 and 25 are histological images (haematoxylin-eosin staining), and 2-6, 8-12, 14-18, 20-24, 26-30 are images of 3D organotypic cultures stained by immunohistochemistry with antibodies against pancytokeratin, CK10, CK13, vimentin and ISH-hAlu.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/6f36dd660d81e6c6a115c67e.jpeg"},{"id":97898587,"identity":"9a652c4f-b673-4929-9be0-112df3fc1f95","added_by":"auto","created_at":"2025-12-10 15:39:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5717020,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/732130d8-128e-4cf6-8bc2-a6c7c6d97d45.pdf"},{"id":96851748,"identity":"b35bbf06-ad45-4f43-97e5-8422229ed442","added_by":"auto","created_at":"2025-11-26 17:50:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":237459,"visible":true,"origin":"","legend":"","description":"","filename":"Fullwetsternblotts.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7842122/v1/995d648534ea26a860ca677a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Xenofree generation of oral keratinocytes from induced pluripotent stem cells derived from adult human fibroblasts for oral mucosal regeneration","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIntraoral surgical wounds occurring post trauma or after tumour excision are either left open or undergo primary closure if small, while they require skin grafts, flaps or delayed closure in case of large defects (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The gold standard for treatment of large defects is the use of skin grafts to prevent infection of the wound bed and promote healing, and this requires usually a second surgical procedure leading to increased post-surgical morbidity. Oral mucosa grafts are best suited, but due to limited availability they are not used for treatment of large defects (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Split-thickness skin grafts are commonly used, but due to presence of various adnexa and the different texture of skin, these are not ideal for the treatment of large oral mucosal defects (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Oral mucosa engineering holds great promise as an alternative to conventional grafting methods, offering a potential solution to overcome these limitations. As a relatively recent field within tissue engineering, it combines cells, materials, and bioactive factors to generate a three-dimensional (3D) model of oral mucosa. It aims to reproduce the real anatomical structure and function of oral mucosa (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStem cells are undifferentiated pluripotent/multipotent cells that can self-renew and differentiate into specialized cells such as epithelial cells, blood cells, adipocytes, \u003cem\u003eetc\u003c/em\u003e (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Based on their origin, stem cells can be classified as embryonic stem cells (ESC), adult stem cells, and induced pluripotent stem cells (iPSC) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). ESCs are a potentially powerful tool for use in tissue engineering, but the use of human ESCs is still not approved ethically. The best source for stem cells is from the patient itself, since this would not involve ethical or immunological concerns. However, the adult stem cells, particularly the epithelial ones, are still very challenging to propagate and amplify in culture for oral epithelial regeneration. The iPSCs generated from adult somatic cells are thus promising candidates for use in cell based therapies (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUse of pluripotent stem cells in regenerative medicine has advanced considerably since Takahashi and Yamanaka developed the iPSCs from somatic cells in 2006, by forced reprogramming of human adult fibroblasts using the transcriptional factors Oct3/4, Sox2, c-Myc, and Klf4 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The iPSCs can self-renew indefinitely without raising any ethical concerns associated with the use of ESC. In the past decade, studies have been focused on numerous aspects of the iPSCs such as optimization methods, mechanisms of reprogramming, differentiation, as well as their clinical applications (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Xenofree expansion of the cells avoids the immunological complications which may be associated with the use of animal derived products like foetal bovine serum (FBS). Studies in the last few years have suggested that the use of human platelet lysate (PL) can support \u003cem\u003ein vitro\u003c/em\u003e clinical grade propagation of human cells, especially for stem cells/progenitor cells. PL contains factors and nutrients which promote cell growth and are essential for wound healing and regeneration of damaged tissues (\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). PL has been proven as a suitable alternative to FBS for propagating human cells in culture for an array of applications in tissue engineering and cell therapy (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo date, several groups, including ours, have reported procedures to differentiate mouse ESCs (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) and human iPSCs derived from skin adult fibroblasts into epidermal keratinocytes (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The iPSCs reprogrammed from normal oral fibroblasts either in FBS containing or xeno-free conditions, have not been used so far for differentiation into oral keratinocytes and for regeneration of either \u003cem\u003ein vitro\u003c/em\u003e or \u003cem\u003ein vivo\u003c/em\u003e oral mucosa.\u003c/p\u003e\u003cp\u003eIn this study, we aimed to investigate if oral/gingival and skin adult fibroblast-derived iPSCs (G-iPSCs and S-iPSCs, respectively) can be induced into oral keratinocytes as a source of cells for oral mucosal regeneration. To achieve this, we tested various conditions and modifications of published protocols known to be efficient in inducing skin keratinocytes from skin fibroblast-derived iPSCs or in inducing epithelial progenitors from mouse iPSCs (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e"},{"header":"MATERIALS \u0026 METHODS","content":"\u003cp\u003eAll experiments were performed in accordance with relevant guidelines and regulations.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003ePrimary cell isolation and culture\u003c/h2\u003e\u003cp\u003eDermal and gingival tissue specimens were obtained from two healthy female volunteers (Donor 1: aged 40\u0026ndash;50 years; Donor 2: aged 50\u0026ndash;60 years) following acquisition of informed consent in accordance with ethical protocols, as previously described (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Two distinct anatomical sites were sampled from each donor: dermal biopsies were harvested from the anterior forearm region, and gingival specimens were excised from the attached gingiva overlying the maxillary first molar. Primary human normal oral fibroblasts (NOF) and normal skin fibroblasts (NSF) were isolated from the attached gingiva and dorsal forearm skin, respectively as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Mycoplasma testing was regularly performed once every two weeks using MycoAlertTM mycoplasma detection kit (Lonza) as per manufacturer\u0026rsquo;s instructions. Normal oral keratinocytes (NOK) were also isolated from the attached gingiva following previously described protocol established in our laboratory (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and were routinely grown on plastic surfaces (Nunc) with no feeding layers, in keratinocyte serum free medium (KSFM) supplemented with 1 ng/ml recombinant human epidermal growth factor (rhEGF, Gibco), 25 \u0026micro;g/ml bovine pituitary extract (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 \u0026micro;g/ml streptomycin (Gibco), 0.25 \u0026micro;g/ml amphotericin B (Gibco). Fibroblasts were grown in Dulbecco\u0026prime;s Modified Eagle\u0026prime;s Medium - high glucose (DMEM) (D6429, Sigma) supplemented with 10% FBS (Fischer scientific), 100 U/ml penicillin, 100 \u0026micro;g/ml streptomycin, 0.25 \u0026micro;g/ml amphotericin B (Gibco). NOF and NSF were also grown in xenofree conditions using Dulbecco\u0026prime;s Modified Eagle\u0026prime;s Medium - high glucose (DMEM) (D6429, Sigma) supplemented with 5% PL (BergenLys, unfiltered, 4-month storage), 1% heparin, 100 U/ml penicillin, 100 \u0026micro;g/ml streptomycin, 0.25 \u0026micro;g/ml amphotericin B (Gibco). NOF and NSF were reprogrammed to gingival and skin iPSCs (G-iPSCs and S-iPSCs respectively) using Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) as described previously (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). They were routinely cultured on plates coated with Geltrex (Gibco) in Stem-flex media (Gibco) with 1% AB/AM (100 U/ml penicillin, 100 \u0026micro;g/ml streptomycin, and 0.25 \u0026micro;g/ml amphotericin B (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eDifferentiation and characterization of human iPSCs\u003c/h3\u003e\n\u003cp\u003eThe iPSCs were differentiated into keratinocytes following slightly modified previously published protocols for differentiation of NSF into skin keratinocytes or mouse iPS into epithelial lineages (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Two of the protocols used were growth factors (GF) based and contained 25 ng/ml bone morphogenic protein 4 (BMP4) and 1uM retinoic acid (RA). RA promotes the differentiation of iPSCs into the ectodermal lineage while BMP4 blocks neural lineage commitment (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). One protocol (Protocol I) involved, in addition, the use of 20 ng/ml rhEGF and 20 ng/ml basic fibroblast growth factor (bFGF), while the other one (Protocol II) involved the use of N2B27 medium. The N2B27 medium combines DMEM/F12 and Neurobasal medium (Gibco) in a 1:1 ratio, supplemented with 0.1 mM nonessential amino acids, 1 mM glutamine, 55 \u0026micro;M 2-mercaptoethanol (2-ME), N2 supplement (100x) (Life Technologies), B27 supplement (50x) (Life Technologies), 50 \u0026micro;g/ml ascorbic acid, 0.05% bovine serum albumin (BSA), 50 U/mL penicillin-streptomycin, 100 ng/ml bFGF (Life Technologies), and 10 \u0026micro;M Y27632 Rock inhibitor (Sigma-Aldrich) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Protocol I lasted for nine days, and protocol II lasted for twenty-four days. The extracellular matrix (ECM) based protocol (Protocol III) used the ECM produced by mitomycin C-metabolically inactivated fibroblasts. This protocol involved also the use of 25 ng/ml BMP4 in the media in addition to ECM and it lasted fourteen days. Protocol III was modified from (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) to minimize the use of xenogeneic derived biological materials as follows: instead of ECM produced by mitomycin-treated mouse PA6 cells as feeder layer, the oral and skin fibroblast-derived iPSCs were grown in this study on a feeder layer produced by mitomycin C - treated human normal primary oral and skin fibroblasts (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe morphology of the iPSCs was observed daily from the first day to the last day of the differentiation protocol using a light microscope. Images were taken every second day when the media was changed. Differentiated cells obtained from all the three protocols were characterized by light microscopy and immunofluorescence and based on the obtained results, Protocol III (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) was further used to produce keratinocytes in both xenofree and xenogeneic conditions.\u003c/p\u003e\u003cp\u003eCollection and use of normal human oral mucosa and normal human skin samples from healthy adult donors were conducted as previously described (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), under the project \u0026ldquo;Modulation of Stem Cell Plasticity for Regenerative Medicine Applications\u0026rdquo;, approved by the Regional Committee for Medical and Health Research Ethics, Western Norway (REK Vest; Approval No. 80005; Approval Date: 17 January 2020). All donors were healthy adults and provided written informed consent for participation and the use of their de-identified samples in research.\u003c/p\u003e\n\u003ch3\u003eMolecular assays\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eDifferentiated iPSCs were cultured on cover slips and fixed with 4% paraformaldehyde (PFA) at room temperature (RT). The cells were then permeabilized with 0.1% Triton X, blocked using 5% BSA in phosphate buffered saline (PBS), and the immunostaining was done using anti-pancytokeratin antibody (DAKO, clone AE1/AE3, 1:100). Alexa Fluor 488 anti-mouse IgG (BioLegend) was used to detect the site of reaction according to the manufacturer`s instructions. The coverslips were then mounted on glass slides with Vectashield containing DAPI (Vector Labs) and the images were captured using a florescence microscope (Axio Imager Z2, Carl Zeiss, Oberkochen, DE). NOK and undifferentiated G-iPSCs were used as positive and negative controls, respectively, as previously described (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). All experiments were repeated at least 3 times in duplicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGene expression analysis\u003c/h2\u003e\u003cp\u003eThe differentiation of iPSCs was assessed also at mRNA level by investigating the expression of several selected pluripotency and epithelial phenotype-related genes, as previously described (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Total RNA was extracted using RNeasy Mini Kit (Qiagen) as per manufacturer\u0026rsquo;s instructions. Measurement of total RNA concentration was done using Nanodrop spectrophotometer (Nanodrop Technologies, USA). Three hundred nanograms of total RNA was converted to cDNA using the High-capacity cDNA kit as per manufacturer\u0026rsquo;s instructions (Applied Biosystems, Carlsbad, US). TaqMan\u003csup\u003e\u0026reg;\u003c/sup\u003e gene expression assays (Applied Biosystems\u003csup\u003e\u0026reg;\u003c/sup\u003e) were used to detect mRNA levels of pluripotency markers (POU5F1-Oct3/4, Nanog, Sox2,) and epithelial markers (cytokeratins 5, 13, 18 and 19 - KR 5, 13, 18, 19). Real-time quantitative polymerase reaction (RT-qPCR) amplification was performed using AB 7500 PCR system for 40 cycles. qPCR was performed on a Step One Plus system, using TaqMan gene expression assays (Applied Biosystems, CA, USA). Data was analysed using the ∆∆Ct method. The expression of the genes was normalized to that of the housekeeping gene, GAPDH. The expression of the pluripotency markers in differentiated iPSCs was presented as fold changes relative to their expression in undifferentiated matched iPSCs. The expression of the epithelial markers in differentiated iPSCs was presented as fold changes relative to NOK. An overview of the primers used for the gene expression analysis is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All experiments were repeated at least 3 times in duplicates.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of TaqMan probes used in the study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene Symbol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTaqMan assay ID\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHs02786624_g1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSOX2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSRY-box 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHs04234836_s1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNANOG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNanog homeobox\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHs02387400_g1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePOU5F1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePOU class 5 homeobox 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHs04260367_gH\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKRT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKeratin 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHs00361185_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKRT13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKeratin 13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHs02558881_s1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKRT18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKeratin 18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHs02827483_g1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKRT19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKeratin 19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHs00761767_s1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eThe differentiated cells were characterized for expression of pluripotency markers: TRA-60 (Stemcell Technologies, clone TRA-1-60R, PE-conjugated, #60064PE, 1:20), SSEA4 (R\u0026amp;D, clone FAB1435A, APC-conjugated, 1:100), Sox2 (R\u0026amp;D, IC2018G, Alexa Fluor\u0026reg; 488-conjugated, 1:100), Oct-3/4 (Santacruz, clone C-10, Alexa Fluor\u0026reg; 488-conjugated, sc-5279), and for the epithelial marker E-cadherin (R\u0026amp;D, clone FAB18381A, APC-conjugated, 1:20). A number of 10⁵ cells were fixed in 4% PFA for 15 min, and permeabilized via 0.1% Triton X, for 15 min. Unspecific binding of antibodies was prevented with blocking buffer (composition 5% BSA in PBS) for 10 min at 4\u0026deg;C. Conjugated monoclonal antibodies were then added to the pellet on ice and the cells were incubated in the dark for 30 min at 4\u0026deg;C before washing. Stained samples were analysed and compared to the corresponding unstained samples (negative control) as well as isotope controls (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The final quantification was performed with a BD Accuri flow cytometer (BD Biosciences\u0026reg;) and the data was analysed using FlowJo (FlowJo\u0026reg;, LLC, Ashland, OR USA). NOK and undifferentiated matched G-iPSCs and S-iPSCs were used as positive controls for epithelial and pluripotency markers, respectively, as described in (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). All experiments were repeated at least 3 times in duplicates.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting\u003c/h3\u003e\n\u003cp\u003eCell lysates for blotting were prepared in RIPA lysis buffer (Sigma-Aldrich, St. Louis, US) containing protease and phosphatase inhibitors (Halt\u003csup\u003e\u0026trade;\u003c/sup\u003e Protease Inhibitor Cocktails, Thermo Scientific). Protein concentration was measured using Pierce bicinchoninic acid (BCA) kit (Thermo Scientific). A total of 25 \u0026micro;g protein was loaded on 4\u0026ndash;12% pre-casted polyacrylamide gels (GenScript), resolved and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). The membranes were blocked with 5% BSA or 5% non-fat dry milk (Biorad) for 1 hr at RT, and incubated with anti-pancytokeratin, (DAKO, clones AE1/AE3, 1.200) and β-actin (Cell Signalling, 1:3000) overnight at 4\u0026ordm; C. The following day the membranes were washed with tris-buffered saline Tween 20 (TBS-T) and then incubated with secondary antibodies anti mouse IgG, HRP-linked (Cell Signalling, 1:3000) anti rabbit IgG, HRP-linked (Cell Signalling, 1:3000) for 1 hour at RT. The reaction was visualized using super signal west femto chemiluminescent substrate (Thermo Scientific, Waltham, US) in SynGene G: Box scanner (Syngene, Frederick, US). The protocol is as describe din (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). All experiments were repeated 3 times.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3D organotypic cultures\u003c/b\u003e\u003c/p\u003e\u003cp\u003e3D organotypic cultures were generated by growing both oral and skin fibroblast-derived iPSCs that were differentiated following Protocol III in both FBS and PL on top of oral fibroblast-populated collagen type I (BD Biosciences) biomatrices, using a well-established protocol in our laboratory (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Simple collagen gels were prepared on ice by mixing 7 vol. (3.40 mg/ml) of rat tail collagen type I (Collaborative Biomedical), 2 vol. reconstitution buffer (261 mM NaHCO3, 150 mM NaOH, 200 mM HEPES) pH 8.15, 1 vol. DMEM 10\u0026times; (Sigma) and 1 vol. FBS (Gibco) or PL. All 3D organotypic cultures were grown in serum free FAD medium (DMEM: Ham\u0026rsquo;s F-12 / 3:1) supplemented with 1 \u0026micro;M hydrocortisone, 10 ng/ml rhEGF, 10 ng/ml KGF, 0.8 \u0026micro;M insulin, 0.25 mM transferrin, 0.25 mM L-ascorbic acid, 15\u0026ndash;30 \u0026micro;M linoleic acid, 15 \u0026micro;M bovine serum albumin, 2 mM L-glutamine (all from Sigma)(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In some cultures, 10ng/ml GM-CSF was also added (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Cultures were grown for three days submerged and for further for seven days at the air-liquid interface on lens paper on metal grids. On the 11th day, the 3D organotypic cultures were harvested, fixed in formalin, embedded in paraffin, cut and hematoxylin-eosin stained for histological analysis, as described in (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). All experiments were repeated 6 times in duplicates or triplicates.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eTo stain the tissues obtained in 3D organotypic cultures, we followed our previously described protocol (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e); 4 \u0026micro;m sections were cut, deparaffinized and rehydrated by immersion in xylene and diminishing concentrations of alcohol. Sections were then incubated for 1 hour at RT with one of the following monoclonal mouse anti-human primary antibodies: anti-pancytokeratin (DAKO, AE1/AE3, 1:100), CK13, CK10 (CRUK, 1:10), and anti-vimentin (DAKO, 1:2000). Envision+\u003csup\u003e\u0026reg;\u003c/sup\u003e anti-mouse (DAKO) was used to detect the site of reaction according to the manufacturer`s instructions. The reaction was visualized using 3,3\u0026prime;-diaminobenzidine tetrahydrochloride (DAB). Sections were then counterstained with hematoxylin (DAKO), dehydrated and cover slipped (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Immunohistochemistry was repeated at least 2 times in duplicates for 4 out of the 6 repetitions of the 3D organotypic assays.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3D printing of poly ɛ-caprolactone (PCL) chambers for xenotransplantation of mucosal sheets\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA 3D CAD model was designed (dimensions in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B) with Magics software (EnvisionTEC, Germany), then sliced into layers at a slice thickness of 80% of the inner needle diameter (ID). In accordance with the manufacturer's guidelines (3D Bioplotter\u0026reg; RP, EnvisionTEC), a slice thickness of 0.32 mm was applied to a stainless- steel needle 0.4 mm in diameter (ID). Before adding 2.5 g of PCL (MW 45 kDa, melting temperature\u0026thinsp;=\u0026thinsp;60\u0026deg;C, Sigma-Aldrich) granules to the cartridge, it was preheated to a preheating temperature of 110\u0026deg;C. The PCL was then kept constant at this temperature during the entire printing time. Chambers were extruded at the predefined designs, as described in (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003emice models\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 16 NSG (NOD/SCID ILgamma2 deficient) mice were used for this study as previously described (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). These mice (aged between 15\u0026ndash;17 weeks, weighing 29-34g) are immunocompromised and lack B and T lymphocytes, as well as natural killer cells. Prior to surgical implantation of PCL chambers and/or subcutaneous cell injection, NSG mice were anesthetized with 3\u0026ndash;4% isoflurane for induction and 1\u0026ndash;2% for maintenance via nose cone, delivered in oxygen. Anesthesia depth was verified by pedal withdrawal reflex. Pre-operative analgesia with buprenorphine (0.05 mg/kg, subcutaneously) was administered. The differentiated iPSCs were both injected subcutaneously within the pre- implanted PCL chambers (6 mice, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), as well as implanted as a mucosal sheet after being pre-assembled in vitro in 3D OT models seven days prior to xenotransplantation and then glued to the lower edge of the PCL chambers with tissue glue Histoacryl\u0026reg; (10mice, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), as previously described (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). After 14 days post-implantation, animals were euthanized using carbon dioxide (CO₂) inhalation in a gradually rising concentration (20\u0026ndash;30% chamber volume/min), followed by cervical dislocation to ensure death. Tissues were harvested immediately thereafter for downstream analysis. All animal experiments are reported in compliance with the ARRIVE 2.0 (Animal Research: Reporting of In Vivo Experiments) guidelines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn situ\u003c/b\u003e \u003cb\u003ehybridization (ISH)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify the transplanted human cells, ISH for the human specific repetitive Alu sequence was used as previously described (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). ISH was performed by RNAscope 2.5 High-Definition Brown Assay according to the manufacturer\u0026rsquo;s instructions (all from Advanced Cell Diagnostics, Newark, CA, United States). Briefly, the tissue sections were baked at 60\u0026deg;C for 1 h followed by deparaffinization in 100% xylene and twice for 5 min each and then two times in 100% ethanol. The slides were then treated with an endogenous peroxidase-blocking reagent, for 15 min by boiling in target retrieval buffer and then treated with protease buffer for 30 min at 40\u0026deg;C. The slides were then incubated with the target Alu probe for 2 h at 40\u0026deg;C, followed by signal amplification. For colorimetric detection, 3,3\u0026prime;- Diaminobenzidine (DAB) was used for 5 min at RT followed by counterstaining with hematoxylin for 5 min. A peptidylprolyl isomerase B (PPIB) Positive Control Probe was used to validate the assay, as previously described (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe statistical analyses were done using GraphPad Prism 9 (GraphPad Software). Data are presented as mean values (+/- standard deviation). Statistical significance was determined by using one-way ANOVA analysis with Tukey\u0026rsquo;s test for multiple comparisons. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003cp\u003eThis work was included in the doctoral thesis of RD, University of Bergen, 2022 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eDifferentiation of G-iPSCs and S-iPSCs into keratinocytes using growth factors (GF) or extracellular matrix (ECM) enriched protocols\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBoth GF enriched (Protocols I and II) and ECM enriched (Protocol III) protocols induced changes in the morphology of G-iPSCs and S-iPSCs, as observed by light microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, A-H). Both G-iPSC and S-iPSC gradually changed their morphology from tight colonies of small, rounded cells with very little cytoplasm, typical for undifferentiated iPSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ) towards larger, flattened, polygonal shape cell morphology, typical for normal oral keratinocytes (NOK, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). When they became confluent, the typical cobble-stone morphology of keratinocytes in monolayer was observed in all conditions, including the condition in which FBS was replaced by PL in the ECM enriched protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H). Immunofluorescence staining using an antibody against pancytokeratin showed various expression in iPSCs differentiated by all the protocols tested. Nearly all iPSC differentiated through ECM enriched protocol (Protocol III) in FBS containing conditions showed a strong and uniform expression of pancytokeratin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), comparable to the expression in NOK (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The iPSCs differentiated through ECM enriched protocol (Protocol III) in xenofree conditions expressed also pancytokeratin, but to a lesser extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F).\u003c/p\u003e\u003cp\u003eFurther tests were performed using iPSC differentiated based on ECM-enriched protocol (Protocol III) only.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCharacterization of differentiated cells obtained from iPSCs in FBS and xenofree conditions\u003c/h2\u003e\u003cp\u003eRT-qPCR analysis showed that differentiated iPSCs (both G-iPSCs and S-iPSCs) still expressed the pluripotency markers Oct3/4, Nanog and Sox2. Expression of Oct3/4 and Nanog in both G-iPSC and S-iPSC was lower in xenofree conditions (PL) than in FBS-containing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), and the difference was statistically significant for Oct3/4 only. However, expression of Sox2 was higher, although not significantly, in PL conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). All differentiated iPSCs expressed the epithelial markers cytokeratins (KR) 5, 13, 18, and 19. The iPSCs differentiated in FBS-containing conditions showed higher expression of these markers when compared to differentiated iPSCs in PL, although the differences were not found to be statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-G).\u003c/p\u003e\u003cp\u003eFlow cytometric analysis showed also decreased expression of pluripotent stem cell markers TRA-60, SSEA4, Sox2 and Oct3/4 of differentiated G-iPSCs and S-iPSCs in both FBS and PL conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-E), although there were no statistically significant differences between iPSCs generated from fibroblasts of different origin (gingival versus skin) or between iPSCs generated in xenofree or FBS containing conditions. E-cadherin expression was higher (although not statistically significant) in differentiated iPSCs in FBS as compared to the differentiated iPSCs in PL conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The western blot for pancytokeratin of the differentiated iPSCs both in presence of FBS or PL showed that the differentiated iPSCs in all conditions expressed pancytokeratin to a certain degree, but the differentiated iPSCs in FBS showed a stronger expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003egeneration of oral mucosal sheets\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBased on the characterization of differentiated iPSCs in monolayers, tests of regeneration of oral mucosal sheets were further performed using only iPSCs differentiated through ECM enriched protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The iPSCs differentiated in FBS-containing conditions formed a cohesive 5\u0026ndash;6 multi-layered epithelial-like tissue on top of collagen gels (both those derived from skin and gingival fibroblasts), with a basal compartment with cells perpendicular on the collagen interface and a more superficial compartment with more flattened cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, F). The iPSCs differentiated in PL also formed a multilayer epithelial-like tissue on top of collagen gels, which in some areas was even thicker (8\u0026ndash;10 cell layers), but the tissue formed showed less distinct basal and superficial cell layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK, P). Immunophenotyping of the tissues developed from differentiated iPSCs in FBS-containing conditions showed strong expression of pancytokeratin, indicating differentiation towards stratified squamous epithelium by iPSCs differentiated in FBS (for both gingival and skin-derived iPSCs). The tissues formed on top of the collagen gels by differentiated iPSCs in PL-containing conditions showed a more patchy expression of pancytokeratin, indicating a limited differentiation towards stratified squamous epithelium when iPSCs were differentiated in xenofree conditions (for both gingival and skin-derived iPSCs). Furthermore, differentiated G-iPSCs gave rise to an epithelium that was negative for CK10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, H,M,R,W), but positive for CK13 in the suprabasal layers both in FBS and PL conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD,N), suggesting differentiation of the epithelium formed in the 3D organotypics on top of NOF-containing collagen gels towards an oral phenotype. The expression was, however, weaker than in the 3D models reconstructed with NOK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eX).\u003c/p\u003e\u003cp\u003eAs expected, vimentin was expressed by the normal oral fibroblasts (NOF) in the collagen matrix but also by few cells in the basal compartment of the tissues regenerated using differentiated iPSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE,J,O,T), similar to the epithelial sheets reconstructed from NOK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eY). Based on our previous findings which showed GM-SCF to be an important differentiation factor (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), GM-SCF was also added to some of the 3D organotypic cultures. No significant differences were observed in differentiation of the epithelium-like tissue formed by differentiated iPSCs with or without GM-CSF (data not shown). However, in presence of GM-CSF a thicker epithelium-like tissue was formed on top of the collagen gels, suggesting a higher proliferation of the epithelial-like tissue generated by differentiated iPSCs in presence of GM-CSF, especially when grown in PL conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eViability of\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003exenotransplanted 3D OTs in a mice model as a proof of principle for clinical use of the oral mucosal sheets derived from differentiated iPSCs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs a proof of principle, we first used an animal model based on previous literature (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) in which differentiated iPSCs admixed with fibroblasts were injected into the inner space of the PCL chambers, as explained in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. The results of that experiment showed inconsistent formation of a single epithelial layer only even in the control mice injected with admixture of NOK and NOF (confirmed with pancytokeratin staining and hAlu ISH \u0026ndash; data not shown). The model has been adapted and improved to xenotransplant differentiated iPSCs already assembled in 3D OT cultures before xenotransplantation, as more detailed explained in Materials and methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). For this model, we first constructed 3D OTs using differentiated iPSCs on top of NOF-populated collagen gels and xenotransplanted them in NGS mice using custom made poly ɛ-caprolactone (PCL) chambers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). As control of the xenotransplantation technique, we constructed 3D OT models using NOK and NOF isolated from normal adult human oral mucosa and xenotransplanted them after 7 days of \u003cem\u003ein vitro\u003c/em\u003e culture on the back of NGS mice, similar to the other iPSC-derived 3D OT cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen cultured in FBS-containing conditions, both gingival- and skin-derived iPSCs generated multilayered epithelial structures lining the inner aspect of the PCL chambers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Immunostaining confirmed epithelial commitment, with pancytokeratin robustly detected across the tissues. Notably, the gingival iPSC-derived epithelium lacked CK10 but displayed CK13, a profile characteristic of oral-type differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, under xenofree (PL) conditions, the epithelial layers derived from both cell sources appeared less compact, with a predominance of basaloid-like cells throughout the thickness, suggesting a reduced degree of stratified squamous maturation compared to FBS-grown counterparts. However, the epithelial-like tissues generated from iPSCs differentiated in xenofree conditions showed expression of pancytokeratin and CK13, although weaker than in FBS-containing conditions.\u003c/p\u003e\u003cp\u003eWhen compared to the epithelium generated by xenotransplanted NOK-containing 3D OTs, the epithelium generated by differentiated iPSCs was less differentiated and expressed weaker CK13, while pancytokeratin expression was comparable for the FBS-containing conditions.\u003c/p\u003e\u003cp\u003eAs anticipated, vimentin staining was detected in the fibroblasts embedded within the collagen matrix of the xenotransplanted constructs, and sporadically in basal cells of the iPSC-derived epithelium, a pattern also observed in NOK-based controls. The human origin of the regenerated tissues was verified through hALU by in situ hybridization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Overall, the in vivo findings were consistent with the epithelial differentiation and tissue organization seen in the in vitro 3D organotypic models.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe introduction of iPSC technology by Takahashi and Yamanaka marked a turning point in regenerative medicine by enabling the reprogramming of somatic fibroblasts into pluripotent cells without major genomic alterations (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This advance addressed key barriers related to donor cell supply, immune compatibility, and ethical concerns (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In the past years tremendous progress has been made in understanding the potential of iPSCs for clinical use (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). iPSC have been differentiated into a number of different cell types, such as cardiomyocytes (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and skin keratinocytes (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Building on protocols established for skin iPSCs, we adapted differentiation strategies to produce keratinocytes from both skin- and gingiva-derived iPSCs, which subsequently formed oral mucosal sheets. Importantly, these cells could be generated under xenofree conditions and further organized into oral-type epithelia with mesenchymal support. While previous reports have described the conversion of skin fibroblast\u0026ndash;derived iPSCs into keratinocytes (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), our findings extend this concept by demonstrating, for the first time to our knowledge, that gingival fibroblast\u0026ndash;derived iPSCs can also be induced into oral keratinocytes using modified culture protocols. Up to now, iPSCs have been successfully used in treatment of burns and other skin disorders for regenerative therapy in preclinical studies and it is foreseen to have a tremendous impact in the field of dermatology based on the 3D \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e animal studies performed during the past decade (\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). However, use of iPSCs for generation of oral mucosa has been lagging. The present study is one of the first where skin and gingival fibroblast derived iPSCs were differentiated into oral keratinocytes and employed to produce 3D mucosal sheets \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eCytokeratins represent key intermediate filament proteins that mark epithelial cell differentiation (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). CK13 is typically distributed across suprabasal layers of non-keratinized oral mucosa, while CK10 is characteristic of epidermis and keratinized oral regions (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In our 3D cultures, gingiva-derived iPSCs differentiated in both FBS and PL conditions displayed CK13 in suprabasal compartments, supporting an oral-type epithelial profile. Interestingly, this pattern was more consistent and pronounced in constructs derived from gingival iPSCs than those from skin iPSCs. Although confirmation with additional lineage-specific markers is needed, these observations suggest that gingival iPSCs may retain an epigenetic memory of their tissue of origin, making them more predisposed to differentiate toward an oral epithelial phenotype.\u003c/p\u003e\u003cp\u003eCompliance with good manufacturing practice (GMP) standards is essential when developing cellular products for therapeutic application. In this context, human platelet lysate (PL) has gained recognition as a clinically acceptable substitute for fetal bovine serum (FBS) (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), with several clinical studies already demonstrating its feasibility in both autologous and allogeneic transplantation settings (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In our experiments, FBS appeared to promote more efficient differentiation of iPSCs into epithelial lineages. Nonetheless, xenofree conditions supplemented with PL were also capable of supporting keratinocyte induction in monolayers as well as epithelial sheet formation in 3D constructs. To enable reliable clinical translation, further optimization is required to improve the robustness of PL-based protocols and to ensure the generation of more uniform keratinocyte populations capable of forming continuous, transplantable oral mucosal sheets.\u003c/p\u003e\u003cp\u003eTo establish proof-of-principle, we developed and refined a murine transplantation system using custom-designed PCL chambers, which allowed stable engraftment of 3D organotypic constructs. These models offered flexibility in controlling extracellular matrix composition and mechanical properties, providing a supportive environment to examine epithelial\u0026ndash;mesenchymal interactions (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Compared to direct injection of mixed cell suspensions, chamber-based transplantation proved more reliable for maintaining stratified epithelial tissues derived from both iPSCs and NOK. The outcomes of these in vivo experiments aligned closely with our in vitro 3D culture findings, reinforcing the potential of iPSC-derived keratinocytes for reconstructing oral mucosa. However, additional refinement will be necessary to enhance reproducibility and efficiency before clinical translation can be realistically pursued.\u003c/p\u003e\u003cp\u003e(\u0026ldquo;Part of this work was included in the doctoral thesis of RD, University of Bergen, 2022\u0026rdquo;).\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eWe show here an optimized ECM based cell culture protocol for inducing efficient differentiation of skin and gingival fibroblast-derived iPSC into oral keratinocytes and successful 3D \u003cem\u003ein vitro\u003c/em\u003e reconstruction of a tissue with a similar phenotype to oral mucosa. Such 3D tissue-engineered oral mucosal sheets might be used for the repair of clinical oral mucosal defects in the future. Further steps should be taken to optimize the differentiation of adult fibroblast-derived iPSC into oral keratinocytes in xenofree conditions for robust reconstruction of oral mucosal tissues \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Helse Vest (Grants No. 912260/2019 and F-13105/2024), The Research Council of Norway through its Center of Excellence funding scheme, (Grant No. 22325).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRidhima Das: Contributed to conception, design, data acquisition, and interpretation, drafted and critically revised the manuscript. Hassan R.W. Ali, Arild Kvalheim: Contributed to generation of fibroblasts and iPSCs. Tarig Osman, Mohammed A. Yassin, Kamal Mustafa, Harsh Dongre, Siren Fromreide, Helge R\u0026aelig;der, Anne Christine Johannessen, Mihaela- Roxana Cimpan, Salwa Suliman, Daniela-Elena Costea: Contributed to conception, design, and critically revised the manuscript. Mihaela Roxana Cimpan, Anne Chr. Johannessen, Salwa Suliman, Daniela-Elena Costea: Supervised the work. All content was critically reviewed and approved by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollection and use of normal human oral mucosa and normal human skin samples from healthy adult donors were conducted under the project \u0026ldquo;Modulation of Stem Cell Plasticity for Regenerative Medicine Applications\u0026rdquo;, approved by the Regional Committee for Medical and Health Research Ethics, Western Norway (REK Vest; Approval No. 80005; Approval Date: 17 January 2020). All donors were healthy adults and provided written informed consent for participation and the use of their de-identified samples in research.\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003eAll animal experiments involving NSG mice were approved by the Norwegian Food Safety Authority (NFSA) under project titles \u003cem\u003e\u0026ldquo;Epi-Trans\u0026mdash;Skin and Mucosa Regeneration Using Induced Pluripotent Stem Cells\u0026rdquo;\u003c/em\u003e (FOTS ID: 22627; Approval Date: 3 September 2020) and \u003cem\u003e\u0026ldquo;Human Tissue Models for Precision Therapy in Head and Neck Cancer\u0026rdquo;\u003c/em\u003e (FOTS ID: 200640; Approval Date: 1 February 2021). All procedures complied with institutional and national ethical guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cins cite=\"mailto:Ridhima%20Das\" datetime=\"2025-07-24T08:58\"\u003e\u0026nbsp;\u003c/ins\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION ON USE OF AI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not used AI-generated content in the preparation of the original manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional data can be made available by the authors upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePurna, S. K. \u0026amp; Babu, M. Collagen based dressings\u0026ndash;a review. \u003cem\u003eBurns\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e (1), 54\u0026ndash;62 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRastogi, S., Modi, M. \u0026amp; Sathian, B. The efficacy of collagen membrane as a biodegradable wound dressing material for surgical defects of oral mucosa: a prospective study. \u003cem\u003eJ. Oral Maxillofac. Surg.\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e (8), 1600\u0026ndash;1606 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIzumi, K. \u0026amp; Feinberg, S. E. Skin and oral mucosal substitutes. \u003cem\u003eOral Maxillofac. Surg. Clin. N. Am.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 61\u0026ndash;71 (2002).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmad, F. \u0026amp; Lata, J. Collagen matrix membrane as a biological dressing in defects of the oral mucosa. \u003cem\u003eNatl. J. Maxillofacial Surg.\u003c/em\u003e ;\u003cb\u003e12\u003c/b\u003e(2). (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKinikoglu, B., Damour, O. \u0026amp; Hasirci, V. Tissue engineering of oral mucosa: a shared concept with skin. \u003cem\u003eJ. Artif. Organs\u003c/em\u003e. \u003cb\u003e18\u003c/b\u003e (1), 8\u0026ndash;19 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeissman, I. L. \u003cem\u003eStem Cells Cell.\u003c/em\u003e ;\u003cb\u003e100\u003c/b\u003e(1):157\u0026ndash;168. (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaz, A. G., Maghaireh, H. \u0026amp; Mangano, F. G. Stem Cells in Dentistry: Types of Intra- and Extraoral Tissue-Derived Stem Cells and Clinical Applications. \u003cem\u003eStem Cells Int.\u003c/em\u003e \u003cb\u003e2018\u003c/b\u003e, 1\u0026ndash;14 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerthiaume, F. \u0026amp; Hsia, H. C. Regenerative Approaches for Chronic Wounds. \u003cem\u003eAnnu. Rev. Biomed. Eng.\u003c/em\u003e ;\u003cb\u003e24\u003c/b\u003e(1). (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakahashi, K. \u0026amp; Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e126\u003c/b\u003e (4), 663\u0026ndash;676 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOkita, K., Ichisaka, T. \u0026amp; Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e448\u003c/b\u003e (7151), 313\u0026ndash;317 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaragiannis, P. \u0026amp; Eto, K. Ten years of induced pluripotency: from basic mechanisms to therapeutic applications. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e143\u003c/b\u003e (12), 2039\u0026ndash;2043 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePiersma, S. R. et al. Proteomics of the TRAP-induced platelet releasate. \u003cem\u003eJ. Proteom.\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e (1), 91\u0026ndash;109 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMacaulay, I. C. Platelet genomics and proteomics in human health and disease. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e115\u003c/b\u003e (12), 3370\u0026ndash;3377 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H. \u0026amp; Tomic-Canic, M. PERSPECTIVE ARTICLE: Growth factors and cytokines in wound healing. \u003cem\u003eWound Repair. Regeneration\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e (5), 585\u0026ndash;601 (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBurnouf, T. et al. Blood-derived biomaterials and platelet growth factors in regenerative medicine. \u003cem\u003eBlood Rev.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e (2), 77\u0026ndash;89 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBurnouf, T., Strunk, D., Koh, M. B. C. \u0026amp; Schallmoser, K. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation? \u003cem\u003eBiomaterials\u003c/em\u003e \u003cb\u003e76\u003c/b\u003e, 371\u0026ndash;387 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAli, H. R. W. et al. Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues. \u003cem\u003eStem Cell. Res. Ther.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 199 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDas, R. et al. Embryonic Stem Cells Can Generate Oral Epithelia under Matrix Instruction. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e ;\u003cb\u003e24\u003c/b\u003e(9). (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. \u003cem\u003eNeuron\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (1), 31\u0026ndash;40 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. \u003cem\u003eNat. Protoc.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (1), 162\u0026ndash;175 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMetallo, C. M., Ji, L., de Pablo, J. J. \u0026amp; Palecek, S. P. Directed differentiation of human embryonic stem cells to epidermal progenitors. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e \u003cb\u003e585\u003c/b\u003e, 83\u0026ndash;92 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKogut, I., Roop, D. R. \u0026amp; Bilousova, G. Differentiation of human induced pluripotent stem cells into a keratinocyte lineage. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e \u003cb\u003e1195\u003c/b\u003e, 1\u0026ndash;12 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoshida, S. et al. Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e (12), e28856 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao, A., Yang, Y., Pan, X., Pan, Y. \u0026amp; Cai, S. Generation of keratinocyte stem-like cells from human fibroblasts via a direct reprogramming approach. \u003cem\u003eBiotechnol. Prog\u003c/em\u003e. \u003cb\u003e36\u003c/b\u003e (3), e2961 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDas, R. \u003cem\u003eNovel Methods and Sources for Regeneration of Oral Mucosa\u003c/em\u003e (University of Bergen, 2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCostea, D. E., Loro, L. L., Dimba, E. A., Vintermyr, O. K. \u0026amp; Johannessen, A. C. Crucial effects of fibroblasts and keratinocyte growth factor on morphogenesis of reconstituted human oral epithelium. \u003cem\u003eJ. Invest. Dermatol.\u003c/em\u003e \u003cb\u003e121\u003c/b\u003e (6), 1479\u0026ndash;1486 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBj\u0026oslash;rlykke, Y. et al. Reprogrammed Cells Display Distinct Proteomic Signatures Associated with Colony Morphology Variability. \u003cem\u003eStem Cells Int.\u003c/em\u003e \u003cb\u003e2019\u003c/b\u003e, 1\u0026ndash;16 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBilousova, G. \u0026amp; Roop, D. R. Generation of Functional Multipotent Keratinocytes from Mouse Induced Pluripotent Stem Cells. In: (eds Has, C. \u0026amp; Sitaru, C.) Molecular Dermatology: Methods and Protocols. Totowa, NJ: Humana; 337\u0026ndash;350. (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDas, R. et al. Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium. \u003cem\u003eEur. J. Oral Sci.\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e (3), e12867 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKataoka, K. et al. Participation of Adult Mouse Bone Marrow Cells in Reconstitution of Skin. \u003cem\u003eAm. J. Pathol.\u003c/em\u003e \u003cb\u003e163\u003c/b\u003e (4), 1227\u0026ndash;1231 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVolarevic, V. et al. Ethical and Safety Issues of Stem Cell-Based Therapy. \u003cem\u003eInt. J. Med. Sci.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (1), 36\u0026ndash;45 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaes, B. C. M. F. et al. Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation. \u003cem\u003eCell Biol. Toxicol.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e (3), 233\u0026ndash;250 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSepac, A. et al. Comparison of Cardiomyogenic Potential among Human ESC and iPSC Lines. \u003cem\u003eCell Transplant.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e (11), 2523\u0026ndash;2530 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoudhury, S., Surendran, N. \u0026amp; Das, A. Recent advances in the induced pluripotent stem cell-based skin regeneration. \u003cem\u003eWound Repair. Regen\u003c/em\u003e. \u003cb\u003e29\u003c/b\u003e (5), 697\u0026ndash;710 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eItoh, M., Kiuru, M., Cairo, M. S. \u0026amp; Christiano, A. M. Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e108\u003c/b\u003e (21), 8797\u0026ndash;8802 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoares, E. \u0026amp; Zhou, H. Pluripotent Stem Cell Differentiation Toward Functional Basal Stratified Epithelial Cells. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e \u003cb\u003e2454\u003c/b\u003e, 297\u0026ndash;304 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYan, Y. et al. Effect of iPSCs-derived keratinocytes on healing of full-thickness skin wounds in mice. \u003cem\u003eExp. Cell. Res.\u003c/em\u003e \u003cb\u003e385\u003c/b\u003e (1), 111627 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSawaf, M. H., Ouhayoun, J. P., Shabana, A. H. \u0026amp; Forest, N. [Cytokeratins, markers of epithelial cell differentiation: expression in normal epithelia]. \u003cem\u003ePathol. Biol. (Paris)\u003c/em\u003e. \u003cb\u003e40\u003c/b\u003e (6), 655\u0026ndash;665 (1992).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOubayoun, J-P., Gosselin, F., Forest, N., Winter, S. \u0026amp; Franke, W. W. Cytokeratin patterns of human oral epithelia: Differences in cytokeratin synthesis in gingival epithelium and the adjacent alveolar mucosa. \u003cem\u003eDifferentiation\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (2), 123\u0026ndash;129 (1985).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchallmoser, K. et al. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. \u003cem\u003eTransfusion\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e (8), 1436\u0026ndash;1446 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEspinosa, A. et al. Implementation of a standardised method for the production of allogeneic serum eye drops from regular blood donors in a Norwegian University Hospital: Some methodological aspects and clinical considerations. \u003cem\u003eTransfus. Apheres. Sci.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e (1), 88\u0026ndash;91 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Ajlouni, J. et al. Safety and Efficacy of Autologous Intra-articular Platelet Lysates in Early and Intermediate Knee Osteoarthrosis in Humans. \u003cem\u003eClin. J. Sport Med.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (6), 524\u0026ndash;528 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMilligan, D. A., Tyler, E. J. \u0026amp; Bishop, C. L. Tissue engineering to better understand senescence: Organotypics come of age. \u003cem\u003eMech. Ageing Dev.\u003c/em\u003e ;\u003cb\u003e190\u003c/b\u003e. (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"induced pluripotent stem cells, xenofree, oral mucosa, keratinocytes","lastPublishedDoi":"10.21203/rs.3.rs-7842122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7842122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the potential of xenofree generation of oral keratinocytes from induced pluripotent stem cells (iPSCs) derived from adult oral and skin fibroblasts for oral mucosal regeneration. Using an extracellular matrix-based protocol, iPSCs were differentiated into cells with a phenotype and molecular profile of oral epithelium, proven through morphological and molecular analyses. Differentiation to epithelial squamous lineage was achieved in serum-containing and xenofree conditions, although the former showed higher efficiency. The differentiated cells successfully formed pluristratified squamous epithelial tissues in 3D organotypic cultures, mimicking oral mucosa. \u003cem\u003eIn vivo\u003c/em\u003e tests using an immunodeficient mouse model validated the viability of multilayered squamous epithelial-like tissues, with distinct oral-specific markers identified in tissues derived from gingival fibroblast iPSCs. These findings demonstrate the feasibility of using iPSCs to create functional oral mucosal sheets, highlighting their potential for clinical applications in regenerative therapies, though further optimization is necessary to enhance differentiation efficiency under xenofree conditions.\u003c/p\u003e","manuscriptTitle":"Xenofree generation of oral keratinocytes from induced pluripotent stem cells derived from adult human fibroblasts for oral mucosal regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 17:49:38","doi":"10.21203/rs.3.rs-7842122/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eb01a98f-4a52-4f3f-b2ea-482d46657bdc","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58504441,"name":"Biological sciences/Biotechnology"},{"id":58504442,"name":"Biological sciences/Cell biology"},{"id":58504443,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-02-01T23:38:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-26 17:49:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7842122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7842122","identity":"rs-7842122","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}