Strong Activation of ID1, ID2, and ID3 Genes Is Coupled with the Formation of Vasculogenic Mimicry Phenotype in Melanoma Cells.

Gene expression patterns are very sensitive to external influences and are reflected in phenotypic changes. It was previously described that transferring melanoma cells from a plastic surface to Matrigel led to formation of de novo vascular networks-vasculogenic mimicry-that are characteristic to a stemness phenotype in aggressive tumors. Up to now there was no detailed data about the gene signature accompanying this process. Here, we show that this transfer shortly led to extremely strong epigenetic changes in gene expression in the melanoma cells. We observed that on Matrigel numerous genes controlling ribosome biogenesis were upregulated. However, most of the activated genes were inhibitors of the differentiation genes (ID1, ID2, and ID3). At the same time, the genes that control differentiation were downregulated. Both the upregulated and the downregulated genes are simultaneously targeted by different transcription factors shaping sets of co-expressed genes. The specific group of downregulated genes shaping contacts with rDNA genes are also associated with the H3K27me3 mark and with numerous lincRNAs and miRNAs. We conclude that the stemness phenotype of melanoma cells is due to the downregulation of developmental genes and formation of dedifferentiated cells.

Accelerated maturation of ARPE-19 cells for the translational assessment of gene therapy.

The human retinal pigment epithelium (RPE) cell line ARPE-19 is widely used as an alternative to primary RPE despite losing many features of primary RPE. We aimed to determine whether a combination of RPE-specific laminin (LN) and nicotinamide (NAM) could improve ARPE-19 redifferentiation to resemble mature RPE and improve the assessment of RPE-specific gene therapy strategies. ARPE-19 cells were propagated on tissue culture plastic supplemented with NAM and human recombinant LN521-coating. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno-associated virus (AAV)1 or AAV2, carrying a VMD2-driven GFP, were assessed at 2- and 4-weeks post-plating in the different culturing conditions with a low multiplicity of infection. The combination of LN521 coating with NAM supplementation promoted cytoskeletal and tight junction protein reorganization. The expression of maturation markers bestrophin-1 and RPE 65 was promoted concomitantly with a reduction of several epithelial-mesenchymal transition markers, such as TNF-α, TGF-ß, CDH2, and vimentin. Redifferentiated ARPE-19 transduced at low multiplicity of infection of both AAV1- and AAV2-VMD2-GFP. Expression of GFP was detected at 2 weeks and increased at 4 weeks post-plating. AAV1 exhibited a greater expression efficacy compared to AAV2 in maturated ARPE-19 cells already after 2 weeks with increased efficiency after 4 weeks. Our study demonstrates an improved maturation protocol for ARPE-19 cells in vitro, mimicking an in vivo phenotype with the expression of signature genes and improved morphology. Viral-mediated RPE-specific gene expression demonstrates that the combination cultures mimic in vivo AAV tropism essential to test new gene therapies for RPE-centered diseases.

A dystroglycan-laminin-integrin axis coordinates cell shape remodeling in the developing Drosophila retina.

Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress continues to be made in our understanding of the pathways that control the apical (top) geometry of epithelial cells, we know comparatively little about those that control cell basal (bottom) geometry. To examine this, we used the Drosophila ommatidium, which is the basic visual unit of the compound eye. The ommatidium is shaped as a hexagonal prism, and generating this 3D structure requires ommatidial cells to adopt specific apical and basal polygonal geometries. Using this model system, we find that generating cell type-specific basal geometries starts with patterning of the basal extracellular matrix, whereby Laminin accumulates at discrete locations across the basal surface of the retina. We find the Dystroglycan receptor complex (DGC) is required for this patterning by promoting localized Laminin accumulation at the basal surface of cells. Moreover, our results reveal that localized accumulation of Laminin and the DGC are required for directing Integrin adhesion. This induces cell basal geometry remodeling by anchoring the basal surface of cells to the extracellular matrix at specific, Laminin-rich locations. We propose that patterning of a basal extracellular matrix by generating discrete Laminin domains can direct Integrin adhesion to induce cell shape remodeling in epithelial morphogenesis.

Integrin signaling in pluripotent cells acts as a gatekeeper of mouse germline entry.

Primordial germ cells (PGCs) are the precursors of gametes and the sole mechanism by which animals transmit genetic information across generations. In the mouse embryo, the transcriptional and epigenetic regulation of PGC specification has been extensively characterized. However, the initial event that triggers the soma-germline segregation remains poorly understood. Here, we uncover a critical role for the basement membrane in regulating germline entry. We show that PGCs arise in a region of the mouse embryo that lacks contact with the basement membrane, and the addition of exogenous extracellular matrix (ECM) inhibits both PGC and PGC-like cell (PGCLC) specification in mouse embryos and stem cell models, respectively. Mechanistically, we demonstrate that the engagement of ß1 integrin with laminin blocks PGCLC specification by preventing the Wnt signaling-dependent down-regulation of the PGC transcriptional repressor, Otx2. In this way, the physical segregation of cells away from the basement membrane acts as a morphogenetic fate switch that controls the soma-germline bifurcation.

Pig-derived ECM-SIS provides a novel matrix gel for tumor modeling.

The absence of effective extracellular matrix to mimic the natural tumor microenvironment remains a significant obstacle in cancer research. Matrigel, abundant in various biological matrix components, is limited in its application due to its high cost. This has prompted researchers to explore alternative matrix substitutes. Here, we have investigated the effects of the extracellular matrix derived from pig small intestinal submucosa (ECM-SIS) in xenograft tumor modeling. Our results showed that the pig-derived ECM-SIS effectively promotes the establishment of xenograft tumor models, with a tumor formation rate comparable to that of Matrigel. Furthermore, we showed that the pig-derived ECM-SIS exhibited lower immune rejection and fewer infiltrating macrophages than Matrigel. Gene sequencing analysis demonstrated only a 0.5% difference in genes between pig-derived ECM-SIS and Matrigel during the process of tumor tissue formation. These differentially expressed genes primarily participate in cellular processes, biological regulation, and metabolic processes. These findings emphasize the potential of pig-derived ECM-SIS as a cost-effective option for tumor modeling in cancer research.

Engineering a 3D Biomimetic Peptides Functionalized-Polyethylene Glycol Hydrogel Model Cocultured with Endothelial Cells and Astrocytes: Enhancing In Vitro Blood-Brain Barrier Biomimicry.

The blood-brain barrier (BBB) poses a significant challenge for drug delivery and is linked to various neurovascular disorders. In vitro BBB models provide a tool to investigate drug permeation across the BBB and the barrier's response to external injury events. Yet, existing models lack fidelity in replicating the BBB's complexity, hindering a comprehensive understanding of its functions. This study introduces a three-dimensional (3D) model using polyethylene glycol (PEG) hydrogels modified with biomimetic peptides that represent recognition sequences of key proteins in the brain. Hydrogels were functionalized with recognition sequences for laminin (IKVAV) and fibronectin peptides (RGD) and chemically cross-linked with matrix metalloprotease-sensitive peptides (MMPs) to mimic the extracellular matrix of the BBB. Astrocytes and endothelial cells were seeded within and on the surface of the hydrogels, respectively. The barrier integrity was assessed through different tests including transendothelial electrical resistance (TEER), the permeability of sodium fluorescence (Na-F), the permeability of Evan's blue bound to albumin (EBA), and the expression of zonula occluden-1 (ZO-1) in seeded endothelial cells. Hydrogels with a combination of RGD and IKVAV peptides displayed superior performance, exhibiting significantly higher TEER values (55.33 ± 1.47 Ω·cm2) at day 5 compared to other 2D controls including HAECs-monoculture and HAECs-cocultured with NHAs seeded on well inserts and 3D controls including RGD hydrogel and RGD-IKVAV monoculture with HAECs and RGD hydrogel cocultured with HAECs and NHAs. The designed 3D system resulted in the lowest Evan's blue permeability at 120 min (0.215 ± 0.055 µg/mL) compared to controls. ZO-1 expression was significantly higher and formed a relatively larger network in the functionalized hydrogel cocultured with astrocytes and endothelial cells compared to the controls. Thus, the designed 3D model effectively recapitulates the main BBB structure and function in vitro and is expected to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

TNF Induces Laminin-332-Encoding Genes in Endothelial Cells and Laminin-332 Promotes an Atherogenic Endothelial Phenotype.

Laminins are essential components of the basement membranes, expressed in a tissue- and cell-specific manner under physiological conditions. During inflammatory circumstances, such as atherosclerosis, alterations in laminin composition within vessels have been observed. Our study aimed to assess the influence of tumor necrosis factor-alpha (TNF), a proinflammatory cytokine abundantly found in atherosclerotic lesions, on endothelial laminin gene expression and the effects of laminin-332 (LN332) on endothelial cells' behavior. We also evaluated the expression of LN332-encoding genes in human carotid atherosclerotic plaques. Our findings demonstrate that TNF induces upregulation of LAMB3 and LAMC2, which, along with LAMA3, encode the LN332 isoform. Endothelial cells cultured on recombinant LN332 exhibit decreased claudin-5 expression and display a loosely connected phenotype, with an elevated expression of chemokines and leukocyte adhesion molecules, enhancing their attractiveness and adhesion to leukocytes in vitro. Furthermore, LAMB3 and LAMC2 are upregulated in human carotid plaques and show a positive correlation with TNF expression. In summary, TNF stimulates the expression of LN332-encoding genes in human endothelial cells and LN332 promotes an endothelial phenotype characterized by compromised junctional integrity and increased leukocyte interaction. These findings highlight the importance of basement membrane proteins for endothelial integrity and the potential role of LN332 in atherosclerosis.

Cardiac Involvement in LAMA2-Related Muscular Dystrophy and SELENON-Related Congenital Myopathy: A Case Series.

Background: LAMA2-related muscular dystrophy (LAMA2-MD) and SELENON-related myopathy (SELENON-RM) are two rare neuromuscular diseases characterized by proximal and axial muscle weakness, scoliosis, spinal rigidity, low bone quality and respiratory impairment. Cardiac involvement has previously been described in retrospective studies and case reports, but large case series and prospective studies in unselected cohorts are lacking. Objective: The objective of this study is to conduct prevalence estimations, perform cardiac phenotyping, and provide recommendations for clinical care. Methods: In this case series including two time points, we conducted comprehensive assessments with electrocardiography (ECG) and transthoracic echocardiography (TTE). ECGs were systematically assessed for a large subset of variables. TTE included left and right ventricular ejection fraction (LVEF/RVEF) and left ventricular global longitudinal strain (GLS), the latter being a more early and sensitive marker of left ventricular dysfunction. Results: 21 LAMA2-MD (M = 5; 20±14 years) and 10 SELENON-RM patients (M = 7; 18±12 years) were included. In most patients, QRS fragmentation and Q waves, markers of heterogeneous ventricular activation, were present both at baseline and at follow-up. GLS was abnormal (age specific in children, > -18% in adults) in 33% of LAMA2-MD and 43% of SELENON-RM patients at baseline. Reduced LVEF (<52% in males, <54% in females and <55% in pediatric population) was observed in three LAMA2-MD patients at baseline and in none of the SELENON-RM patients. GLS and LVEF did not change between baseline and follow-up. RVEF was normal in all patients. Conclusion: ECG abnormalities and abnormal GLS are prevalent in LAMA2-MD and SELENON-RM, yet abnormal LVEF was only seen in LAMA2-MD patients. One LAMA2-MD patient had a clinically relevant deterioration in LVEF during 1.5-year follow-up. We advise routine screening of all patients with LAMA2-MD or SELENON-RM with ECG and echocardiography at diagnosis, minimally every two years from second decade of life and if new cardiac signs arise.

A Multicenter Cross-Sectional Study of the Swiss Cohort of LAMA2-Related Muscular Dystrophy.

Background: LAMA2-related muscular dystrophy (LAMA2-RD) is an autosomal-recessive disorder and one of the most common congenital muscular dystrophies. Due to promising therapies in preclinical development, there is an increasing effort to better define the epidemiology and natural history of this disease. Objective: The present study aimed to describe a well-characterized baseline cohort of patients with LAMA2-RD in Switzerland. Methods: The study used data collected by the Swiss Registry for Neuromuscular Disorders (Swiss-Reg-NMD). Diagnostic findings were derived from genetics, muscle biopsy, creatine kinase-level and electrophysiological testing, as well as from brain MRIs. Further clinical information included motor assessments (CHOP INTEND, MFM20/32), joint contractures, scoliosis, ophthalmoplegia, weight gain, feeding difficulties, respiratory function, cardiac investigations, EEG findings, IQ and schooling. Results: Eighteen patients with LAMA-RD were included in the Swiss-Reg-NMD as of May 2023 (age at inclusion into the registry: median age 8.7 years, range 1 month - 31 years F = 8, M = 10). Fourteen patients presented with the severe form of LAMA2-RD (were never able to walk; CMD), whereas four patients presented with the milder form (present or lost walking capability; LGMD). All patients classified as CMD had symptoms before 12 months of age and 11/14 before the age of six months. 15 carried homozygous or compound heterozygous pathogenic or likely pathogenic variants in LAMA2 and two were homozygous for a variant of unknown significance (one patient unknown). Brain MRI was available for 14 patients, 13 had white matter changes and 11 had additional structural abnormalities, including cobblestone malformations, pontine hypoplasia and an enlarged tegmento-vermial angle not reported before. Conclusion: This study describes the Swiss cohort of patients with LAMA2-RD and gives insights into measuring disease severity and disease progression, which is important for future clinical trials, as well as for a better clinical understanding and management of patients with LAMA2-RD.

Moonlighting on the Fasciola hepatica tegument: Enolase, a glycolytic enzyme, interacts with the extracellular matrix and fibrinolytic system of the host.

Enolase is a 47 kDa enzyme that functions within the glycolysis and gluconeogenesis pathways involved in the reversible conversion of D-2-phosphoglycerate (2PGA) to phosphoenolpyruvate (PEP). However, in the context of host-pathogen interactions, enolase from different species of parasites, fungi and bacteria have been shown to contribute to adhesion processes by binding to proteins of the host extracellular matrix (ECM), such as fibronectin (FN) or laminin (LM). In addition, enolase is a plasminogen (PLG)-binding protein and induces its activation to plasmin, the main protease of the host fibrinolytic system. These secondary 'moonlighting' functions of enolase are suggested to facilitate pathogen migration through host tissues. This study aims to uncover the moonlighting role of enolase from the parasite Fasciola hepatica, shedding light on its relevance to host-parasite interactions in fasciolosis, a global zoonotic disease of increasing concern. A purified recombinant form of F. hepatica enolase (rFhENO), functioning as an active homodimeric glycolytic enzyme of ~94 kDa, was successfully obtained, fulfilling its canonical role. Immunoblotting studies on adult worm extracts showed that the enzyme is present in the tegument and the excretory/secretory products of the parasite, which supports its key role at the host-parasite interface. Confocal immunolocalisation studies of the protein in newly excysted juveniles and adult worms also localised its expression within the parasite tegument. Finally, we showed by ELISA that rFhENO can act as a parasitic adhesin by binding host LM, but not FN. rFhENO also binds PLG and enhances its conversion to plasmin in the presence of the tissue-type and urokinase-type PLG activators (t-PA and u-PA). This moonlighting adhesion-like function of the glycolytic protein enolase could contribute to the mechanisms by which F. hepatica efficiently invades and migrates within its host and encourages further research efforts that are designed to impede this function by vaccination or drug design.

Extracellular matrix substrates differentially influence enteric glial cell homeostasis and immune reactivity.

Introduction: Enteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed. Methods: We examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice. Results: We identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1ß treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation. Discussion: Together, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.

Biologically Relevant Laminin-511 Moderates the Derivation and Proliferation of Human Lens Epithelial Stem/Progenitor-Like Cells.

Purpose: The role of specific extracellular matrix (ECM) molecules in lens cell development and regeneration is poorly understood, as appropriate cellular models are lacking. Here, a laminin-based lens cell in vitro induction system was developed to study the role of laminin in human lens epithelial stem/progenitor cell (LES/PC) development. Methods: The human embryonic stem cell-based lens induction system followed a three-stage protocol. The expression profile of laminins during lens induction was screened, and laminin-511 (LN511) was tested as a candidate substitute. LN511 induction system cellular and molecular features, including induction efficiency, transcription factor expression related to different lens development stages, ECM alterations, and Hippo/YAP signaling, were evaluated. Results: LAMA5, LAMB1, and LAMC1 were highly expressed around the time of LES/PC derivation. We chose LN511 (product of LAMA5, LAMB1, and LAMC1) and found that it considerably enhanced lens cell induction efficiency, compared to that in Matrigel-coated culture, as more and larger lentoid bodies were detected. Notably, LES/PC induction efficiency improved by promoting lens specification-related transcription factor expression and cell proliferation. Transcriptome analysis revealed that compared to those with Matrigel, ECM accumulation and cell adhesion were downregulated in the LN511 system. Hippo/YAP signaling was hypoactive during LES/P-like cell generation, and small molecule inhibitors of YAP/TAZ activity upregulated LES/PC marker expression and promoted the efficiency of LES/P-like cell derivation. Conclusions: The laminin isoform LN511 is a reliable substitute for the LES/P-like cell induction system, and LN511-YAP acted as efficient modulators of LES/PC derivation; this contributes to knowledge of the role of the ECM in human lens development.

Development of Phase-Separating Microfiber Network Hydrogels to Promote In Vitro Vascularization.

Engineered vascularized tissues in vitro exhibit the potential for transplantation therapy and disease modeling. Despite efforts to design hydrogels as cell culture platforms for in vitro vascularization, development of vascularized tissues recapitulating the natural structures and functions remains difficult due to a poor understanding of the relationships between the matrix microstructures and tube formation of endothelial cells. Herein, we developed microfiber network hydrogels with microporous structures by controlling the liquid-liquid phase separation (LLPS) of proteins and matrix structures in hydrogels. Extracellular matrix protein gelatin was modified with hydrogen-bonding moieties and mixed with hyaluronic acid sodium salt to form microfiber network structures. Gelatin gelation and hyaluronic acid sodium salt dissolution led to the formation of a microporous microfiber network hydrogel formation. Matrix structures of hydrogels were modified by controlling LLPS that affects endothelial cell tube formation. Vascularization was improved using laminin peptides and coculturing with mesenchymal stem cells. Overall, our approach exhibits the potential to induce in vitro vascularization for regenerative medicine and disease modeling applications.

Lamc1 promotes osteogenic differentiation and inhibits adipogenic differentiation of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) exhibit multi-lineage differentiation potential and robust proliferative capacity. The late stage of differentiation signifies the functional maturation and characterization of specific cell lineages, which is crucial for studying lineage-specific differentiation mechanisms. However, the molecular processes governing late-stage BMSC differentiation remain poorly understood. This study aimed to elucidate the key biological processes involved in late-stage BMSC differentiation. Publicly available transcriptomic data from human BMSCs were analyzed after approximately 14 days of osteogenic, adipogenic, and chondrogenic differentiation. Thirty-one differentially expressed genes (DEGs) associated with differentiation were identified. Pathway enrichment analysis indicated that the DEGs were involved in extracellular matrix (ECM)-receptor interactions, focal adhesion, and glycolipid biosynthesis, a ganglion series process. Subsequently, the target genes were validated using publicly available single-cell RNA-seq data from mouse BMSCs. Lamc1 exhibited predominant distribution in adipocytes and osteoblasts, primarily during the G2/M phase. Tln2 and Hexb were expressed in chondroblasts, osteoblasts, and adipocytes, while St3gal5 was abundantly distributed in stem cells. Cell communication analysis identified two receptors that interact with LAMCI. q-PCR results confirmed the upregulation of Lamc1, Tln2, Hexb, and St3gal5 during osteogenic differentiation and their downregulation during adipogenic differentiation. Knockdown of Lamc1 inhibited adipogenic and osteogenic differentiation. In conclusion, this study identified four genes, Lamc1, Tln2, Hexb, and St3gal5, that may play important roles in the late-stage differentiation of BMSCs. It elucidated their interactions and the pathways they influence, providing a foundation for further research on BMSC differentiation.

Mechanism and application of fibrous proteins in diabetic wound healing: a literature review.

Diabetic wounds are more complex than normal chronic wounds because of factors such as hypoxia, reduced local angiogenesis, and prolonged inflammation phase. Fibrous proteins, including collagen, fibrin, laminin, fibronectin, elastin etc., possess excellent inherent properties that make them highly advantageous in the area of wound healing. Accumulating evidence suggests that they contribute to the healing process of diabetic wounds by facilitating the repair and remodel of extracellular matrix, stimulating the development of vascular and granulation tissue, and so on. However, there is currently a lack of a comprehensive review of the application of these proteins in diabetes wounds. An overview of fibrous protein characteristics and the alterations linked to diabetic wounds is given in this article's initial section. Next is a summary of the advanced applications of fibrous proteins in the last five years, including acellular dermal matrix, hydrogel, foam, scaffold, and electrospun nanofibrous membrane. These dressings have the ability to actively promote healing in addition to just covering wounds compared to traditional wound dressings like gauze or bandage. Research on fibrous proteins and their role in diabetic wound healing may result in novel therapeutic modalities that lower the incidence of diabetic wounds and thereby enhance the health of diabetic patients.

Cross-sectional survey study of the natural history of LAMA2-related dystrophy.

BACKGROUND: LAMA2-related dystrophies (LAMA2-RD) are a rare group of neuromuscular disorders with a broad spectrum of phenotype severity, ranging from mild to severe. We performed a cross-sectional study of LAMA2-RD through motor function and pulmonary tests to establish the disease's natural history. METHODS: Forty-four individuals with LAMA2-RD were included and evaluated once through functional outcome measures including Motor Function Measure 32 (MFM32), Revised Upper Limb Module (RULM), goniometry, and Forced Vital Capacity (FVC). Fixed Effect Regression Model (ERM) and Kaplan-Meier curve were used for calculating the rate of the disease progression RESULTS: Patients were between 2 and 25 years old (mean 11.4), the most frequent phenotype presentation was non-ambulant (N=36, 81.8%) while eight patients (18,2 %) were ambulant. The non-ambulant group presented a more severe progression of the disease. Non-ambulant patients had a 1.85 % decrease in FVC/year against 1.32 %/year among ambulant patients. In the non-ambulant group, there was a 4.2 % drop/year in the MFM32-D2 domain (p<0.00001), a 2.6 % drop/year in the D3 domain (p<0.0001), and a 2.7 % drop/year in the MFM32 global assessment (p<0.0001). However, the non-ambulant group's evaluation of upper limb function through the RULM scale did not show a statistically significant reduction. In the non-ambulant group, elbow and knee retractions worsened 3.22 degrees/year (p=0.00087) and 1.92 degrees/year, respectively. While in those patients who acquired gait, elbow and knee retractions worsened 2.45 degrees/year (p=0.0003) and 1.73 degrees/year (p=0.01), respectively. CONCLUSION: This study confirmed the progressive nature of LAMA2-RD, both in ambulant and non-ambulant patients. MFM32, FVC, and goniometry were identified as promising outcome measures for natural history studies and clinical trials in LAMA2-RD.

The potential of hydrogel-free tumoroids in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck malignancy, and in particular squamous cell carcinoma (SCC), is responsible for a significant disease burden globally. The lack of an optimal in vitro model system to accurately recapitulate in vivo response to therapy in HNSCC remains a challenge. The development of patient-derived three-dimensional tumour cultures, or tumoroids, has enabled improved modelling of the tumour microenvironment through simulation of important characteristics such as tumour hypoxia, cell-cell interactions and nutrient diffusion characteristics. METHODS: We performed a comprehensive English-language literature review of current methods of tumoroid development utilising Matrigel and Cultrex Basement Membrane Extract 2 (key terms: tumour organoids, tumoroids, hydrogels, Matrigel, Cultrex, squamous cell carcinoma, head and neck)-two common proprietary murine-derived hydrogels containing extracellular matrix proteins. Nascent literature on the establishment of a novel hydrogel-free platform for tumoroid development as distinct from these existing methods was also explored. RESULTS: Whilst useful for facilitating cell-matrix interactions and providing a scaffold for three-dimensional cell growth and organisation, murine-derived cell matrix methods were noted to have notable limitations including temperature sensitivity and the medium forming a barrier to analysis of the supernatant. A novel hydrogel-free method of establishing in vitro tumoroid cultures has been subject to experimentation in colorectal but not head and neck malignancy. The absence of a hydrogel provides for the de novo synthesis of extracellular matrix native to the tumour and self-organisation of cells within this scaffold through the use of ultralow attachment plates. This model demonstrates similar structural and physiological properties to native tissue, whilst enabling more accurate biomimicry of the tumour microenvironment for drug testing. CONCLUSIONS: In the absence of prior experimentation on a hydrogel-free method for establishing HNSCC tumoroids, and comparisons between hydrogel and hydrogel-free models, the future development of a comparative protocol encompassing recruitment, collection, processing and analysis represents a valuable opportunity.

Salivary gland organoid transplantation as a therapeutic option for radiation-induced xerostomia.

BACKGROUND: Xerostomia is a pathological condition characterized by decreased salivation due to salivary gland dysfunction and is frequently attributed to irreversible damage as a side effect of radiation therapy. Stem cell-derived organoid therapy has garnered attention as a promising avenue for resolving this issue. However, Matrigel, a hydrogel commonly used in organoid culture, is considered inappropriate for clinical use due to its undefined composition and immunogenicity. In this study, we aimed to develop a method for culturing collagen-based human salivary gland organoids (hSGOs) suitable for clinical applications and evaluated their therapeutic effectiveness. METHODS: Human salivary gland stem cells were isolated from the salivary gland tissues and cultured in both Matrigel and collagen. We compared the gene and protein expression patterns of salivary gland-specific markers and measured amylase activity in the two types of hSGOs. To evaluate the therapeutic effects, we performed xenogeneic and allogeneic transplantation using human and mouse salivary gland organoids (hSGOs and mSGOs), respectively, in a mouse model of radiation-induced xerostomia. RESULTS: hSGOs cultured in Matrigel exhibited self-renewal capacity and differentiated into acinar and ductal cell lineages. In collagen, they maintained a comparable self-renewal ability and more closely replicated the characteristics of salivary gland tissue following differentiation. Upon xenotransplantation of collagen-based hSGOs, we observed engraftment, which was verified by detecting human-specific nucleoli and E-cadherin expression. The expression of mucins, especially MUC5B, within the transplanted hSGOs suggested a potential improvement in the salivary composition. Moreover, the allograft procedure using mSGOs led to increased salivation, validating the efficacy of our approach. CONCLUSIONS: This study showed that collagen-based hSGOs can be used appropriately in clinical settings and demonstrated the effectiveness of an allograft procedure. Our research has laid the groundwork for the future application of collagen-based hSGOs in allogeneic clinical trials.

Harnessing Decellularized Extracellular Matrix for Enhanced Fidelity in Colorectal Cancer Organoid and Cell-Derived Xenograft Models.

This study evaluates the efficacy of a decellularized intestine tissue-derived extracellular matrix (Intestine ECM) as a scaffold for culturing colorectal cancer (CRC) organoids and establishing cell-derived xenograft (CDX) models, comparing its performance to traditional Matrigel. Intestine ECM demonstrates comparable support for organoid formation and cellular function, highlighting its potential as a more physiologically relevant and reproducible platform. Our findings suggest that Intestine ECM enhances the mimetic environment for colon epithelium, supporting comparable growth and improved differentiation compared to Matrigel. Moreover, when used as a delivery carrier, Intestine ECM significantly increases the growth rate of CDX models using patient-derived primary colorectal cancer cells. This enhancement demonstrates Intestine ECM's role not only as a scaffold but also as a vital component of the tumor microenvironment, facilitating more robust tumorigenesis. These findings advocate for the broader application of Intestine ECM in cancer model systems, potentially leading to more accurate preclinical evaluations and the development of targeted cancer therapies.