Generation of mesenchymal stromal cells from urine-derived iPSCs of pediatric brain tumor patients

Generation of mesenchymal stromal cells from urine-derived iPSCs of pediatric brain tumor patients Carmen Baliña-Sa ´nchez 1 † , Yolanda Aguilera 1 † , Norma Ada ´n 1 ‡ , Jesu ´s Marı ´aSierra-Pa ´rraga 1 ‡ , Laura Olmedo-Moreno 1 , Concepcio ´nPanadero-Moro ´n 1 , Rosa Cabello-Laureano 2 , Catalina Ma ´rquez-Vega 3 , Alejandro Martı ´n-Montalvo 1,4 and Vivian Capilla-Gonza ´lez 1 * 1 Department of Regeneration and Cell Therapy, Andalusian Molecular Biology and Regenerative Medicine Centre (CABIMER)-CSIC-US-UPO, Seville, Spain, 2 Pediatric Surgery Service, Hospital Virgen del Rocio, Seville, Spain, 3 Pediatric Oncology Service, Hospital Virgen del Rocio, Seville, Spain, 4 Centro de Investigacio ´n Biome ´dica en Red de Diabetes y Enfermedades Metabo ´licas Asociadas (CIBERDEM), Madrid, Spain Human induced pluripotent stem cells (iPSCs) provide a virtually inexhaustible source of starting material for next generation cell therapies, offering new opportunities for regenerative medicine. Among different cell sources for the generation of iPSCs, urine cells are clinically relevant since these cells can be repeatedly obtained by non-invasive methods from patients of any age and health condition. These attributes encourage patients to participate in preclinical and clinical research. In particular, the use of urine-derived iPSC products is a convenient strategy for children with brain tumors, which are medically fragile patients. Here, we investigate the feasibility of using urine samples as a source of somatic cells to generate iPSC lines from pediatric patients with brain tumors (BTiPSC). Urinary epithelial cells were isolated and reprogrammed using nonintegrative Sendai virus vectors harboring the Yamanaka factors KLF4, OCT3/4, SOX2 and C-MYC. After reprogramming, BT-iPSC lines were subject to quality assessment and were compared to iPSCs obtained from urine samples of nontumor pediatric patients (nonT-iPSC). We demonstrated that iPSCs can be successfully derived from a small volume of urine obtained from pediatric patients. Importantly, we showed that BT-iPSCs are equivalent to nonT-iPSCs in terms of morphology, pluripotency, and differentiation capacity into the three germ layers. In addition, both BT-iPSCs and nonT-iPSCs efficiently differentiated into functional mesenchymal stem/stromal cells (iMSC) with immunomodulatory properties. Therefore, this study provides an attractive approach to non-invasively generate personalized iMSC products intended for the treatment of children with brain tumors. KEYWORDS iPSC, mesenchymal stem/stromal cells (MSC), central nervous system cancer, children, cell therapy, cell reprogramming, oncology, cancer Frontiers in Immunology frontiersin.org01 OPEN ACCESS EDITED BY Raghavan Chinnadurai, Mercer University, United States REVIEWED BY Paula Schiapparelli, Mayo Clinic Florida, United States Giuliana Minani Bertolino, Universite ´de Montpellier, France *CORRESPONDENCE Vivian Capilla-Gonza ´lez [email protected] † These authors have contributed equally to this work ‡ These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology RECEIVED 18 August 2022 ACCEPTED 03 January 2023 PUBLISHED 26 January 2023 CITATION Baliña-Sa ´nchez C, Aguilera Y, Ada ´nN, Sierra-Pa ´rraga JM, Olmedo-Moreno L, Panadero-Moro ´nC,Cabello-Laureano R, Ma ´rquez-Vega C, Martı ´n-Montalvo A and Capilla-Gonza ´lez V (2023) Generation of mesenchymal stromal cells from urinederived iPSCs of pediatric brain tumor patients. Front. Immunol. 14:1022676. doi: 10.3389/fimmu.2023.1022676 COPYRIGHT ©2023Baliña-Sa ´nchez, Aguilera, Ada ´n, Sierra-Pa ´rraga, Olmedo-Moreno, Panadero-Moro ´n, Cabello-Laureano, Ma ´rquez-Vega, Martı ´n-Montalvo and Capilla-Gonza ´lez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 26 January 2023 DOI 10.3389/fimmu.2023.1022676 Introduction In 2006, there was a breakthrough in the field of regenerative medicine, when Takahashi and Yamanaka developed the technology to transform any somatic cell into a pluripotent stem cell. These reprogrammed cells, called induced Pluripotent Stem Cells (iPSCs), can be generated by ectopic expression of four transcription factors (i.e. OSKM factors): octamer binding transcription factor 3/4 (OCT3/ 4), sex determining region Y-box 2 (SOX2), Krüppel-line factor 4 (KLF4) and cellular-myelocytomatosis (C-MYC)(1,2). Similar to embryonic stem cells, iPSCs have the ability to self-renew and differentiate into any specialized cell of the body. In addition, iPSCs have certain advantages over other stem cell types for cell-based therapies. Firstly, iPSCs avoid the ethical concerns about the use of embryos to generate pluripotent stem cells. Secondly, they provide a virtually unlimited supply of human cells, bringing the possibility of generating personalized cells for autologous treatment, preventing immune rejection. Importantly, iPSC technology allows the manufacturing of next generation cells, such as iPSC-derived mesenchymal stem/stromal cells (iMSCs), which have been shown to have increased therapeutic efficacy when compared to tissuederived mesenchymal stem/stromal cells (MSCs) in pre-clinical studies (3–8). Thirdly, these cells can be harvested from easily accessible sources, such as skin, blood or urine. Particularly, urinederived iPSCs are obtained by simple, pain-free methods, reducing the risk of adverse effects associated with invasive collection procedures. Therefore, urine offers an interesting option to collect cells repeatedly from patients of any age and under any medical condition, such as pediatric cancer patients (9–11). Brain tumors are the most common solid tumor in children and represent the leading cause of pediatric cancer-related deaths. Latest advances in diagnosis and treatments have improved survival rates of children suffering brain tumors. However, adverse effects of cancer therapies are still affecting the health of many brain tumor survivors. For this reason, researchers are focusing on the development of new strategies aimed to reduce toxicity of cancer treatments. The majority of brain tumor patients that receive radiotherapy, one of the most common treatments for cancer (12), exhibit cognitive dysfunction, including deficits in learning, memory, language, attention and executive function (13). These neurological complications are frequently associated with radiation-induced damage to healthy brain tissue, such as neuroinflammation and cell death (12,14–17). Despite the fact that neurocognitive sequelae of radiotherapy may occur in patients of any age, these adverse effects particularly affect pediatric patients because the developing brain is more sensitive to radiation. Recent reports have demonstrated the neuroprotective effects of cell-based therapies to prevent neurological complications of radiotherapy, thus promoting a healthy cancer-free life (18–24). In particular, the administration of MSCs has been shown to efficiently rescue behavioral deficits in mice following cranial radiation, by reducing neuroinflammation and cell death (18,22–24). However, the clinical translation of MSC-based therapies as a neuroprotective strategy is hampered by challenges related to manufacturing and cell availability. In this context, the use of iPSCs as an unlimited source of MSCs (i.e., iMSCs) emerges as an interesting option that enables large-scale production of cellular products for both autologous and allogenic therapies. Furthermore, iMSCs can generate from a single iPSC clone, thus reducing the heterogeneity acquired by tissuederived MSCs (25–27). These attributes facilitate the obtaining of consistent and robust final products that could be used for the prevention of radiation-related neurological complications. However, the generation of iMSCs from brain tumor pediatric patients remains to be achieved. In this study, we demonstrated that urine-derived epithelial cells (UDCs) is a feasible source to generate iPSCs from children with brain tumors following a non-invasive cell collection procedure. In addition, we show that iPSCs differentiate into functional iMSCs. To our knowledge, this is the first study involving brain tumor pediatric patients that successfully generate iMSCs. The establishment of iPSC lines offers a stable source of MSCs (i.e., iMSCs), boosting their clinical application in a variety of diseases, for both children and adults. Methods Obtaining of urine samples from pediatric patients Urine samples were collected from pediatric patients (age <60 months old) at the Hospital Universitario Virgen del Rocı  o of Seville, after obtaining written informed consent. Samples with volumes ranging from 12 to 40 ml were collected in a sterile container, kept at 4° C, and processed within 1 hour. Urine obtained from brain tumor patients was collected prior to oncological treatment (e.g. surgery, radiotherapy or chemotherapy). Urine obtained from nononcological patients was used as control samples (Table 1,Figure 1). Isolation and expansion of UDCs Urine was centrifuged at 400 × g for 10 min at room temperature. Cell pellets were washed with Dulbecco’s phosphate buffered saline (DPBS), resuspended in isolation medium (DMEM/F-12 with 15mM HEPES, 10% fetal bovine serum (FBS), 1% non-essential amino acids, 10 ng/mL recombinant human EFG, 36 ng/mL hydrocortisone, 5 µg/ mL recombinant human insulin, 500 ng/mL epinephrine, 5 µg/mL human holo-transferrin, 4 pg/mL triodo-L-thyronine, 434.4 µg/mL alanyl-glutamine, 100 µg/mL penicillin/streptomycin, 2.5 µg/mL amphotericin B and 0.1% rock inhibitor) and seeded in gelatincoated plates. Half-medium changes were performed every day to avoid unnecessary cellular stress. Once the first colonies were observed (day 7-15), total medium was replaced. The first passage was performed when cells were approximately 30% confluent using trypsin (day 12-20). Then, UDCs were seeded at a density of 10.000 cells/cm 2 with a 1:1 mix of isolation medium and expansion media (i.e., isolation medium with reduced FBS concentration to 5% and antibiotic free). From passage 2, only expansion medium was used to boost UDC amplification prior to cell reprograming. UDCs were incubated at 37°C in a humidified atmosphere with 20% O 2 and 5% CO 2 . Baliña-Sa ´nchez et al. 10.3389/fimmu.2023.1022676 Frontiers in Immunology frontiersin.org02 Reprogramming of UDCs into iPSCs UDCs at passage below 5 were reprogrammed when cells reached a 30-60% confluency. To reduce the risk of genetic abnormalities, UDCs were reprogrammed to iPSCs using the non-integrative CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher, Waltham, MA, USA) (Figure 2A). After a 7-day period, transfected cells were transferred onto a matrigel-coated plate with mTeSR plus basal medium (STEMCELL Technologies, Grenoble, France) and allowed to grow in a humidified incubator with 37°C, 20% O 2 and 5% CO 2 . After 5 days, iPSC colonies emerged. The first 4 passages were carried out mechanically to specifically select colonies with an iPSC morphology. Then, cell passages were performed using the ReLeSR reagent (STEMCELL Technologies), following the manufacturer’s guidelines. iPSCs were expanded for full characterization and banking. All established cell lines will be deposited at the Banco Nacional de Lı  neas Celulares (BNLC) of the Institute of Health Carlos III, following the Spanish legislation. Characterization of the generated iPSCs Alkaline phosphatase staining Alkaline phosphatase activity was evaluated in fixed iPSC colonies using the SIGMAFAST™BCIP®/NBT kit (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer’s guidelines. Stained iPSCs were visualized and imaged using an Olympus IX71 microscope equipped with an DPController and DPManager software (Center Valley, PA, USA). Three lineage differentiation For the differentiation of iPSCs into the three germ layers (endoderm, mesoderm and ectoderm), we used the specific induction media of the STEMdiff™Trilineage Differentiation Kit (STEMCELL Technologies). According to the manufacturer’s instructions, a 5-day period was required for endoderm and mesoderm differentiation, while ectoderm induction needed 7 days. Media was changed daily. Differentiated cells were harvested and used to isolate RNA for reverse transcriptase‐polymerase chain reaction (RT-PCR) and to perform immunofluorescence assays. Immunofluorescence staining Cells were fixed with 4% paraformaldehyde (PFA), washed with phosphate buffered saline (PBS), incubated in blocking solution for 1 hour, and incubated with primary antibodies at 4°C overnight (see Supplemental Table 2 for antibodies information). Then, cells were washed and incubated with the appropriate secondary antibodies conjugated with fluorophores. Hoechst 33342 nucleic acid stain (Sigma-Aldrich) was used to detect cell nuclei. Fluorescence labeling was examined using an Olympus IX71 microscope equipped with DPController and DPManager software. Molecular karyotyping DNA from iPSCs was extracted using the DNeasy Blood and Tissue kit (QIAGEN, Gilden, Germany). Then, molecular karyotyping was performed with an Affymetrix CytoScan 750k Array (Affymetrix, Santa Clara, CA, USA) by trained experts of the Genome Core Facility of CABIMER to detect chromosomal variations (log2 ratio intensities and copy number). Data viewing and analysis was performed with the Affymetrix Chromosome Analysis Suite (ChAS) software, using the standard setting filters (400 kbp with a marker count of ≥50 for gains; 400 kbp with a marker count of ≥25 for losses) and compared to control data from the Database of Genomic Variants (DGV). The Online Mendelian Inheritance in Man (OMIM) database was used to study associations of genetic alterations with disease susceptibility. All samples and processes fulfilled the following TABLE 1 Clinical characteristics of pediatric patients. Patient Age (months) Sex Diagnosis Sample Volume (urine) hPSCreg name Sample ID* nonT Patient 1 7 male Inguinal hernia 12.6 mL ESi089-A nonT-iPSCs 1 nonT Patient 2 56 male Cryptorchidism 13.6 mL ESi090-A nonT-iPSCs 2 BT Patient 1 43 female Low grade Glioma 32 mL ESi087-A BT-iPSCs 1 BT Patient 2 24 female Metastatic brain tumor 40 mL ESi088-A BT-iPSCs 2 * Sample ID relates to the name given to each iPSC line in this manuscript. FIGURE 1 Representative magnetic resonance images of pediatric patients with brain tumors included in the study. Saggital and axial images reveal a low grade glioma (BT Patient 1, 43-month-old) and a metastatic brain tumor (BT Patient 2, 24-month-old). Red arrows point to the tumor. Baliña-Sa ´nchez et al. 10.3389/fimmu.2023.1022676 Frontiers in Immunology frontiersin.org03 quality criteria: MAPD ≤0.25, Waviness SD ≤0.12 and SNPQC ≥15. Affymetrix data are deposited in Gene Expression Omnibus (GEO) database repository (accession number: GSE213813). Fingerprinting The identity of the established cell lines was analyzed by the cell line authentication service qGenomics (Barcelona, Spain). DNA analysis of the iPSCs and their parental UDCs was performed by genotyping the following human-specific short tandem repeat (STR) markers: TH01,D21S11,D5S828,D13S317,D7S820,D16S539, CSF1PO,vWA and TPOX. The combination of these nine genetic markers results in an allele profile with a random match probability of 1 in 2.9 x 10 9 . A gender-specific marker (AMEL) was also analyzed to establish the presence of sex chromosomes, as well as a specific marker to detect the presence of murine sequences that could result from a possible cross-contamination of the cultured iPSCs with any independent culture of murine cells. Differentiation of iPSCs into MSCs and characterization Differentiation of iPSCs into iMSCs was induced with the STEMdiff™Mesenchymal Progenitor Kit (STEMCELL Technologies), following the manufacturer’s instructions. Briefly, iPSCs were differentiated into early mesoderm progenitor cells for four days. Then, cells were plated in animal component-free (ACF) precoated wells to derive early mesenchymal progenitor cells. By day 21, differentiated cells exhibited MSC phenotype that was verified by morphological analysis, flow cytometry and multilineage differentiation potential, based on previously published methodology (28,29). For the flow cytometry study, the surface markers CD13, CD14, CD73, CD105, CD29, CD31, CD34, CD45, CD90 and HLA II were used (see Supplemental Table 2 for antibody information). Differentiation of iMSCs into adipocytes was performed using the MesenCult™Adipogenic Differentiation Kit (STEMCELL A B FIGURE 2 Isolation of UDCs and reprogramming process. (A) Schematic timeline of the process for isolation and expansion of UDCs and iPSC generation. (B) Microscope images showing the morphological aspect of cultured UDCs (scale bar 200 µm), iPSC colonies obtained after selective passaging postreprogramming (scale bar 1 mm), iPSCs at higher magnifications (scale bar 100 µm) and alkaline phosphatase staining of iPSC colonies (scale bar 400 µm). Baliña-Sa ´nchez et al. 10.3389/fimmu.2023.1022676 Frontiers in Immunology frontiersin.org04 technologies). After 21 days, the presence of lipid droplets was determined by staining the cells with Oil red O (Sigma-Aldrich) after fixation with 4% PFA for 5 minutes. Differentiation of iMSCs into osteoblasts was performed using the MesenCult™Osteogenic Differentiation Kit (STEMCELL technologies). After 15 days, the presence of osteoblasts was determined by staining the cells with Alizarin Red S sodium salt (Alfa Aesar, Haverhill, MA, USA), after fixation with 4% PFA for 5 minutes. Differentiation of iMSCs into chondrocytes was performed using the MesenCult™-ACF Chondrogenic Differentiation kit (STEMCELL technologies). After 21 days, cell pellets were fixed overnight with 4% PFA, paraffinembedded and sectioned with a microtome (Leica RM 2255) to 5 µm thickness. Then, sections were stained with Alcian-Blue solution (Sigma-Aldrich). Images were visualized with an Olympus IX71 microscope and a Nikon ECLIPSE Si microscope. Inflammatory cytokines secreted by iMSCs Culture medium was collected after 48 hours of cell growth to analyze cytokines secreted by iMSCs. Interleukin 8 (IL-8), interleukin 12p70 (IL-12p70), monocyte chemoattractant protein-1 (MCP-1, also named CCL2), platelet-derived growth factor BB (PDGF-BB) and tumor necrosis factor alpha (TNFa) were examined using the Quantibody Human Inflammation Array-3 (Raybiotech, Inc., Norcross, GA, USA), according to the manufacturer´s protocol. Fluorescence signals were detected at 532 nm by a laser scanner (Axon GenePix; Molecular Devices, Sunny-vale, CA, USA). iMSC priming Cells were incubated during 48 hours with (primed iMSC) or without (non-primed iMSC) an inflammatory cytokine mixture containing recombinant human TNFa(10 ng/mL) and recombinant human Interferon gamma (IFNg;10ng/mL)in DMEM supplemented with 15% FBS. For gene expression analysis, a set of cells was thoroughly washed after priming to remove these cytokines and collected for RNA isolation either inmediatly or 48 hours after priming. For secretome generation, another set of cells was thoroughly washed after priming and then fresh culture medium was added. After a 48 hours period, conditioned medium (CM) was collected and stored at -80° until use. RNA extraction and RT-PCR Total RNA was isolated from cultured cells with the Easy-Blue™ kit (iNtRON Biotechnology, Inc., Seongnam, Korea). cDNA was obtained from 1 mg of total RNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Conventional RTPCR was used to evaluate the expression of pluripotencyand differentiation-associated genes using the primers described in Supplemental Table 1. The clearance of the Sendai virus (SeV) vector and the OSKM reprogramming factors was also confirmed by conventional RT-PCR. Agarose gel electrophoresis was used to resolve PCR products. Presence of mycoplasma was analyzed using the commercial kit Venor GeM (Minerva Biolabs GmbH, Berlin, Germany). For quantitative RT-PCR (RT-qPCR), the iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) was used following the manufacturer guidelines, using a total volume of 10 µL and 50 ng of cDNA per reaction, in triplicates. RT-qPCR was performed using a ViiA™7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and analyzed with the ViiA™7 Software (Applied Biosystems), using the standard instrument protocol. The expression of immunomodulatory genes was studied using the primers described in Supplemental Table 1 and the relative gene expression was normalized using TATA box binding protein (TBP) as the housekeeping gene. Data was collected from three independent experiments. iMSC and PBMC cocultures Peripheral blood mononuclear cells (PBMCs) from two donors with different blood types were purchased from STEMCELL Technologies (Cat# 70025.2). PBMCs were thawed and cultured overnight in RPMI medium supplemnted with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine to allow resumption of metabolism, as suggested by the manufacturer. ImmunoCult™Human CD3/CD28 T Cell Activator reagent (Cat# 10971, STEMCELL Technologies) was used to stimulate PBMCs, following the manufacturer’s instructions (25 µl of reagent per ml of medium). For direct cocultures, 1·10 5 activated PBMCs were added to cultured iMSCs at different ratios (PBMC:iMSC of 2:1, 4:1, 8:1, 16:1) in a 96 wellplatefor5days(30). For indirect cocultures, 1·10 5 activated PBMCs were incubated for 72h either in RPMI or in a combination of RPMI and CM from primed/non-primed iMSC (volume ration RPMI : RPMI:CM of 1:1) in a 96 well plate. The CM was generated from 1·10 6 iMSCs cultured for 2 days in 10 ml of medium. To study the proliferation of CD3+ T cell, PBMCs were labelled with the cell division tracker 5chloromethylfluorescein diacetate (C7025, Thermo Fisher) and then subjected to flow cytometry (31). To evaluate the proliferation of CD4+ and CD8+ T cell subpopulations, a Ki-67 proliferation test was performed following the manufacturer´s protocol (32). Flow cytometry Cultured cells were harvested and incubated for 20 minutes with the appropriated primary antibodies in the dark at room temperature (see Supplemental Table 2 for antibody information). For Ki-67 staining, cells were fixed and permeabilized with ethanol at -20° C for 2 hours before incubation with primary antibody. After primary antibody incubation, cells were washed with PBS, centrifuged at 2000 rpm for 5 minutes and analyzed using an LSRFortessa X-20 flow cytometer (BD Biosciences, San Diego, CA, USA) and the BD FACSDiva software (BD Biosciences). Determination of superoxide dismutase activity The antioxidant action of the iMSC secretome was measured by the Superoxide Dismutase (SOD) Activity Assay Kit (Cayman Baliña-Sa ´nchez et al. 10.3389/fimmu.2023.1022676 Frontiers in Immunology frontiersin.org05 Chemical, Ann Arbor, MI, USA). Samples were assayed according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a Varioskan Flash microplate reader (Thermo Electron, Vantaa, Finland). Units of SOD activity were calculated from a standard curve using purified bovine erythrocyte SOD enzyme. All measurements were performed in duplicate. Statistical analysis Data expressed as mean ± SEM was analyzed using the GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA). Parametric ANOVA followed by a Tukey´s or Dunnett’spost-hoc test was performed to compare more than two experimental groups. Comparison between two experimental groups were performed with Student’s t-test. All differences were considered significant at a P value <0.05. Results UDCs from children with brain tumors can be successfully cultured and reprogrammed into iPSCs The isolation of epithelial cells was carried out from urine samples of four pediatric patients, two with brain tumors (BT patients) and two with non-tumor conditions (nonT patients) (Figure 1,Table 1). Adherent UDCs were observed during the first week of culture, showing spindle-shaped or round-shaped morphologies. First UDC colonies emerged on day 11 and, then, UDCs were expanded for a maximum of 5 passages to assure enough material for reprogramming (Figure 2). To generate iPSCs, we used the non-integrative Sendai viral vectors to reduce the risk of genetic abnormalities in the generated cell lines (Figure 2A). The reprogramming efficiency was similar in all patient-derived samples. At day 7 post-reprogramming, colonies with typical iPSC morphology (i.e. dense, roundly shaped colonies with sharp edges, containing small cells with a high nucleus-to-cytoplasm ratio) were selected and manually picked to achieve highest culture purity. Alkaline phosphatase activity staining confirmed the identification of pluripotent iPSC colonies (Figure 2B). Passaged iPSCs were seeded onto matrigel-coated plates and expanded for full characterization. To further validate the identity of the generated cell lines, gene expression of pluripotency markers was evaluated. RT-PCR analysis evidences that, in contrast to UDCs, iPSC colonies from BT patients and nonT patients had a robust expression of the key pluripotency genes OCT3/4, SOX2, NANOG and TERT (Figure 3A). We also evaluated the expression of pluripotency markers at protein level by immunofluorescence, using the intracellular marker OCT3/4 and the surface markers SSEA4, TRA-1-60 and TRA-1-81. Cultured iPSCs stained positive for all the markers assessed, confirming their pluripotency (Figure 3B). Multilineage differentiation potential was confirmed in patient-derived iPSCs To further demonstrate the pluripotency of iPSCs, tri-lineage differentiation was performed using specific induction media. Reprogrammed cells efficiently differentiated into endodermal, A B FIGURE 3 Analysis of the pluripotency markers in the generated iPSC lines. (A) RT-PCR analysis of the expression of pluripotency-associated genes. Note that all iPSC lines highly express OCT3/4, SOX2, NANOG and TERT, while UDCs do not. (B) Immunofluorescence staining showing the presence of pluripotency markers OCT3/4 (magenta), SSEA-4 (green), TRA-1-60 (green) and TRA-1-81 (green) in the iPSCs. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar 100 µm. Baliña-Sa ´nchez et al. 10.3389/fimmu.2023.1022676 Frontiers in Immunology frontiersin.org06 mesodermal and ectodermal lineage cells. The differentiation potential of the generated iPSCs was confirmed through expression of specific genes for endoderm (SOX17, FOXA2), mesoderm (CXCR4, BRACH)andectoderm(NGN3)(Figure 4A). In addition, differentiated iPSCs were assessed for immunofluorescence using AFP (endoderm marker), SMA (mesoderm marker) and Nestin (ectoderm marker) (Figure 4B). Both RT-PCR and immunofluorescence results denoted similar tri-lineage differentiation potential in all patient-derived iPSC lines. Authentication, molecular karyotyping, virus clearance and mycoplasma testing of established iPSC DNA fingerprinting analysis indicates that iPSCs from BT and nonT patients shared identity with their parental UDCs, demonstrating that we generated four new iPSC lines (Figure 5A). The safety of the reprogramming method was evaluated by molecular karyotyping, which evidences minimal chromosomal abnormalities (<1.1% for autosomes) in all urine-derived iPSCs, based on Log2 ratio and copy number (Figure 5B). Importantly, genomic alterations related to tumorigenesis were absent in the iPSCs from brain tumor pediatric patients (Supplemental Table 3). Finally, the absence of exogenous reprogramming vectors and mycoplasma contamination was verified in the generated iPSC lines by negative PCR (Figures 5C,D). These results support that the production of iPSCs from pediatric patients with brain tumors may be suitable for clinical applications. Differentiation of iPSCs into iMSCs In order to determine whether iPSCs can serve as a platform to obtain next generation MSCs, we used specific induction media to generate iMSCs (Figure 6A). During the course of the protocol, iPSCs from BT and nonT patients acquired a fibroblast-like morphology (Figure 6B). By day 21, differentiated iPSCs expressed typical MSC markers, such as CD13, CD29, CD73, CD90 and CD105, while they lacked the expression of CD14, CD31, CD34, CD45, and HLA-II (Figure 6C,Supplemental Figure 1). In addition, the absence of undifferentiated cells (i.e. iPSCs) was confirmed by the lack of expression of TRA-1-60 and SSEA4, supporting successful differentiation of iPSCs into MSCs (Supplemental Figure 2). iMSC identity was further confirmed based on their multilineage differentiation potential. Both BT and nonT patient-derived iMSC possessed the ability to differentiate into adipocytes, osteocytes and chondrocytes, as demonstrated by the positive staining for Oil Red O, Alizarin Red S and Alcian Blue, respectively (Figure 6D, Supplementary Figure 1). These data demonstrate that urinederived iPSCs from pediatric patients can be efficiently differentiated into iMSCs, irrespective of brain tumor diagnosis. Immunomodulatory potential of the generated iMSCs To evaluate the immunomodulatory phenotype of the generated iMSCs, we examined the secretion of immunoregulatory cytokines in the media of cultured cells. We analyzed the presence of IL-8, ILA B FIGURE 4 Analysis of the differentiation capacity of the generated iPSCs into the three embryonic layers. (A) RT-PCR analysis showing the expression of specific differentiation markers, including markers for endoderm (SOX17 and FOXA2), mesoderm (CXCR4 and BRACH) and ectoderm (NGN3). Note that differentiated iPSCs highly express all the trilineage differentiation markers, while undifferentiated iPSCs do not. (B) Immunofluorescence staining showing the presence of alfa-fetoprotein (AFP) for endoderm, Smooth Muscle Actin (SMA) for mesoderm and Nestin for ectoderm in the differentiated iPSCs. Nuclei were counterstained with Hoechst 33342. Scale bar 50 µm. Baliña-Sa ´nchez et al. 10.3389/fimmu.2023.1022676 Frontiers in Immunology frontiersin.org07 12p70, MCP-1, PDGF-BB and TNFaand observed that all the iMSCs secrete at least two of the analyzed cytokines (Figure 7A). Then, we assessed the capacity of iMSCs to reduce T cell proliferation using activated PBMCs from different donors, in both direct and indirect cocultures (Figures 7B,C). We showed that the generated iMSCs cultured in direct contact with PBMCs inhibited CD3+ T cell proliferation in the PBMC of the two donors tested (Figure 7B). In addition, we also determined that the proliferation of CD3+ T cells was diminished by the secretome of BT-iMSC and nonT-iMSC, with an overall similar trend in both CD4+ and CD8+ T cell subsets (Figure 7C). Despite the fact that comparison of iMSCs with adipose tissue-derived MSCs (adMSCs) was not the focus of this study, we would like to mention that the secretome of iMSCs exhibited higher capacity to decrease CD3+ T cell proliferation and showed enhanced antioxidant capacity when compared to adMSCs (Supplemental Figures 3A-C). Furthermore, the secretome of primed iMSCs was used to interrogate the immunomodulatory plasticity of MSCs after stimulation with inflammatory cytokines (33,34). We found that cell priming with TNFaand IFNgmodulated the immunomodulatory potential of iMSC secretome, showing in several instances a predisposition to dampen the inhibition of T cell proliferation when compared to the CM of non-primed iMSCs (Figure 7C). These results demonstrated that urine-derived iPSCs from BT and nonT patients are a viable source to obtain iMSCs with immunomodulatory properties. In order to investigate how the priming with inflammatory cytokines regulates BT-iMSC immunomodulation, we examined themRNA levels of several immunomodulatory genes, including TNFa, TGFb, IL-6, IL-1b, IDO and COX2, in BT-iMSCs. While an overall increase of these factors was found immediately after cell priming (Figure 7D), the expression of the immunomodulatory genes tested reverted to basal levels after 48 hours of priming, with minimal residual effects on IL-6 expression in primed cells (Figure 7E). These data suggest that a transient upregulation of immunomodulatory genes in iMSCs is sufficient to improve their immunomodulatory secretome. Discussion Reprogramming somatic cells into iPSCs has emerged as an innovative strategy to improve manufacturing of cellular products for regenerative medicine. Among the different somatic cell types, A BCD FIGURE 5 Authentication, molecular karyotyping, virus clearance and mycoplasma testing of established iPSCs. (A) DNA fingerprint analysis showing that the allele pattern in the iPSCs generated is 100% concordant with the patients’UDCs and it is not concordant with any commercial cell line whose genotype is posted in public databases. The STR locations studied were: TH01, D21S11, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, TPOX and AMEL. The percentage of matching between iPSCs and their parental UDCs is indicated for each sample. (B) Whole genome view of the iPSC lines which displays all somatic and sex chromosomes in one frame. The smooth signal plot (right y-axis) is the smoothing of the Log2 ratios (left y-axis), which depict the signal intensities of probes on the microarray and represents the number of copies of each chromosome. The pink, green and yellow colors represent the raw signal for each individual chromosome probe, and the blue signal represents the normalized probe signal, used to identify copy number and any aberrations. The percentage of autosomal loss of heterozygosity (LOH) is indicated for each cell line. (C) RT-PCR analysis showing the absence of expression of Sendai virus (SeV) genome and OSKM transgenes in the established iPSC lines. UDCs served as negative control and recently transfected iPSCs served as positive control. (D) PCR test for mycoplasma detection, showing the absence of contamination in cultured iPSC lines. Baliña-Sa ´nchez et al. 10.3389/fimmu.2023.1022676 Frontiers in Immunology frontiersin.org08 UDCs represent a convenient source of iPSCs for personalized cell therapies, particularly for those patients with a severe disability affecting their health. Among medically fragile patients, children with cancer are at a high risk of frailty due to the progression of the disease and to the side effects of cancer treatments. For instance, brain tumor pediatric patients experience multiple neurological complications, including deficits in learning, memory, language, attention and processing speed, even after overcoming the illness (35). These debilitating conditions impede in many cases their participation in clinical trials, which is a major challenge for the translation of pediatric cancer research into clinical practice. Unlike solid biopsies, urine collection represents an easy, safe, pain-free and inexpensive way of obtaining large quantities of somatic cells to engineer cell-based therapeutics for a variety of diseases (11,36,37). A B C FIGURE 6 Directed differentiation of iPSCs towards iMSCs. (A) Schematic timeline of the process for differentiation into iMSCs. (B) Microscope images showing the morphological aspect of differentiated iMSCs over time (scale bar 100 µm). (C) Flow cytometry analysis of differentiated iMSCs showing that cells were positive for the MSC-specific markers CD13, CD73, CD90 and CD105, whereas they were negative for CD14, CD34, CD45, and HLA-II. (D) Representative images of the differentiated iMSCs into osteocytes (scale bar 100µm), adipocytes (scale bar 50µm) and chondrocytes (scale bar 100µm) identified by Alizarin Red, Oil Red O and Alcian Blue staining, respectively. Baliña-Sa ´nchez et al. 10.3389/fimmu.2023.1022676 Frontiers in Immunology frontiersin.org09