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Objective: To conduct a proof-of-concept study of the detection of two synthetic models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using polarimetric imaging. Approach: Two SARS-CoV-2 models were prepared as engineered lentiviruses pseudotyped with the G protein of the vesicular stomatitis virus, and with the characteristic Spike protein of SARS-CoV-2. Samples were prepared in two biofluids (saline solution and artificial saliva), in four concentrations, and deposited as 5-µL droplets on a supporting plate. The angles of maximal degree of linear polarization (DLP) of light diffusely scattered from dry residues were determined using Mueller polarimetry from87 samples at 405 nm and 514 nm. A polarimetric camera was used for imaging several samples under 380–420 nm illumination at angles similar to those of maximal DLP. Per-pixel image analysis included quantification and combination of polarization feature descriptors in 475 samples. Main results: The angles (from sample surface) of maximal DLP were 3° for 405 nm and 6° for 514 nm. Similar viral particles that differed only in the characteristic spike protein of the SARS-CoV-2, their corresponding negative controls, fluids, and the sample holder were discerned at 10-degree and 15-degree configurations. Significance: Polarimetric imaging in the visible spectrum may help improve fast, non-contact detection and identification of viral particles, and/or other microbes such as tuberculosis, in multiple dry fluid samples simultaneously, particularly when combined with other imaging modalities. Further analysis including realistic concentrations of real SARS-CoV-2 viral particles in relevant human fluids is required. Polarimetric imaging under visible light may contribute to a fast, cost-effective screening of SARS-CoV-2 and other pathogens when combined with other imaging modalities. © 2023 The Author(s)

Background: There remains much interest in improving cryopreservation techniques for advanced therapy medicinal products (ATMPs). Recently, human platelet lysate (hPL) has emerged as a promising candidate to replace fetal bovine serum (FBS) as a xeno-free culture supplement for the expansion of human cell therapy products. Whether hPL can also substitute for FBS in cryopreservation procedures remains poorly studied. Here, we evaluated several cryoprotective formulations based on a proprietary hPL for the cryopreservation of bioengineered tissues and cell therapy products. Methods: We tested different xenogeneic-free, pathogen-inactivated hPL (ihPL)- and non-inactivated-based formulations for cryopreserving bioengineered tissue (cellularized nanostructured fibrin agarose hydrogels (NFAHs)) and common cell therapy products including bone marrow-derived mesenchymal stromal cells (BM-MSCs), human dermal fibroblasts (FBs) and neural stem cells (NSCs). To assess the tissue and cellular properties post-thaw of NFAHs, we analyzed their cell viability, identity and structural and biomechanical properties. Also, we evaluated cell viability, recovery and identity post-thaw in cryopreserved cells. Further properties like immunomodulation, apoptosis and cell proliferation were assessed in certain cell types. Additionally, we examined the stability of the formulated solutions. The formulations are under a bidding process with MD Bioproducts (Zurich, Switzerland) and are proprietary. Results: Amongst the tissue-specific solutions, Ti5 (low-DMSO and ihPL-based) preserved the viability and the phenotype of embedded cells in NFAHs and preserved the matrix integrity and biomechanical properties similar to those of the standard cryopreservation solution (70% DMEM + 20% FBS + 10% DMSO). All solutions were stable at − 20 °C for at least 3 months. Regarding cell-specific solutions, CeA maintained the viability of all cell types > 80%, preserved the immunomodulatory properties of BM-MSCs a

The prompt management of patients with massive burns is essential to maximize survival by preventing infection, hemorrhage, fluid and heat loss, and to optimally prepare the wound bed for the application of autografts or cultured tissue-engineered artificial autologous skin. Acute treatments are typically based on temporary bio-dressings, commonly cadaveric skin allografts, but supply challenges, high costs and increasingly stringent regulatory requirements preclude their widespread use. Nanostructured fibrin-agarose hydrogels (NFAH) have been proven to be safe and effective biomaterials in preclinical and clinical studies, and show good hemostatic and biomechanical properties. Here we generated and tested NFAH with embedded allogeneic dermal fibroblasts (NFAH-F) under Good Manufacturing Practice (GMP) conditions. Fibroblasts were first expanded and characterized to create a GMP bank and the NFAH-F was manufactured on demand. Three patients with major burns were treated with this product as a temporary bio-dressing under compassionate use. Our results suggest that NFAH-F product was a safe product and no adverse reactions were observed. In all cases, the patients survived until definitive treatment. Therefore, the application of NFAH-F might be a temporary bio-dressing for patients with massive burns when cadaveric skin allografts are not available.

[Objective] To conduct a proof-of-concept study of the detection of two synthetic models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using polarimetric imaging., [Approach] Two SARS-CoV-2 models were prepared as engineered lentiviruses pseudotyped with the G protein of the vesicular stomatitis virus, and with the characteristic Spike protein of SARS-CoV-2. Samples were prepared in two biofluids (saline solution and artificial saliva), in four concentrations, and deposited as 5-µL droplets on a supporting plate. The angles of maximal degree of linear polarization (DLP) of light diffusely scattered from dry residues were determined using Mueller polarimetry from87 samples at 405 nm and 514 nm. A polarimetric camera was used for imaging several samples under 380–420 nm illumination at angles similar to those of maximal DLP. Per-pixel image analysis included quantification and combination of polarization feature descriptors in 475 samples., [Main results] The angles (from sample surface) of maximal DLP were 3° for 405 nm and 6° for 514 nm. Similar viral particles that differed only in the characteristic spike protein of the SARS-CoV-2, their corresponding negative controls, fluids, and the sample holder were discerned at 10-degree and 15-degree configurations., [Significance] Polarimetric imaging in the visible spectrum may help improve fast, non-contact detection and identification of viral particles, and/or other microbes such as tuberculosis, in multiple dry fluid samples simultaneously, particularly when combined with other imaging modalities. Further analysis including realistic concentrations of real SARS-CoV-2 viral particles in relevant human fluids is required. Polarimetric imaging under visible light may contribute to a fast, cost-effective screening of SARS-CoV-2 and other pathogens when combined with other imaging modalities.

Effective testing is essential to control the coronavirus disease 2019 (COVID-19) transmission. Here we report a-proof-of-concept study on hyperspectral image analysis in the visible and near-infrared range for primary screening at the point-of-care of SARS-CoV-2. We apply spectral feature descriptors, partial least square-discriminant analysis, and artificial intelligence to extract information from optical diffuse reflectance measurements from 5 µL fluid samples at pixel, droplet, and patient levels. We discern preparations of engineered lentiviral particles pseudotyped with the spike protein of the SARS-CoV-2 from those with the G protein of the vesicular stomatitis virus in saline solution and artificial saliva. We report a quantitative analysis of 72 samples of nasopharyngeal exudate in a range of SARS-CoV-2 viral loads, and a descriptive study of another 32 fresh human saliva samples. Sensitivity for classification of exudates was 100% with peak specificity of 87.5% for discernment from PCR-negative but symptomatic cases. Proposed technology is reagent-free, fast, and scalable, and could substantially reduce the number of molecular tests currently required for COVID-19 mass screening strategies even in resource-limited settings.

Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies.

Recent years have witnessed the introduction of ex vivo expanded dermal fibroblasts for several cell therapy and tissue-engineering applications, including the treatment of facial scars and burns, representing a promising cell type for regenerative medicine. We tested different in-house produced human platelet lysate (HPL) solutions against fetal bovine serum as supplements for in vitro fibroblast expansion by comparing cell yield, molecular marker expression, extracellular matrix (ECM) generation, genomic stability and global gene expression. Our in-house produced HPL supported fibroblast growth at levels similar to those for FBS and commercial HPL products and was superior to AB human serum. Cells grown in HPL maintained a fibroblast phenotype (VIM+, CD44+, CD13+, CD90+), ECM generation capacity (FN+, COL1+) and a normal karyotype, although gene expression profiling revealed changes related to cell metabolism, adhesion and cellular senescence. The HPL manufacturing process was validated within a GMP compliant system and the solution was stable at -80ºC and -20ºC for 2 years. Dermal fibroblasts expanded in vitro with HPL maintain a normal karyotype and expression of fibroblast markers, with only minor changes in their global gene expression profile. Our in-house produced GMP-HPL is an efficient, safe and economical cell culture supplement that can help increase the healthcare activity of blood transfusion centers through the re-use of transfusional plasma and platelets approaching their expiration date. Currently, our HPL solution is approved by the Spanish Agency of Medicines and Medical Devices and is being used in the manufacture of cell therapy products.Abbreviations: AB plasma: plasma group AB; ABHS: AB Human Serum; ABHS+GF: AB Human Serum supplemented with growth factors; ANOVA: Analysis of variance; ATMPs: Advanced Therapies for Medicinal Products; CPE: cytopathic effect; DEGs: Differentially expressed genes; DMEM: Dulbecco's modified Eagle's Medium; ECM: Ex

[Simple Summary] Animal models are routinely used in pre-clinical studies to evaluate the safety and efficacy of novel therapies, such as cell transplantation, but have limited predictive value. In this study, we set up an experimental model using human stem cells grown in 3D, which form rudimentary brain structures in vitro, called brain organoids. We investigated the possibility of using these brain organoids to evaluate the safety of a cell therapy product, by comparing the results obtained in our model with the standard mouse model. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used in regulatory studies., Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies.

Effective testing is essential to control the coronavirus disease 2019 (COVID-19) transmission. Here we report a-proof-of-concept study on hyperspectral image analysis in the visible and near-infrared range for primary screening at the point-of-care of SARS-CoV-2. We apply spectral feature descriptors, partial least square-discriminant analysis, and artificial intelligence to extract information from optical diffuse reflectance measurements from 5 µL fluid samples at pixel, droplet, and patient levels. We discern preparations of engineered lentiviral particles pseudotyped with the spike protein of the SARS-CoV-2 from those with the G protein of the vesicular stomatitis virus in saline solution and artificial saliva. We report a quantitative analysis of 72 samples of nasopharyngeal exudate in a range of SARS-CoV-2 viral loads, and a descriptive study of another 32 fresh human saliva samples. Sensitivity for classification of exudates was 100% with peak specificity of 87.5% for discernment from PCR-negative but symptomatic cases. Proposed technology is reagent-free, fast, and scalable, and could substantially reduce the number of molecular tests currently required for COVID-19 mass screening strategies even in resource-limited settings.

Optical spectroscopic techniques have been commonly used to detect the presence of biofilm-forming pathogens (bacteria and fungi) in the agro-food industry. Recently, near-infrared (NIR) spectroscopy revealed that it is also possible to detect the presence of viruses in animal and vegetal tissues. Here we report a platform based on visible and NIR (VNIR) hyperspectral imaging for non-contact, reagent free detection and quantification of laboratory-engineered viral particles in fluid samples (liquid droplets and dry residue) using both partial least square-discriminant analysis and artificial feed-forward neural networks. The detection was successfully achieved in preparations of phosphate buffered solution and artificial saliva, with an equivalent pixel volume of 4 nL and lowest concentration of 800 TU.mu L-1. This method constitutes an innovative approach that could be potentially used at point of care for rapid mass screening of viral infectious diseases and monitoring of the SARS-CoV- 2 pandemic.

With an expanding elderly population, an increasing number of older adults will experience spinal cord injury (SCI) and might be candidates for cell-based therapies, yet there is a paucity of research in this age group. The objective of the present study was to analyze how aged rats tolerate behavioral testing, sur- gical procedures, post-operative complications, intra-spinal cell transplantation and immunosuppres- sion, and to examine the effectiveness of human iPSC-derived Neural Progenitor Cells (IMR90-hiPSC- NPCs) in a model of SCI. We performed behavioral tests in rats before and after inducing cervical hemi-contusions at C4 level with a fourth-generation Ohio State University Injury Device. Four weeks later, we injected IMR90-hiPSC-NPCs in animals that were immunosuppressed by daily cyclosporine injection. Four weeks after injection we analyzed locomotor behavior and mortality, and histologically assessed the survival of transplanted human NPCs. As rats aged, their success at completing behavioral tests decreased. In addition, we observed high mortality rates during behavioral training (41.2%), after cervical injury (63.2%) and after cell injection (50%). Histological analysis revealed that injected cells sur- vived and remained at and around the grafted site and did not cause tumors. No locomotor improvement was observed in animals four weeks after IMR90-hiPSC-NPC transplantation. Our results show that elderly rats are highly vulnerable to interventions, and thus large groups of animals must be initially established to study the potential efficacy of cell-based therapies in age-related chronic myelopathies.

Neural stem cells represent an attractive tool for the development of regenerative therapies and are being tested in clinical trials for several neurological disorders. Human neural stem cells can be isolated from the central nervous system or can be derived in vitro from pluripotent stem cells. Embryonic sources are ethically controversial and other sources are less well characterized and/or inefficient. Recently, isolation of NSC from the cerebrospinal fluid of patients with spina bifida and with intracerebroventricular hemorrhage has been reported. Direct reprogramming may become another alternative if genetic and phenotypic stability of the reprogrammed cells is ensured. Here, we discuss the advantages and disadvantages of available sources of neural stem cells for the production of cell-based therapies for clinical applications. We review available safety and efficacy clinical data and discuss scalability and quality control considerations for manufacturing clinical grade cell products for successful clinical application.

With an expanding elderly population, an increasing number of older adults will experience spinal cord injury (SCI) and might be candidates for cell-based therapies, yet there is a paucity of research in this age group. The objective of the present study was to analyze how aged rats tolerate behavioral testing, surgical procedures, post-operative complications, intra-spinal cell transplantation and immunosuppression, and to examine the effectiveness of human iPSC-derived Neural Progenitor Cells (IMR90-hiPSC-NPCs) in a model of SCI. We performed behavioral tests in rats before and after inducing cervical hemi-contusions at C4 level with a fourth-generation Ohio State University Injury Device. Four weeks later, we injected IMR90-hiPSC-NPCs in animals that were immunosuppressed by daily cyclosporine injection. Four weeks after injection we analyzed locomotor behavior and mortality, and histologically assessed the survival of transplanted human NPCs. As rats aged, their success at completing behavioral tests decreased. In addition, we observed high mortality rates during behavioral training (41.2%), after cervical injury (63.2%) and after cell injection (50%). Histological analysis revealed that injected cells survived and remained at and around the grafted site and did not cause tumors. No locomotor improvement was observed in animals four weeks after IMR90-hiPSC-NPC transplantation. Our results show that elderly rats are highly vulnerable to interventions, and thus large groups of animals must be initially established to study the potential efficacy of cell-based therapies in age-related chronic myelopathies.

INTRODUCCIÓN: La pérdida de sangre en pacientes sometidos a resección o traumatismo hepático es una de las principales causas de morbilidad y mortalidad perioperatoria. En este contexto, los agentes hemostáticos tópicos resultan una herramienta esencial, aunque cualquier mejora de éstos en términos de reducción del tiempo de hemostasia y/o resangrado, facilidad de uso y aplicabilidad, y mejora de respuesta inflamatoria y tisular, podría representar un gran impacto no sólo en los costes hospitalarios sino también en la calidad de vida de los pacientes. Durante los últimos años, los hidrogeles de fibrina y agarosa nanoestructurados (NFAH) se han utilizado como sustitutos artificiales de tejidos. Los resultados preclínicos obtenidos en animales despiertan especial interés en biomedicina al demostrar cualidades de biocompatilibilidad y biodegradabilidad. Estos NFAH fabricados con plasma humano, agarosa tipo VII, ácido tranexámico y cloruro cálcico están compuesto principalmente de fibrina, un polímero cuya función principal es bloquear la pérdida masiva de sangre y participar en la reparación de tejidos dañados tras la ruptura vascular. Por tanto, surge la hipótesis que el NFAH tenga efecto hemostático, no sólo por un potencial efecto bioactivo debido a su composición sino también por un efecto biomecánico protector de la herida. OBJETIVO: El objetivo general de esta Tesis Doctoral es estudiar la eficacia y la seguridad de este NFAH como parche hemostático en cirugía hepática. METODOLOGÍA: Para ello se fabricaron hidrogeles de fibrina y agarosa nanoestructurados con célula (c-NFAH) y sin células (a-NAH) que se aplicaron a dos modelos experimentales de resección hepática en rata: un modelo de resección moderado (con una incisión de 0,5 cm de longitud) y uno severo (con una incisión de 1,5 cm de longitud). Dos agentes hemostáticos comerciales ampliamente utilizados en cirugía hepática, Hemopatch y Tachosil, fueron incluidos en los estudios. Las variables analizadas fueron

Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology.

Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology.

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin‐agarose hydrogel patch, with (c‐NFAH) or without cells (a‐NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post‐operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a‐NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a‐NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a‐NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures.

Damage to the retinal pigment epithelium (RPE) in age-related macular degeneration and other diseases results in photoreceptor cell death and blindness. Replacement of RPE is therefore being explored as a therapy for several retinal diseases. To move toward a future personalized autologous transplant approach, we have prepared a biocompatible implant using RPE derived from induced pluripotent stem cells (iPSCs) reprogrammed from a healthy donor's monocytes. The correct positioning of the polarized RPE is essential to fulfill its role and protect photoreceptors from degeneration. Hence, we have used a biocompatible hydrogel matrix of fibrin and agarose that allows the surgical placement of an RPE sheet in the subretinal space, keeping its functional orientation. Our aim was to demonstrate safety and viability of the transplant in preclinical models. Pigs were used to test the feasibility of a regular vitreoretinal surgery. Our results show that this implant is suitable for subretinal transplantation allowing human RPE cells to survive and maintain their phenotype and orientation without any local or systemic adverse events. The ability to transplant the iPSC-derived RPE sheet in its natural orientation will surely increase the chance to obtain a therapeutic effect in future translational studies. In the promising field of cellular therapy for retinal degenerative diseases, a new biomaterial is proposed as a scaffold to grow and surgically introduce a monolayer of retinal pigment epithelial cells into the subretinal space, keeping the orientation of the cells for a proper functional integration of the transplant. The use of induced pluripotent stem cells as the starting material for retinal pigment epithelial cells is intended to advance toward a personalized medicine approach.

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin‐agarose hydrogel patch, with (c‐NFAH) or without cells (a‐NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post‐operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a‐NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a‐NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a‐NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures.

Podoplanin is a small mucin-like transmembrane protein expressed in several adult tissues and with an important role during embryogenesis. It is needed for the proper development of kidneys and lungs as well as accurate formation of the lymphatic vascular system. In addition, it is involved in the physiology of the immune system. A wide variety of tumors express podoplanin, both in the malignant cells and in the stroma. Although there are exceptions, the presence of podoplanin results in poor prognosis. The main consequence of forced podoplanin expression in established and tumor-derived cell lines is an increase in cell migration and, eventually, the triggering of an epithelial–mesenchymal transition, whereby cells acquire a fibroblastoid phenotype and increased motility. We will examine the current status of the role of podoplanin in the induction of epithelial–mesenchymal transition as well as the different interactions that lead to this program.

Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.