Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.

Llgl1 mediates timely epicardial emergence and establishment of an apical laminin sheath around the trabeculating cardiac ventricle.

During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall.

Niche cells regulate primordial germ cell quiescence in response to basement membrane signaling.

Stem cell quiescence, proliferation and differentiation are controlled by interactions with niche cells and a specialized extracellular matrix called basement membrane (BM). Direct interactions with adjacent BM are known to regulate stem cell quiescence; however, it is less clear how niche BM relays signals to stem cells that it does not contact. Here, we examine how niche BM regulates Caenorhabditis elegans primordial germ cells (PGCs). BM regulates PGC quiescence even though PGCs are enwrapped by somatic niche cells and do not contact the BM; this can be demonstrated by depleting laminin, which causes normally quiescent embryonic PGCs to proliferate. We show that following laminin depletion, niche cells relay proliferation-inducing signals from the gonadal BM to PGCs via integrin receptors. Disrupting the BM proteoglycan perlecan blocks PGC proliferation when laminin is depleted, indicating that laminin functions to inhibit a proliferation-inducing signal originating from perlecan. Reducing perlecan levels in fed larvae hampers germline growth, suggesting that BM signals regulate germ cell proliferation under physiological conditions. Our results reveal how BM signals can regulate stem cell quiescence indirectly, by activating niche cell integrin receptors.

The alpha7 integrin subunit in astrocytes promotes endothelial blood-brain barrier integrity.

The blood-brain barrier (BBB) is a vascular endothelial cell boundary that partitions the circulation from the central nervous system to promote normal brain health. We have a limited understanding of how the BBB is formed during development and maintained in adulthood. We used quantitative transcriptional profiling to investigate whether specific adhesion molecules are involved in BBB functions, with an emphasis on understanding how astrocytes interact with endothelial cells. Our results reveal a striking enrichment of multiple genes encoding laminin subunits as well as the laminin receptor gene Itga7, which encodes the alpha7 integrin subunit, in astrocytes. Genetic ablation of Itga7 in mice led to aberrant BBB permeability and progressive neurological pathologies. Itga7-/- mice also showed a reduction in laminin protein expression in parenchymal basement membranes. Blood vessels in the Itga7-/- brain showed separation from surrounding astrocytes and had reduced expression of the tight junction proteins claudin 5 and ZO-1. We propose that the alpha7 integrin subunit in astrocytes via adhesion to laminins promotes endothelial cell junction integrity, all of which is required to properly form and maintain a functional BBB.

Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.

A developmental landscape of 3D-cultured human pre-gastrulation embryos.

Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms1,2 that do not recapitulate the in vivo conditions3-5. Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior-posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.

Functional diversity of laminins.

Laminins are a large family of conserved, multidomain trimeric basement membrane proteins that contribute to the structure of extracellular matrix and influence the behavior of associated cells, such as adhesion, differentiation, migration, phenotype stability, and resistance to anoikis. In lower organisms such as Hydra there is only one isoform of laminin, but higher organisms have at least 16 trimeric isoforms with varying degrees of cell/tissue specificity. In vitro protein and cell culture studies, gene manipulation in animals, and laminin gene mutations in human diseases have provided insight into the specific functions of some laminins, but the biological roles of many isoforms are still largely unexplored, mainly owing to difficulties in isolating them in pure form from tissues or cells. In this review, we elucidate the evolution of laminins, describe their molecular complexity, and explore the current knowledge of their diversity and functional aspects, including laminin-mediated signaling via membrane receptors, in vitro cell biology, and involvement in various tissues gained from animal model and human disease studies. The potential use of laminins in cell biology research and biotechnology is discussed.

Cancer-Associated Fibroblast Induces Acinar-to-Ductal Cell Transdifferentiation and Pancreatic Cancer Initiation Via LAMA5/ITGA4 Axis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplastic stroma surrounding most tumors. Activated stromal fibroblasts, namely cancer-associated fibroblasts (CAFs), play a major role in PDAC progression. We analyzed whether CAFs influence acinar cells and impact PDAC initiation, that is, acinar-to-ductal metaplasia (ADM). ADM connection with PDAC pathophysiology is indicated, but not yet established. We hypothesized that CAF secretome might play a significant role in ADM in PDAC initiation. METHODS: Mouse and human acinar cell organoids, acinar cells cocultured with CAFs and exposed to CAF-conditioned media, acinar cell explants, and CAF cocultures were examined by means of quantitative reverse transcription polymerase chain reaction, RNA sequencing, immunoblotting, and confocal microscopy. Data from liquid chromatography with tandem mass spectrometry analysis of CAF-conditioned medium and RNA sequencing data of acinar cells post-conditioned medium exposure were integrated using bioinformatics tools to identify the molecular mechanism for CAF-induced ADM. Using confocal microscopy, immunoblotting, and quantitative reverse transcription polymerase chain reaction analysis, we validated the depletion of a key signaling axis in the cell line, acinar explant coculture, and mouse cancer-associated fibroblasts (mCAFs). RESULTS: A close association of acino-ductal markers (Ulex europaeus agglutinin 1, amylase, cytokeratin-19) and mCAFs (α-smooth muscle actin) in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1Cre (KPC) and LSL-KrasG12D/+; Pdx1Cre (KC) autochthonous progression tumor tissue was observed. Caerulein treatment-induced mCAFs increased cytokeratin-19 and decreased amylase in wild-type and KC pancreas. Likewise, acinar-mCAF cocultures revealed the induction of ductal transdifferentiation in cell line, acinar-organoid, and explant coculture formats in WT and KC mice pancreas. Proteomic and transcriptomic data integration revealed a novel laminin α5/integrinα4/stat3 axis responsible for CAF-mediated acinar-to-ductal cell transdifferentiation. CONCLUSIONS: Results collectively suggest the first evidence for CAF-influenced acino-ductal phenotypic switchover, thus highlighting the tumor microenvironment role in pancreatic carcinogenesis inception.

Timely Schwann cell division drives peripheral myelination in vivo via the laminin/cAMP pathway.

Schwann cells (SCs) migrate along peripheral axons and divide intensively to generate the right number of cells prior to axonal ensheathment; however, little is known regarding the temporal and molecular control of their division and its impact on myelination. We report that Sil, a spindle pole protein associated with autosomal recessive primary microcephaly, is required for temporal mitotic exit of SCs. In sil-deficient cassiopeia (csp-/-) mutants, SCs fail to radially sort and myelinate peripheral axons. Elevation of cAMP, but not Rac1 activity, in csp-/- restores myelin ensheathment. Most importantly, we show a significant decrease in laminin expression within csp-/- posterior lateral line nerve and that forcing Laminin 2 expression in csp-/- fully restores the ability of SCs to myelinate. Thus, we demonstrate an essential role for timely SC division in mediating laminin expression to orchestrate radial sorting and peripheral myelination in vivo.

Laminin 511 and WNT signalling sustain prolonged expansion of hiPSC-derived hippocampal progenitors.

Using the timely re-activation of WNT signalling in neuralizing human induced pluripotent stem cells (hiPSCs), we have produced neural progenitor cells with a gene expression profile typical of human embryonic dentate gyrus (DG) cells. Notably, in addition to continuous WNT signalling, a specific laminin isoform is crucial to prolonging the neural stem state and to extending progenitor cell proliferation for over 200 days in vitro. Laminin 511 is indeed specifically required to support proliferation and to inhibit differentiation of hippocampal progenitor cells for extended time periods when compared with a number of different laminin isoforms assayed. Global gene expression profiles of these cells suggest that a niche of laminin 511 and WNT signalling is sufficient to maintain their capability to undergo typical hippocampal neurogenesis. Moreover, laminin 511 signalling sustains the expression of a set of genes responsible for the maintenance of a hippocampal neurogenic niche. Finally, xenograft of human DG progenitors into the DG of adult immunosuppressed host mice produces efficient integration of neurons that innervate CA3 layer cells spanning the same area of endogenous hippocampal neuron synapses.

Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila.

The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. Here, we systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. We find that the Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, we show that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy we show that basement membrane stiffness mainly depends on Collagen IV. Our analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

Release of miR-29 Target Laminin C2 Improves Skin Repair.

miRNAs are small noncoding RNAs that regulate mRNA targets in a cell-specific manner. miR-29 is expressed in murine and human skin, where it may regulate functions in skin repair. Cutaneous wound healing model in miR-29a/b1 gene knockout mice was used to identify miR-29 targets in the wound matrix, where angiogenesis and maturation of provisional granulation tissue was enhanced in response to genetic deletion of miR-29. Consistently, antisense-mediated inhibition of miR-29 promoted angiogenesis in vitro by autocrine and paracrine mechanisms. These processes are likely mediated by miR-29 target mRNAs released upon removal of miR-29 to improve cell-matrix adhesion. One of these, laminin (Lam)-c2 (also known as laminin γ2), was strongly up-regulated during skin repair in the wound matrix of knockout mice. Unexpectedly, Lamc2 was deposited in the basal membrane of endothelial cells in blood vessels forming in the granulation tissue of knockout mice. New blood vessels showed punctate interactions between Lamc2 and integrin α6 (Itga6) along the length of the proto-vessels, suggesting that greater levels of Lamc2 may contribute to the adhesion of endothelial cells, thus assisting angiogenesis within the wound. These findings may be of translational relevance, as LAMC2 was deposited at the leading edge in human wounds, where it formed a basal membrane for endothelial cells and assisted neovascularization. These results suggest a link between LAMC2, improved angiogenesis, and re-epithelialization.

Characterization of the dystrophin-associated protein complex by mass spectrometry.

The dystrophin-associated protein complex (DAPC) is a highly organized multiprotein complex that plays a pivotal role in muscle fiber structure integrity and cell signaling. The complex is composed of three distinct interacting subgroups, intracellular peripheral proteins, transmembrane glycoproteins, and extracellular glycoproteins subcomplexes. Dystrophin protein nucleates the DAPC and is important for connecting the intracellular actin cytoskeletal filaments to the sarcolemma glycoprotein complex that is connected to the extracellular matrix via laminin, thus stabilizing the sarcolemma during muscle fiber contraction and relaxation. Genetic mutations that lead to lack of expression or altered expression of any of the DAPC proteins are associated with different types of muscle diseases. Hence characterization of this complex in healthy and dystrophic muscle might bring insights into its role in muscle pathogenesis. This review highlights the role of mass spectrometry in characterizing the DAPC interactome as well as post-translational glycan modifications of some of its components such as α-dystroglycan. Detection and quantification of dystrophin using targeted mass spectrometry are also discussed in the context of healthy versus dystrophic skeletal muscle.

Androgenic steroids induce pathologic scarring in a preclinical porcine model via dysfunctional extracellular matrix deposition.

Hypertrophic scarring is a major source of morbidity. Sex hormones are not classically considered modulators of scarring. However, based on increased frequency of hypertrophic scarring in patients on testosterone, we hypothesized that androgenic steroids induce abnormal scarring and developed a preclinical porcine model to explore these effects. Mini-swine underwent castration, received no testosterone (noT) or biweekly testosterone therapy (+T), and underwent excisional wounding. To create a delayed wound healing model, a subset of wounds were re-excised at 2 weeks. Scars from postoperative day 42 (POD42) and delayed wounds (POD28) were harvested 6 weeks after initial wounding for analysis via histology, bulk RNA-seq, and mechanical testing. Histologic analysis of scars from +T animals showed increased mean fibrosis area (16 mm2noT, 28 mm2+T; p = .007) and thickness (0.246 mm2noT, 0.406 mm2+T; p < .001) compared to noT. XX+T and XY+T scars had greater tensile burst strength (p = .024 and p = .013, respectively) compared to noT swine. Color deconvolution analysis revealed greater deposition of type I and type III collagen as well as increased collagen type I:III ratio in +T scars. Dermatopathologist histology scoring showed that +T exposure was associated with worse overall scarring (p < .05). Gene ontology analysis found that testosterone exposure was associated with upregulation of cellular metabolism and immune response gene sets, while testosterone upregulated pathways related to keratinization and laminin formation on pathway analysis. In conclusion, we developed a preclinical porcine model to study the effects of the sex hormone testosterone on scarring. Testosterone induces increased scar tissue deposition and appears to increase physical strength of scars via supraphysiologic deposition of collagen and other ECM factors. The increased burst strength seen in both XX and XY animals suggests that hormone administration has a strong influence on scar mechanical properties independent of chromosomal sex. Anti-androgen topical therapies may be a promising future area of research.

LAMA3 Promotes Tumorigenesis of Oral Squamous Cell Carcinoma by METTL3-Mediated N6-Methyladenosine Modification.

Laminin subunit alpha 3 (LAMA3) is a cancer regulator. However, its effects and regulatory pathways in oral squamous cell carcinoma (OSCC) progression remain unknown. This research aimed to determine the influence of LAMA3 regulation via methyltransferase-like 3 (METTL3) on OSCC progression. Using quantitative real-time polymerase chain reaction and bioinformatics analysis, the expression levels of LAMA3 and METTL3 in OSCC tissues were examined. The functional roles of LAMA3 and METTL3 were analyzed using cell functional experiments. Using methylated RNA immunoprecipitation and mRNA stability assays, LAMA3 and METTL3 regulation was investigated. In OSCC tissues, LAMA3 was upregulated. LAMA3 inhibition hampered OSCC cell proliferation, invasion, and migration while its overexpression facilitated OSCC cell progression. METTL3 serves as a crucial upstream regulator of LAMA3 in OSCC and upregulates LAMA3 expression via an m6A-dependent mechanism. The low METTL3 expression partially restored the enhanced malignant phenotype induced by LAMA3 overexpression. Our findings indicate that METTL3 and LAMA3 act as pro-oncogenic factors in OSCC, with METTL3 promoting OSCC malignancy via m6A modification-dependent stabilization of LAMA3 transcripts, representing a novel regulatory mechanism in OSCC.

Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene.

Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease1. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy2j/dy2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy2j/dy2j mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR-dCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.

Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Bovine and human endometrium-derived hydrogels support organoid culture from healthy and cancerous tissues.

Organoid technology has provided unique insights into human organ development, function, and diseases. Patient-derived organoids are increasingly used for drug screening, modeling rare disorders, designing regenerative therapies, and understanding disease pathogenesis. However, the use of Matrigel to grow organoids represents a major challenge in the clinical translation of organoid technology. Matrigel is a poorly defined mixture of extracellular matrix proteins and growth factors extracted from the Engelbreth-Holm-Swarm mouse tumor. The extracellular matrix is a major driver of multiple cellular processes and differs significantly between tissues as well as in healthy and disease states of the same tissue. Therefore, we envisioned that the extracellular matrix derived from a native healthy tissue would be able to support organoid growth akin to organogenesis in vivo. Here, we have developed hydrogels from decellularized human and bovine endometrium. These hydrogels supported the growth of mouse and human endometrial organoids, which was comparable to Matrigel. Organoids grown in endometrial hydrogels were proteomically more similar to the native tissue than those cultured in Matrigel. Proteomic and Raman microspectroscopy analyses showed that the method of decellularization affects the biochemical composition of hydrogels and, subsequently, their ability to support organoid growth. The amount of laminin in hydrogels correlated with the number and shape of organoids. We also demonstrated the utility of endometrial hydrogels in developing solid scaffolds for supporting high-throughput, cell culture-based applications. In summary, endometrial hydrogels overcome a major limitation of organoid technology and greatly expand the applicability of organoids to understand endometrial biology and associated pathologies.