Xenogeneic Animals Research Articles

Establishment of Canine Oral Mucosal Melanoma Cell Lines and Their Xenogeneic Animal Models.

Canine oral melanoma is the most prevalent malignant tumor in dogs and has a poor prognosis due to its high aggressiveness and high metastasis and recurrence rates. More research is needed into its treatment and to understand its pathogenic factors. In this study, we isolated a canine oral mucosal melanoma (COMM) cell line designated as COMM6605, which has now been stably passaged for more than 100 generations, with a successful monoclonal assay and a cell multiplication time of 22.2 h. G-banded karyotype analysis of the COMM6605 cell line revealed an abnormal chromosome count ranging from 45 to 74, with the identification of a double-armed chromosome as the characteristic marker chromosome of this cell line. The oral intralingual and dorsal subcutaneous implantation models of BALB/c-nu mice were successfully established; Melan-A (MLANA), S100 beta protein (S100β), PNL2, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) were stably expressed positively in the canine oral tumor sections, tumor cell lines, and tumor sections of tumor-bearing mice. Sublines COMM6605-Luc-EGFP and COMM6605-Cherry were established through lentiviral transfection, with COMM6605-Luc-EGFP co-expressing firefly luciferase (Luc) and enhanced green fluorescent protein (EGFP) and COMM6605-Cherry expressing the Cherry fluorescent protein gene. The COMM6605-Luc-EGFP fluorescent cell subline was injected via the tail vein and caused lung and lymph node metastasis, as detected by mouse live imaging, which can be used as an animal model to simulate the latter steps of hematogenous spread during tumor metastasis. The canine oral melanoma cell line COMM6605 and two sublines isolated and characterized in this study can offer a valuable model for studying mucosal melanoma.

Generation of allogeneic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected Dmdmdx mouse induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Notably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Regulation of Immune Checkpoint Antigen CD276 (B7-H3) on Human Placenta-Derived Mesenchymal Stromal Cells in GMP-Compliant Cell Culture Media.

Therapies utilizing autologous mesenchymal cell delivery are being investigated as anti-inflammatory and regenerative treatments for a broad spectrum of age-related diseases, as well as various chronic and acute pathological conditions. Easily available allogeneic full-term human placenta mesenchymal stromal cells (pMSCs) were used as a potential pro-regenerative, cell-based therapy in degenerative diseases, which could be applied also to elderly individuals. To explore the potential of allogeneic pMSCs transplantation for pro-regenerative applications, such cells were isolated from five different term-placentas, obtained from the dissected maternal, endometrial (mpMSCs), and fetal chorion tissues (fpMSCs), respectively. The proliferation rate of the cells in the culture, as well as their shape, in vitro differentiation potential, and the expression of mesenchymal lineage and stem cell markers, were investigated. Moreover, we studied the expression of immune checkpoint antigen CD276 as a possible modulation of the rejection of transplanted non-HLA-matched homologous or even xeno-transplanted pMSCs. The expression of the cell surface markers was also explored in parallel in the cryosections of the relevant intact placenta tissue samples. The expansion of pMSCs in a clinical-grade medium complemented with 5% human platelet lysate and 5% human serum induced a significant expression of CD276 when compared to mpMSCs expanded in a commercial medium. We suggest that the expansion of mpMSCs, especially in a medium containing platelet lysate, elevated the expression of the immune-regulatory cell surface marker CD276. This may contribute to the immune tolerance towards allogeneic pMSC transplantations in clinical situations and even in xenogenic animal models of human diseases. The endurance of the injected comparably young human-term pMSCs may promote prolonged effects in clinical applications employing non-HLA-matched allogeneic cell therapy for various degenerative disorders, especially in aged adults.

Functional calcium-responsive parathyroid glands generated using single-step blastocyst complementation

Patients with permanent hypoparathyroidism require lifelong replacement therapy to avoid life-threatening complications, The benefits of conventional treatment are limited, however. Transplanting a functional parathyroid gland (PTG) would yield better results. Parathyroid gland cells generated from pluripotent stem cells in vitro to date cannot mimic the physiological responses to extracellular calcium that are essential for calcium homeostasis. We thus hypothesized that blastocyst complementation (BC) could be a better strategy for generating functional PTG cells and compensating loss of parathyroid function. We here describe generation of fully functional PTGs from mouse embryonic stem cells (mESCs) with single-step BC. Using CRISPR-Cas9 knockout of Glial cells missing2 (Gcm2), we efficiently produced aparathyroid embryos for BC. In these embryos, mESCs differentiated into endocrinologically mature PTGs that rescued Gcm2-/- mice from neonatal death. The mESC-derived PTGs responded to extracellular calcium, restoring calcium homeostasis on transplantation into mice surgically rendered hypoparathyroid. We also successfully generated functional interspecies PTGs in Gcm2-/- rat neonates, an accomplishment with potential for future human PTG therapy using xenogeneic animal BC. Our results demonstrate that BC can produce functional endocrine organs and constitute a concept in treatment of hypoparathyroidism.

Evaluation of the Immunopathological Response to BCG Vaccine in a Xenogeneic Immunocompetent Animal

Evaluation of the Immunopathological Response to BCG Vaccine in a Xenogeneic Immunocompetent Animal

Differentiating Human Pluripotent Stem Cells to Cardiomyocytes Using Purified Extracellular Matrix Proteins.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hESC-CMs and iPSC-CMs, respectively), which hold great promise for cardiac regenerative medicine and disease modeling efforts. However, the most widely employed differentiation protocols require undefined substrates that are derived from xenogeneic (animal) products, contaminating resultant hESC- and iPSC-CM cultures with xenogeneic proteins and limiting their clinical applicability. Additionally, typical hESC- and iPSC-CM protocols produce CMs that are significantly contaminated by non-CMs and that are immature, requiring lengthy maturation procedures. In this review, we will summarize recent studies that have investigated the ability of purified extracellular matrix (ECM) proteins to support hESC- and iPSC-CM differentiation, with a focus on commercially available ECM proteins and coatings to make such protocols widely available to researchers. The most promising of the substrates reviewed here include laminin-521 with laminin-221 together or Synthemax (a synthetic vitronectin-based peptide coating), which both resulted in highly pure CM cultures. Future efforts are needed to determine whether combinations of specific purified ECM proteins or derived peptides could further improve CM maturation and culture times, and significantly improve hESC- and iPSC-CM differentiation protocols.

How mesenchymal stem cell cotransplantation with hematopoietic stem cells can improve engraftment in animal models.

BACKGROUNDBone marrow transplantation (BMT) can be applied to both hematopoietic and nonhematopoietic diseases; nonetheless, it still comes with a number of challenges and limitations that contribute to treatment failure. Bearing this in mind, a possible way to increase the success rate of BMT would be cotransplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) to improve the bone marrow niche and secrete molecules that enhance the hematopoietic engraftment. AIMTo analyze HSC and MSC characteristics and their interactions through cotransplantation in murine models.METHODSWe searched for original articles indexed in PubMed and Scopus during the last decade that used HSC and MSC cotransplantation and in vivo BMT in animal models while evaluating cell engraftment. We excluded in vitro studies or studies that involved graft versus host disease or other hematological diseases and publications in languages other than English. In PubMed, we initially identified 555 articles and after selection, only 12 were chosen. In Scopus, 2010 were identified, and six were left after the screening and eligibility process.RESULTSOf the 2565 articles found in the databases, only 18 original studies met the eligibility criteria. HSC distribution by source showed similar ratios, with human umbilical cord blood or animal bone marrow being administered mainly with a dose of 1 × 107 cells by intravenous or intrabone routes. However, MSCs had a high prevalence of human donors with a variety of sources (umbilical cord blood, bone marrow, tonsil, adipose tissue or fetal lung), using a lower dose, mainly 106 cells and ranging 104 to 1.5 × 107 cells, utilizing the same routes. MSCs were characterized prior to administration in almost every experiment. The recipient used was mostly immunodeficient mice submitted to low-dose irradiation or chemotherapy. The main technique of engraftment for HSC and MSC cotransplantation evaluation was chimerism, followed by hematopoietic reconstitution and survival analysis. Besides the engraftment, homing and cellularity were also evaluated in some studies. CONCLUSIONThe preclinical findings validate the potential of MSCs to enable HSC engraftment in vivo in both xenogeneic and allogeneic hematopoietic cell transplantation animal models, in the absence of toxicity.

Adverse effect of vaccination in xenogeneic animals

Lumpy skin disease (LSD) is a devastating, emerging viral disease of cattle. It causes significant economic losses due to trade restrictions that are placed on infected animals and the biological effects of the disease: infertility, dramatic loss in milk production, induction of abortion and mortality. It is caused by lumpy skin disease virus (LSDV), which belongs to the Poxviridae family. Vaccination has been determined to be the most effective way to control LSD infection among livestock. However, some adverse effects have been reported in animals vaccinated with live vaccines. To the best of our knowledge, this is the first study to report the systemic lesions that are associated with LSD vaccination in xenogeneic animals. The aim of our study was to compare the immunogenicity and pathogenicity of a live attenuated vaccine of Romanian strain of sheeppox virus (SPPV) through study of two different routes of administration in xenogeneic animals (mice). Swiss male mice were inoculated with two doses of SPPV vaccine by two different routes intranasal (IN, through nebulisation), and intraperitoneal (IP) injection) and the levels of immunoglobulins and histopathological findings were reported. Our results showed marked increases in levels of immunoglobulins (Ig) dependent on the administration route: IgG in IP-inoculated mice and IgA in IN-vaccinated mice. IgM levels became markedly high after vaccination via both routes. Histologically, nebulisation of mice with SPPV vaccine caused more pulmonary lesions than did IP injection and promoted the proliferation of megakaryocytes in splenic tissues. In contrast, IP injection had less effect on pulmonary tissues and induced activation of extramedullary haematopoiesis (EH) in the hepatic tissues. LSD vaccination in xenogeneic animals caused serious systemic complications and the severity of the lesions caused to tissue depended on the route of administration.

Human oral mucosa-derived neural crest-like stem cells differentiate into functional osteoprogenitors that contribute to regeneration of critical size calvaria defects.

Regeneration of large bony defects is an unmet medical need. The therapeutic effect of fully developed bony constructs engineered in vitro from mineralized scaffold and adult stem cells is hampered by deficient long-term graft integration. The purpose of the present study was to investigate the regenerative capacity of a bony primordial construct consisting of human oral mucosa stem cells (hOMSC)-derived osteoprogenitors and absorbable Gelfoam® sponges. Gingiva and alveolar mucosa-derived hOMSC were differentiated into osteoprogenitors (Runx2 and osterix positive) and loaded into Gelfoam® sponges to generate primordial hOMSC constructs. These were implanted into critical size calvaria defects in the rat. Defects treated with human dermal fibroblasts (HDF) constructs; Gelfoam® sponges and untreated defects served as controls. After 120-day post-implantation defects treated with hOMSC constructs, HDF constructs and gelatin and untreated defects exhibited 86%, 30%, 21%, and 9% of new bone formation, respectively. Immunofluorescence analysis for human nuclear antigen (HNA), bone sialoprotein (BSP), and osteocalcin (OCN) revealed viable hOMSC-derived osteoblasts and osteocytes that formed most of the cell population of the newly formed bone at 30 and 120days post surgery. Few HNA-positive HDF that were negative for BSP and OCN were identified together with inflammatory cells in the soft tissue adjacent to new bone formation only at 30days post implantation. Collectively, the results demonstrate that primordial in vitro engineered constructs consisting of hOMSC-derived osteoprogenitors and absorbable gelatin almost completely regenerate critical size defects in an immunocompetent xenogeneic animal by differentiating into functional osteoblasts that retain the immunomodulatory ability of naïve hOMSC.

Abstract 12382: Evaluation of Tissue Engineered Human Acellular Vessels as a Blalock-Taussig-Thomas Shunt in a Juvenile Primate Model

Cyanotic congenital heart disease (CCHD) due to one or more structural abnormalities causes insufficient blood oxygenation. Approximately 2 out of every 1000 infants are born with CCHD and palliative treatment for many patients involves shunting blood from the systemic to the pulmonary circuits, often through a modified Blalock-Taussig-Thomas shunt (BTTs). Synthetic vascular grafts typically used for BTTs have associated risks for intimal hyperplasia, thrombosis, and infection. Humacyte, Inc. has developed a 6mm diameter tissue-engineered blood vessel, the Human Acellular Vessel (HAV), currently being studied in adults suffering from vascular trauma, peripheral arterial disease, and renal failure requiring hemodialysis. In addition to restoring blood flow and providing arteriovenous access in these patients, the HAV has demonstrated the capacity for host cellular remodeling that transformed it into a native-like living tissue. Here the HAV production platform was modified to generate small diameter (3.5mm) vessels for preclinical evaluation. BTTs were placed from the right subclavian artery to the pulmonary artery in juvenile rhesus macaques. Post-operative imaging from 1 week to 6 months using angiography and MRI was used to confirm HAV patency, structure, and quantify blood flow. At explantation, the HAV and surrounding tissues were collected and processed for histological analyses. Results confirmed the feasibility of using the HAV BTTs, but also showed that all macaques had some immunological reactivity to the HAV human extracellular matrix in this xenogeneic animal model. Nonetheless, the HAV BTTs remained patent and performed well for up to six months in animals that had only mild host reactivity. HAV repopulation by host cells expressing smooth muscle and endothelial markers as well as the formation of neoadventitial tissue was also observed. These findings may support use of the HAV as a BTTs in CCHD and potentially other pediatric indications.

Failure of decellularized porcine small intestinal submucosa as a heart valved conduit

ObjectiveDecellularized extracellular matrix made from porcine small intestinal submucosa, commercially available as CorMatrix (CorMatrix Cardiovascular, Inc, Roswell, Ga) is used off-label to reconstruct heart valves. Recently, surgeons experienced failures and words of caution were raised. The aim of this study was to evaluate decellularized porcine small intestinal submucosa as right-sided heart valved conduit in a xenogeneic animal model. MethodsA pulmonary valve replacement was performed with custom-made valved conduits in 10 lambs and 10 sheep (1 month [3 lambs and 3 sheep], 3 months [3 lambs and 3 sheep], 6 months [4 lambs and 4 sheep]). Valve function was assessed after implantation and before the animal was put to death. Explanted conduits were inspected macroscopically and analyzed using immunohistochemistry and scanning electron microscopy. They also underwent mechanical testing and testing for biochemical composition. ResultsAll valved conduits were successfully implanted. Five sheep and 2 lambs died due to congestive heart failure within 2 months after surgery. In the animals that died, the valve leaflets were thickened with signs of inflammation (endocarditis in 4). Five sheep and 8 lambs (1 month: 6 out of 6 animals, 3 months: 4 out of 6 animals, 6 months: 3 out of 8 animals) survived planned follow-up. At the time they were put to death, 5 lambs had significant pulmonary stenosis and 1 sheep showed severe regurgitation. A well-functioning valve was seen in 4 sheep and 3 lambs for up to 3 months. These leaflets showed limited signs of remodeling. ConclusionsFifty percent of sheep and 20% of lambs died due to valve failure before the planned follow-up period was complete. A well-functioning valve was seen in 35% of animals, albeit with limited signs of tissue remodeling at ≤3 months after implantation. Further analysis is needed to understand the disturbing dichotomous outcome before clinical application can be advised.

Auxiliary xenotransplantation as an in vivo bioreactor—Development of a transplantable liver graft from a tiny partial liver

We established a completely novel method of auxiliary xenogeneic partial liver transplantation and examined whether liver grafts procured from Syrian hamsters regenerated in nude rats, which were used as in vivo bioreactors. The hamsters and the rats were all males (n=10). Partial liver grafts from hamsters were transplanted into nude rats in an auxiliary manner. We evaluated liver graft injury, rejection, and regeneration during 7days after auxiliary xenogeneic partial liver transplantation. All rats survived until sacrifice on post-operative day (POD) 1, 3, and 7. HE-staining showed normal at POD1, mild periportal edema, and slight bile duct and venous endothelial inflammation at POD3, and moderate acute cellular rejection at POD7 without parenchymal necrosis. The liver regeneration rates at POD3 and 7 were 1.54±0.23 and 2.54±0.43, respectively. The Ki-67 labeling index was also elevated at POD3 (27.5±4.1%). Serum HGF and VEGF were elevated at POD1 and 3. ATP levels of liver grafts recovered at POD7. These results revealed that with appropriate immunosuppressive therapy, partial liver graft regeneration occurred in a xenogeneic animal, which suggests liver grafts regenerated in xenogeneic environments, such as an in vivo bioreactor, have potential to be transplantable liver grafts for humans.

Hurdles to Generating Human Islets in Animals via Blastocyst Complementation

To clarify the hurdles to generation of human islets via blastocyst complementation and to identify techniques to overcome them. Blastocyst complementation is a promising method for generating functional islets from pluripotent stem cells which are identical to in vivo islets. Studies have reported successful generation of mouse pancreas in rats and rat pancreas in mice via interspecies blastocyst complementation and have shown the possibility for generation of human organs in xenogeneic animals. However, there remain hurdles to generating human islets in animals. The major hurdles to generating human islets include difficulty in engineering human-animal chimeras due to the cellular status of human pluripotent stem cells, immunological rejection of donor tissue in xenogeneic animals, and ethical concerns.

Immunoregulatory properties of I.A. mesenchymal stem cells are maintained in a xenogenic animal model

Purpose: Osteoarthritis is a chronic joint disease afflicting a significant portion of the world’s population with no currently available cure. Clinical trials using mesenchymal stem cell-based therapies have a positive effect on pain and quality of life in osteoarthritis but the mechanism of action remains unclear. This study aimed to identify the lymphocyte response to a xenogeneic human umbilical cord-derived MSC-based cell therapy in the face of osteoarthritis progression. Methods: A unilateral medial meniscal release model was employed in 14 sheep to create OA where n=7 sheep received I.A. cell therapy (OACT) in the operated knee and n=7 received saline as controls (OA). Another unoperated group (n=7) received cell therapy (CT Control). Three months later IA cell therapy consisting of 1.0× 107 Human SHC-MSCs* (Small Mesenchymal Stem Cells primed with Hypoxia and Calcium ions) was administered into the medial knee joint space. Synovial fluid was aspirated periodically for 13 weeks for flow cytometry analysis. At termination of the study, the stifle joints underwent macroscopic and histologic assessments using the OARSI histology scoring system for subchondral bone, cartilage and synovial membrane. Flow cytometry data was analyzed with a linear mixed effects model while pathology scores were analyzed by the Mann-Whitney U test and Wilcoxon signed rank test, with the Bonferroni correction for multiple comparisons. Results: There were no adverse reactions to the I.A. therapy. Cell therapy administration induced an influx of CD4+ leukocytes (Fig 1). Of these there was a significant increase in the proportion of CD4+CD25+ and CD4+CD25hi leukocytes; the CD4+CD25hi being subset that may be composed of regulatory T cells. There was no significant effect of the cell therapy treatment on the proportion of synovial fluid CD8+ cells or B cells (BAQ44A+), and B cells occurred in small numbers. Conclusions: Xenogeneic cell therapy did not trigger a classical host versus graft reaction leading to tissue injury. Cell therapy induced chemotaxis of CD4+ cells to the joint space but this was not associated with pathological change in tissues. Despite expression of activation markers (CD25+) these cells may regulate the immune response since CD4+cells can be sources of anti-inflammatory cytokines such as IL-4 and IL-10, as well as inhibiting the release of pro-inflammatory cytokines such as IL-1β or TNF-α. These findings support the notion that phenomena unrelated to long-term survival and engraftment of injected cells may have clear immunoregulatory effects.

Development of xenogeneic decellularized biotubes for off-the-shelf applications.

In earlier studies, we developed in vivo tissue-engineered, autologous, small-caliber vascular grafts, called "biotubes," which withstand systemic blood pressure and exhibit excellent performance as small-caliber vascular prostheses in animal models. However, biotube preparation takes 4weeks; therefore, biotubes cannot be applied in emergency situations. Moreover, for responses to various types of surgery, grafts should ideally be readily available in advance. The aim of this study was to develop novel, off-the-shelf, small-caliber vascular grafts by decellularizing in vivo tissue-engineered xenogeneic tubular materials. Silicone rod molds (diameter: 2mm, length: 70mm) placed in subcutaneous pouches of a beagle dog for 4weeks were harvested with their surrounding connective tissues. Tubular connective tissues were obtained after pulling out the impregnated molds. Subsequently, they were decellularized by perfusion with sodium dodecyl sulfate and Triton X-100. They were stored as off-the-shelf grafts at -20°C for 1week. The decellularized grafts derived from the beagle dog were xenogeneically transplanted to the abdominal aortas of rats (n=3). No signs of abnormal inflammation or immunological problems due to the xenogeneic material were observed. Echocardiography confirmed the patency of the grafts at 1month after implantation. Histological evaluation revealed that the grafts formed neointima on the luminal surface, and that the graft walls had cell infiltration. Little accumulation of CD68-positive macrophages in the graft wall was observed. Xenogeneic decellularized tubular tissues functioned as small-caliber vascular grafts, as well as autologous biotubes. This technology enables the easy fabrication of grafts from xenogeneic animals in advance and their storage for at least a week, satisfying the conditions for off-the-shelf grafts.

Genipin-crosslinked adipose stem cell derived extracellular matrix-nano graphene oxide composite sponge for skin tissue engineering.

Autologous skin grafts, which can cause donor site morbidity, are currently used to treat deep wounds. To improve the regeneration of poorly healing wounds, cell-derived extracellular matrix (ECM) scaffolds are garnering great research interest due to their associated lower risks of pathogen transfer and immune rejection. However, the mechanical properties of cell-derived ECM scaffolds are inferior when compared to those of tissue-derived ECM scaffolds. To overcome this drawback, different amounts (10, 20, 50, and 100 μg mL-1) of graphene oxide (GO) and genipin (1% w/v) were applied to adipose stem cell (ASC)-derived ECM sponges. There are still only a few studies employing cell-derived extracellular matrices as biomimetic scaffolds for biomedical applications. The aim of our study was to develop biocompatible, biodegradable, low immunogenic, and genipin-crosslinked ASC-derived ECM sponges containing a suitable amount of GO for skin-tissue engineering. Sponges were fabricated using cultures of ASCs, cell sheets, and decellularization of an ASC cell sheet, freeze-thawing, and crosslinking in a sequential manner. Scanning electron microscopic analyses of the sponges demonstrated a highly porous microstructure with a pore size of 71.22 ± 19.52 μm. The in vitro degradation rate was found to be significantly higher in the non-crosslinked ECM sponges and pure ECM sponges than in the genipin-crosslinked ECM sponges. During an in vivo study, we investigated the material feasibilities and degradability of the constructed ECM sponges as a suitable skin tissue-engineering scaffold in a xenogenic animal (rat) model for 4 weeks. After subcutaneous implantation, the ECM sponges containing a medium amount of GO showed appropriate biodegradation with a lower inflammatory reaction. Hence, the fabricated ECM sponges might be a suitable xenogenous skin substitute for full-thickness skin defects and in other future soft-tissue engineering applications, such as healing partial tears of the anterior cruciate ligament.

P08.34 Targeting glioma stem cells with interferon-β

Glioblastoma is the most common malignant primary brain tumor in adults. Despite surgery, radiotherapy and chemotherapy, patients suffering from this tumor have a poor prognosis with a median survival still limited to approximately one year in population-based studies. Current standard of care is only moderately effective and patients eventually all succumb to their disease. Therefore, the main effort in the field consists in finding new and better therapies based on increasing knowledge of the complex tumor microenvironment and the signalling pathways that drive this devastating disease. A population of cells within the tumor with stem cell features, now commonly referred to as “glioma-initiating cells” (GIC), has been attributed a central role in the escape of glioblastomas from the current clinical approaches of radio- and chemotherapy. We have recently demonstrated that long-term glioma cell lines (LTC) as well as GIC cultures express receptors for interferon (IFN)-β, IFNAR1 and IFNAR2, and respond to IFN-β with induction of STAT-3 phosphorylation and MxA protein accumulation. Single exposure to IFN-β inhibits cell cycle progression, induces a minor loss of viability, and strongly interferes with sphere formation in GIC cultures. Modelling acquired IFN-β resistance in GIC in vitro induces a stable resistant phenotype defined by clonogenic survival assays which allows to identify resistance mechanisms in more detail. Transcriptomic and proteomic profiling of IFN-β-treated GIC in vitro already revealed candidate molecules potentially mediating these biological responses. We also examined the anti-glioma activity of type I interferons in clinically relevant orthotopic xenogeneic animal models in vivo. Treatment with the more stable pegylated IFN-β significantly prolongs survival in the LN-229 and ZH-161 mouse models. In vivo, IFN-β induces a significant increase in MxA expression in the tumor cells but does not change vessel density. Since IFN-β is widely used for the treatment of human patients with multiple sclerosis, there is considerable knowledge on its safety in patients with brain disease, allowing for rapid translation of novel IFN-β-based strategies into the clinic.

Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls. The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as "bio-scaffolds" for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications. In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin <em>In Vitro</em> Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls. The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as "bio-scaffolds" for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications. In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.