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Uncontrolled bleeding during surgery is associated with high mortality and prolonged hospital stay, necessitating the use of hemostatic agents. Fibrin sealant patches offer an efficient solution to achieve hemostasis and improve patient outcomes in liver resection surgery. We have previously demonstrated the efficacy of a nanostructured fibrin-agarose hydrogel (NFAH). However, for the widespread distribution and commercialization of the product, it is necessary to develop an optimal preservation method that allows for prolonged stability and facilitates storage and distribution. We investigated cryopreservation as a potential method for preserving NFAH using trehalose. Structural changes in cryopreserved NFAH (Cryo-NFAH) were investigated and comparative in vitro and in vivo efficacy and safety studies were performed with freshly prepared NFAH. We also examined the long-term safety of Cryo-NFAH versus TachoSil in a rat partial hepatectomy model, including time to hemostasis, intra-abdominal adhesion, hepatic hematoma, inflammatory factors, histopathological variables, temperature and body weight, hemocompatibility and cytotoxicity. Structural analyses demonstrated that Cryo-NFAH retained most of its macro- and microscopic properties after cryopreservation. Likewise, hemostatic efficacy assays showed no significant differences with fresh NFAH. Safety evaluations indicated that Cryo-NFAH had a similar overall profile to TachoSil up to 40 days post-surgery in rats. In addition, Cryo-NFAH demonstrated superior hemostatic efficacy compared with TachoSil while also demonstrating lower levels of erythrolysis and cytotoxicity than both TachoSil and other commercially available hemostatic agents. These results indicate that Cryo-NFAH is highly effective hemostatic patch with a favorable safety and tolerability profile, supporting its potential for clinical use., (© 2024. The Author(s).)

Background: In preterm birth germinal matrix hemorrhages (GMHs) and the consequent posthemorrhagic hydrocephalus (PHH), the neuroepithelium/ependyma development is disrupted. This work is aimed to explore the possibilities of ependymal repair in GMH/PHH using a combination of neural stem cells, ependymal progenitors (EpPs), and mesenchymal stem cells., Methods: GMH/PHH was induced in 4-day-old mice using collagenase, blood, or blood serum injections. PHH severity was characterized 2 weeks later using magnetic resonance, immunofluorescence, and protein expression quantification with mass spectrometry. Ependymal restoration and wall regeneration after stem cell treatments were tested in vivo and in an ex vivo experimental approach using ventricular walls from mice developing moderate and severe GMH/PHH. The effect of the GMH environment on EpP differentiation was tested in vitro. Two-tailed Student t or Wilcoxon-Mann-Whitney U test was used to find differences between the treated and nontreated groups. ANOVA and Kruskal-Wallis tests were used to compare >2 groups with post hoc Tukey and Dunn multiple comparison tests, respectively., Results: PHH severity was correlated with the extension of GMH and ependymal disruption (means, 88.22% severe versus 19.4% moderate). GMH/PHH hindered the survival rates of the transplanted neural stem cells/EpPs. New multiciliated ependymal cells could be generated from transplanted neural stem cells and more efficiently from EpPs (15% mean increase). Blood and TNFα (tumor necrosis factor alpha) negatively affected ciliogenesis in cells committed to ependyma differentiation (expressing Foxj1 [forkhead box J1] transcription factor). Pretreatment with mesenchymal stem cells improved the survival rates of EpPs and ependymal differentiation while reducing the edematous (means, 18% to 0.5% decrease in severe edema) and inflammatory conditions in the explants. The effectiveness of this therapeutical strategy was corroborated in vivo (means, 29% to 0% in severe edema)., Conclusions: In GMH/PHH, the ependyma can be restored and edema decreased from either neural stem cell or EpP transplantation in vitro and in vivo. Mesenchymal stem cell pretreatment improved the success of the ependymal restoration., Competing Interests: Disclosures Dr Sánchez-Pernaute is consultant researcher for Bayer and Novartis. Dr Fernández-Muñoz has research grants from Instituto de Salud Carlos III and Fundación Alicia Koplowitz. The other authors report no conflicts.

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., Competing Interests: Declaration of Competing Interest MSG, BFM and ILR are authors of a patent application for the use of different media based on HPL [58] (nº application Spanish Patent Office: P201730713). MA is author of a patent for the use of nanostructured fibrin agarose hydrogels [59] (Patent number PCT/ES2010/070569). RCC. and BFM. are authors of a patent application for the use of NFAH as a haemostatic agent (N° application Spanish Patent Office: P201830346). The authors have no other conflicts interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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 and promoted good recovery post-thaw. Besides, both tested solutions were stable at - 20 °C for 18 months. Finally, we established that there is a 3-h window in which thawed NFAHs and FBs maintain optimum viability immersed in the formulated solutions and at least 2 h for BM-MSCs., Conclusions: Our results show that pathogen-inactivated solutions Ti5 allocated for bioengineered tissues and CeA allocated for cells are efficient and safe candidates to cryopreserve ATMPs and offer a xenogeneic-free and low-DMSO alternative to commercially available cryoprotective solutions., (© 2023. The Author(s).)

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: Extracellular matrix; ELISA: enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; FDR: False discovery rate; FGF: Fibroblast growth factor; GMP: Good manufacturing practice; HPL: Human platelet lysate; HPL-CM: commercial human platelet lysate; MSCs: mesenchymal stem cells; NEAA: non-essential amino acids; P/S: penicillin/streptomycin; PBS: phosphate buffered saline; PC: leukodepleted platelet concentrate; PCR: polymerase chain reaction; PDGF: Platelet-derived growth factor; PDGFRA: Platelet-derived growth factor receptor alpha; qPCR: quantitative polymerase chain reaction; RNA: Ribonucleic acid; RT: Room temperature; TAC: Transcriptome analysis console; TGF-β: Transforming growth factor beta.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background Aims: Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me 2 SO)., Methods: In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me 2 SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality., Results: Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me 2 SO., Conclusions: Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients., Competing Interests: Declaration of Competing Interest RH and NG are employees of Cellulis S.L. DG is a member of the advisory board of TiGenix. DG and MG have applied for two patents related to the studies titled “Identification and isolation of multipotent cells from nonosteochondral mesenchymal tissue” (WO 2006/057649) and “Use of adipose tissue-derived stromal stem cells in treating fistula” (WO 2006/136244). DG and MG are shareholders of Biosurgery, an educational company providing services to Takeda., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Impact Statement: 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., (© 2019 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Podoplanin (PDPN) is a mucin-like transmembrane glycoprotein that plays an important role in development and cancer. Here, we provide evidence that the intracellular domain (ICD) of podoplanin is released into the cytosol following a sequential proteolytic processing by a metalloprotease and γ-secretase. Western blotting and cell fractionation studies revealed that HEK293T and MDCK cells transfected with an eGFP-tagged podoplanin construct (PDPNeGFP, 50-63kDa) constitutively express two C-terminal fragments (CTFs): a ∼33kDa membrane-bound PCTF33, and a ∼29kDa cytosolic podoplanin ICD (PICD). While pharmacological inhibition of metalloproteases reduced the expression of PCTF33, treatment of cells with γ-secretase inhibitors resulted in enhanced PCTF33 levels. PCTF33 processing by γ-secretase depends on presenilin-1 (PS1) function: cells expressing a dominant negative form of PS1 (PS1 D385N), and mouse embryonic fibroblasts (MEFs) genetically deficient in PS1, but not in PS2, show higher levels of PCTF33 expression with respect to wild-type MEFs. Furthermore, transfection of PS1 deficient MEFs with wild-type PS1 (PS1 wt) decreased PCTF33 levels. N-terminal amino acid sequencing of the affinity purified PICD revealed that the γ-secretase cleavage site was located between valines 150 and 151, but these residues are not critical for proteolysis. We found that podoplanin CTFs are also generated in cells expressing podoplanin mutants harboring heterologous transmembrane regions. Taken together, these results indicate that podoplanin is a novel substrate for PS1/γ-secretase., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin-CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.

Podoplanin/PA2.26 antigen is a small transmembrane mucin expressed in different types of cancer where it is associated with increased cell migration, invasiveness and metastasis. Little is known about the mechanisms that control podoplanin expression. Here, we show that podoplanin synthesis can be controlled at different levels. We analyzed podoplanin expression in a wide panel of tumour cell lines. The podoplanin gene (PDPN) is transcribed in cells derived from sarcomas, embryonal carcinomas, squamous cell carcinomas and endometrial tumours, while cell lines derived from colon, pancreatic, ovarian and ductal breast carcinomas do not express PDPN transcripts. PDPN is expressed as two mRNAs of approximately 2.7 and approximately 0.9 kb, both of which contain the coding sequence and arise by alternative polyadenylation. Strikingly, in most of the cell lines where PDPN transcripts were found, no podoplanin or only very low levels of the protein could be detected in Western blot. Treatment of several of these cell lines with the calpain inhibitor calpeptin resulted in podoplanin accumulation, whereas lactacystin, a specific inhibitor of the proteasome, had no effect. In vitro experiments showed that podoplanin is a substrate of calpain-1. These results indicate that at least in some tumour cells absence or reduced podoplanin protein levels are due to post-translational calpain-mediated proteolysis. We also report in this article the identification of a novel podoplanin isoform that originates by alternative splicing and differs from the standard form in lacking two cytoplasmic residues (YS). YS dipeptide is highly conserved across species, suggesting that it might be functionally relevant.