Diversity and host interaction of the gut microbiota in specific pathogen-free pigs.

Pigs are widely used as animal models in various studies related to humans. The interaction between the gut microbiota and the host has significant effects on the host's health and disease status. However, although there have been many studies investigating the pig gut microbiota, the findings have been inconsistent due to variations in rearing conditions. Interactions between the gut microbiota and host have not been fully explored in pigs. Specific pathogen-free (SPF) pigs are ideal non-primate large animals to study the interactions between the gut microbiota and the host. In this study, we performed high-throughput sequencing analysis of the gut microbiota and the gut tissue transcriptome of six SPF pigs to provide a systematic understanding of the composition, function, and spatial distribution of gut microbiota in SPF pigs. We identified significant differences in microbial diversity and functionality among different gastrointestinal tract sites. Metagenomics data analysis revealed significant differences in alpha diversity and beta diversity of microbiota in different gastrointestinal sites of SPF pigs. Additionally, transcriptomic data indicated significant differences in gene expression as well as KEGG and GO functional enrichment between the small intestine and large intestine. Furthermore, by combining microbial metagenomics and host transcriptomics analyses, specific correlations were found between gut microbiota and host genes. These included a negative correlation between the TCN1 gene and Prevotella dentalis, possibly related to bacterial metabolic pathways involving vitamin B12, and a positive correlation between the BDH1 gene and Roseburia hominis, possibly because both are involved in fatty acid metabolism. These findings lay the groundwork for further exploration of the co-evolution between the microbiota and the host, specifically in relation to nutrition, metabolism, and immunity. In conclusion, we have elucidated the diversity of the gut microbiota in SPF pigs and conducted a detailed investigation into the interactions between the gut microbiota and host gene expression. These results contribute to our understanding of the intricate dynamics between the gut microbiota and the host, offering important references for advancements in life science research, bioproduct production, and sustainable development in animal husbandry.

Exploring functional metabolites and proteomics biomarkers in late-preterm and natural-born pigs.

Introduction: Pigs are often used to study the intestinal development of newborns, particularly as preterm pig models that mimic the intestinal growth of human preterm infants. Neonatology's study of delivery mode's impact on neonatal development is crucial. Methods: We established 14 newborn pigs delivered via cesarean sections (C-section, at 113 days of gestational age, CS group) and 8 naturally born pigs were used as controls (at 114 days of gestational age, NF group). The impact of two alternative delivery procedures (C-section and natural birth) on the levels of short-chain fatty acids (SCFAs) and organic acids in the hepatic and intestines of newborn pigs were compared using metabolomics. The underlying molecular pathways are examined at the "protein-metabolite" level by integrating proteomic data. Results: The findings demonstrated that the mode of delivery changed the metabolism of SCFAs in newborn pigs, perhaps by affecting the physiology levels of cyclic intermediates such as lactate and malate in the pyruvate metabolic pathway. Additionally, by participating in the fatty acid metabolism pathway, two distinct proteins (FASN and HSD17B4) may impact the physiological concentration of these tiny metabolites. Discussion: In conclusion, this study provided reliable animal model data for understanding the physiological SCFA metabolic information and its affecting mechanism of large-gestational age preterm infants.

Commensal microbiota modulates phenotypic characteristics and gene expression in piglet Peyer's patches.

The gastrointestinal tract contains a complex microbial community. Peyer's patches (PPs) play an important role in inducing mucosal immune responses in the gastrointestinal tract. However, little is known about the effect of commensal microbiota on the host's PPs. Here, we analyzed the phenotypic-to-transcriptome changes in the intestine PPs of specific pathogen-free (SPF) and germ-free (GF) piglets (fed in an environment with and without commensal microbiota, respectively) to elucidate the role of commensal microbiota in host intestine mucosal immunity. Analyses of anatomical and histological characteristics showed that commensal microbiota deficiency led to PP hypoplasia, especially regarding B and T cells. A total of 12,444 mRNAs were expressed in 12 libraries; 2,156 and 425 differentially expressed (DE) mRNAs were detected in the jejunal PP (JPP) and ileal PP (IPP), respectively (SPF vs. GF). The shared DE mRNAs of the JPP and IPP were mainly involved in basic physiological and metabolic processes, while the specific DE mRNAs were enriched in regulating immune cells in the JPP and microbial responses and cellular immunity in the IPP. Commensal microbiota significantly modulated the expression of genes related to B-cell functions, including activation, proliferation, differentiation, apoptosis, receptor signaling, germinal center formation, and IgA isotype class switching, particularly in the JPP. TLR4 pathway-related genes were induced in response to microbial colonization and in LPS/SCFA-treated B cells. We also detected 69 and 21 DE lncRNAs in the JPP and IPP, respectively, and four one-to-one lncRNA-mRNA pairs were identified. These findings might represent key regulatory axes for host intestine mucosal immunity development during microbial colonization. Overall, the findings of this study revealed that commensal microbiota modulated phenotypic characteristics and gene expression in the piglet intestine PPs and underscored the importance of early microbial colonization for host mucosal immunity development.

Thymosin beta 4 prevents systemic lipopolysaccharide-induced plaque load in middle-age APP/PS1 mice.

Lipopolysaccharide (LPS) produced by the gut during systemic infections and inflammation is thought to contribute to Alzheimer's disease (AD) progression. Since thymosin beta 4 (Tß4) effectively reduces LPS-induced inflammation in sepsis, we tested its potential to alleviate the impact of LPS in the brain of the APPswePS1dE9 mouse model of AD (APP/PS1) and wildtype (WT) mice. 12.5-month-old male APP/PS1 mice (n = 30) and their WT littermates (n = 29) were tested for baseline food burrowing performance, spatial working memory and exploratory drive in the spontaneous alternation and open-field tests, prior to being challenged with LPS (100ug/kg, i.v.) or its vehicle phosphate buffered saline (PBS). Tß4 (5 mg/kg, i.v.) or PBS, was administered immediately following and at 2 and 4 h after the PBS or LPS challenge, and then once daily for 6 days (n = 7-8). LPS-induced sickness was assessed though monitoring of changes in body weight and behaviour over a 7-day period. Brains were collected for the determination of amyloid plaque load and reactive gliosis in the hippocampus and cortex. Treatment with Tß4 alleviated sickness symptoms to a greater extent in APP/PS1 than in WT mice by limiting LPS-induced weight loss and inhibition of food burrowing behaviour. It prevented LPS-induced amyloid burden in APP/PS1 mice but increased astrocytic and microglial proliferation in the hippocampus of LPS-treated WT mice. These data show that Tß4 can alleviate the adverse effects of systemic LPS in the brain by preventing exacerbation of amyloid deposition in AD mice and by inducing reactive microgliosis in aging WT mice.

An open source pipeline for quantitative immunohistochemistry image analysis of inflammatory skin disease using artificial intelligence.

BACKGROUND: The application of artificial intelligence (AI) to whole slide images has the potential to improve research reliability and ultimately diagnostic efficiency and service capacity. Image annotation plays a key role in AI and digital pathology. However, the work-streams required for tissue-specific (skin) and immunostain-specific annotation has not been extensively studied compared with the development of AI algorithms. OBJECTIVES: The objective of this study is to develop a common workflow for annotating whole slide images of biopsies from inflammatory skin disease immunostained with a variety of epidermal and dermal markers prior to the development of the AI-assisted analysis pipeline. METHODS: A total of 45 slides containing 3-5 sections each were scanned using Aperio AT2 slide scanner (Leica Biosystems). These slides were annotated by hand using a commonly used image analysis tool which resulted in more than 4000 images blocks. We used deep learning (DL) methodology to first sequentially segment (epidermis and upper dermis), with the exclusion of common artefacts and second to quantify the immunostained signal in those two compartments of skin biopsies and the ratio of positive cells. RESULTS: We validated two DL models using 10-fold validation runs and by comparing to ground truth manually annotated data. The models achieved an average (global) accuracy of 95.0% for the segmentation of epidermis and dermis and 86.1% for the segmentation of positive/negative cells. CONCLUSIONS: The application of two DL models in sequence facilitates accurate segmentation of epidermal and dermal structures, exclusion of common artefacts and enables the quantitative analysis of the immunostained signal. However, inaccurate annotation of the slides for training the DL model can decrease the accuracy of the output. Our open source code will facilitate further external validation across different immunostaining platforms and slide scanners.

Interleukin-22 regulates neutrophil recruitment in ulcerative colitis and is associated with resistance to ustekinumab therapy.

The function of interleukin-22 (IL-22) in intestinal barrier homeostasis remains controversial. Here, we map the transcriptional landscape regulated by IL-22 in human colonic epithelial organoids and evaluate the biological, functional and clinical significance of the IL-22 mediated pathways in ulcerative colitis (UC). We show that IL-22 regulated pro-inflammatory pathways are involved in microbial recognition, cancer and immune cell chemotaxis; most prominently those involving CXCR2+ neutrophils. IL-22-mediated transcriptional regulation of CXC-family neutrophil-active chemokine expression is highly conserved across species, is dependent on STAT3 signaling, and is functionally and pathologically important in the recruitment of CXCR2+ neutrophils into colonic tissue. In UC patients, the magnitude of enrichment of the IL-22 regulated transcripts in colonic biopsies correlates with colonic neutrophil infiltration and is enriched in non-responders to ustekinumab therapy. Our data provide further insights into the biology of IL-22 in human disease and highlight its function in the regulation of pathogenic immune pathways, including neutrophil chemotaxis. The transcriptional networks regulated by IL-22 are functionally and clinically important in UC, impacting patient trajectories and responsiveness to biological intervention.

Mfn2 is responsible for inhibition of the RIG-I/IRF7 pathway and activation of NLRP3 inflammasome in Seneca Valley virus-infected PK-15 cells to promote viral replication.

Seneca Valley virus (SVV), a non-enveloped positive single-stranded virus can cause vesicular disease in swine. However, the mechanisms by which SVV activates an innate immune response remain unknown. Mitofusin-2 (MFN2), a mitochondria-shaping protein regulating mitochondrial fusion and fission, plays a crucial role in innate immune responses. But, the roles of Mfn2 in SVV infection have not been elucidated. Here, we show that SVV inhibited Mfn2 expression and NLRP3 inflammasome, activating RIG-I/IRF7 signaling pathway to increase IFN-λ3 expression. Overexpression of Mfn2 inhibited RIG-I/IRF7 signaling pathway, thus decreasing IFN-λ3 expression and promoting SVV replication. Interestingly, overexpression of Mfn2 also activated NLRP3 inflammasome but did not inhibit SVV proliferation. That may mean the RIG-I/IRF7 signaling pathway plays a more important role in SVV proliferation in PK-15 cells. This study could provide important insights into the modulation of host metabolism during SVV infection and provide a strong theoretical basis for a better understanding of the pathogenic mechanism and immune activation mechanism of SVV.

Generation of immunodeficient pig with hereditary tyrosinemia type 1 and their preliminary application for humanized liver.

BACKGROUND: Mice with humanized livers are important models to study drug toxicology testing, development of hepatitis virus treatments, and hepatocyte transplantation therapy. However, the huge difference between mouse and human in size and anatomy limited the application of humanized mice in investigating human diseases. Therefore, it is urgent to construct humanized livers in pigs to precisely investigate hepatocyte regeneration and human hepatocyte therapy. CRISPR/Cas9 system and somatic cell cloning technology were used to generate two pig models with FAH deficiency and exhibiting severe immunodeficiency (FAH/RAG1 and FAH/RAG1/IL2RG deficiency). Human primary hepatocytes were then successfully transplanted into the FG pig model and constructed two pigs with human liver. RESULTS: The constructed FAH/RAG1/IL2RG triple-knockout pig models were characterized by chronic liver injury and severe immunodeficiency. Importantly, the FG pigs transplanted with primary human hepatocytes produced human albumin in a time dependent manner as early as 1 week after transplantation. Furthermore, the colonization of human hepatocytes was confirmed by immunochemistry staining. CONCLUSIONS: We successfully generated pig models with severe immunodeficiency that could construct human liver tissues.

[Preliminary Experimental Study of Establishing a Lung Development Model with Specific Pathogen Free Preterm Pigs].

OBJECTIVE: To determine the best time for conducting cesarean section for the establishment of an animal model of lung development with specific pathogen free (SPF) preterm Bama minipigs under the condition of not making medical interventions such as hyperoxia, mechanical ventilation, or medication. METHODS: SPF Bama sows at gestational day (GD) 113, GD107, GD104, GD101, and GD98 were selected and cesarean sections were performed. Then, the viability of the preterm piglets were observed. Based on their general data, viability, and paraffin sections stained with hematoxylin and eosin, the best time for performing cesarean section in order to build a SPF preterm pig model of lung development was determined. RESULTS: Cesarean sections were performed on a total of 7 sows and 55 piglets were delivered, among which 25 were still alive 3 hours after delivery. Seven piglets of GD104 and all piglets of GD107 and GD113 survived, while piglets of GD98 and GD101 all died. The survival rate of piglets of GD104 was 33.33% (7/21). Piglets of GD98 already possessed fully developed physical appearance and lung shape. Piglets from GD104 had better lung expansion and higher density of thin-walled alveoli. The lungs of GD107 piglets were basically fully expanded, and the density of thin-walled alveoli was almost the same as that of normal full-term piglets. CONCLUSIONS: Findings of this study suggest that SPF preterm piglets of GD104 with no specific pathogen exposure and no medical intervention can be used to establish a SPF preterm pig model of lung development.

Reduced Expression of the Co-regulator TLE1 in Type 2 Diabetes Is Associated with Increased Islet α-Cell Number.

ß-Cell dysfunction in type 2 diabetes (T2D) is associated with loss of cellular identity and mis-expression of alternative islet hormones, including glucagon. The molecular basis for these cellular changes has been attributed to dysregulation of core ß-cell transcription factors, which regulate ß-cell identity through activating and repressive mechanisms. The TLE1 gene lies near a T2D susceptibility locus and, recently, the glucagon repressive actions of this transcriptional coregulator have been demonstrated in vitro. We investigated whether TLE1 expression is disrupted in human T2D, and whether this is associated with increased islet glucagon-expressing cells. Automated image analysis following immunofluorescence in donors with (n = 7) and without (n = 7) T2D revealed that T2D was associated with higher islet α/ß cell ratio (Control: 0.7 ± 0.1 vs T2D: 1.6 ± 0.4; P < .05) and an increased frequency of bihormonal (insulin+/glucagon+) cells (Control: 0.8 ± 0.2% vs T2D: 2.0 ± 0.4%, P < .05). In nondiabetic donors, the majority of TLE1-positive cells were mono-hormonal ß-cells (insulin+/glucagon-: 98.2 ± 0.5%; insulin+/glucagon+: 0.7 ± 0.2%; insulin-/glucagon+: 1.1 ± 0.4%; P < .001). TLE1 expression was reduced in T2D (Control: 36 ± 2.9% vs T2D: 24 ± 2.6%; P < .05). Reduced islet TLE1 expression was inversely correlated with α/ß cell ratio (r = -0.55; P < .05). TLE1 knockdown in EndoC-ßH1 cells was associated with a 2.5-fold increase in glucagon gene mRNA and mis-expression of glucagon in insulin-positive cells. These data support TLE1 as a putative regulator of human ß-cell identity, with dysregulated expression in T2D associated with increased glucagon expression potentially reflecting ß- to α-cell conversion.

In Situ Analysis Reveals That CFTR Is Expressed in Only a Small Minority of ß-Cells in Normal Adult Human Pancreas.

CONTEXT: Although diabetes affects 40% to 50% of adults with cystic fibrosis, remarkably little is known regarding the underlying mechanisms leading to impaired pancreatic ß-cell insulin secretion. Efforts toward improving the functional ß-cell deficit in cystic fibrosis-related diabetes (CFRD) have been hampered by an incomplete understanding of whether ß-cell function is intrinsically regulated by cystic fibrosis transmembrane conductance regulator (CFTR). Definitively excluding meaningful CFTR expression in human ß-cells in situ would contribute significantly to the understanding of CFRD pathogenesis. OBJECTIVE: To determine CFTR messenger ribonucleic acid (mRNA) and protein expression within ß-cells in situ in the unmanipulated human pancreas of donors without any known pancreatic pathology. DESIGN: In situ hybridization for CFTR mRNA expression in parallel with insulin immunohistochemical staining and immunofluorescence co-localization of CFTR with insulin and the ductal marker, Keratin-7 (KRT7), were undertaken in pancreatic tissue blocks from 10 normal adult, nonobese deceased organ donors over a wide age range (23-71 years) with quantitative image analysis. RESULTS: CFTR mRNA was detectable in a mean 0.45% (range 0.17%-0.83%) of insulin-positive cells. CFTR protein expression was co-localized with KRT7. One hundred percent of insulin-positive cells were immunonegative for CFTR. CONCLUSIONS: For the first time, in situ CFTR mRNA expression in the unmanipulated pancreas has been shown to be present in only a very small minority (<1%) of normal adult ß-cells. These data signal a need to move away from studying endocrine-intrinsic mechanisms and focus on elucidation of exocrine-endocrine interactions in human cystic fibrosis.

Developing a simple method to enhance the generation of cone and rod photoreceptors in pluripotent stem cell-derived retinal organoids.

Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases such as Stargardt's disease, age-related macular degeneration, and retinitis pigmentosa. The final impact of all retinal dystrophies is the loss of photoreceptors; hence, there is a pressing need for research into replacement. Seminal work has shown that a simple three-dimensional culture system enables differentiation of human pluripotent stem cells to retinal organoids containing large numbers of photoreceptors developing alongside retinal neurons and Müller glia cells in a laminated structure that resembles the native retina. Despite these promising developments, current protocols show different efficiencies across pluripotent stem cells and result in retinal organoids with a mixture of photoreceptor cells at varying maturation states, along with nonphotoreceptor cell types. In this study, we investigated the impact of stage-specific addition of retinoic acid (RA), 9-cis-retinal, 11-cis-retinal, levodopa (l-DOPA), triiodothyronine (T3), and γ-secretase inhibitor ((2S)-N-[(3,5-Difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine1,1-dimethylethyl ester2L [DAPT]) in the generation of cone and rod photoreceptors. Our results indicate that addition of RA + T3 during days 90 to 120 of differentiation enhanced the generation of rod and S-cone photoreceptor formation, while the combined addition of DAPT from days 28 to 42 with RA during days 30 to 120 of differentiation led to enhanced generation of L/M-cones at the expense of rods. l-DOPA when added together with RA during days 90 to 120 of differentiation also promoted the emergence of S-cones at the expense of rod photoreceptors. Collectively, these data represent an advance in our ability to direct generation of rod and cone photoreceptors in vitro.

Identification of the core bacteria in rectums of diarrheic and non-diarrheic piglets.

Porcine diarrhea is a global problem that leads to large economic losses of the porcine industry. There are numerous factors related to piglet diarrhea, and compelling evidence suggests that gut microbiota is vital to host health. However, the key bacterial differences between non-diarrheic and diarrheic piglets are not well understood. In the present study, a total of 85 commercial piglets at three pig farms in Sichuan Province and Chongqing Municipality, China were investigated. To accomplish this, anal swab samples were collected from piglets during the lactation (0-19 days old in this study), weaning (20-21 days old), and post-weaning periods (22-40 days), and fecal microbiota were assessed by 16S rRNA gene V4 region sequencing using the Illumina Miseq platform. We found age-related biomarker microbes in the fecal microbiota of diarrheic piglets. Specifically, the family Enterobacteriaceae was a biomarker of diarrheic piglets during lactation (cluster A, 7-12 days old), whereas the Bacteroidales family S24-7 group was found to be a biomarker of diarrheic pigs during weaning (cluster B, 20-21 days old). Co-correlation network analysis revealed that the genus Escherichia-Shigella was the core component of diarrheic microbiota, while the genus Prevotellacea UCG-003 was the key bacterium in non-diarrheic microbiota of piglets in Southwest China. Furthermore, changes in bacterial metabolic function between diarrheic piglets and non-diarrheic piglets were estimated by PICRUSt analysis, which revealed that the dominant functions of fecal microbes were membrane transport, carbohydrate metabolism, amino acid metabolism, and energy metabolism. Remarkably, genes related to transporters, DNA repair and recombination proteins, purine metabolism, ribosome, secretion systems, transcription factors, and pyrimidine metabolism were decreased in diarrheic piglets, but no significant biomarkers were found between groups using LEfSe analysis.

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids.

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.

Production of F0 mice from embryonic stem cells injected eight-cell stage embryos which stored at refrigeration temperature.

At refrigeration temperature, mouse embryos can retain their developmental ability for a couple of days. Previous research reports have focused on the effect of cool temperature on the development of 2-cell stage embryos, morulae or blastocysts and determined that the embryo still has the ability to produce offspring after about 48 h storage at refrigeration temperature. Here we examined whether refrigeration temperature affects the development of the eight-cell stage and if the stored eight-cell stage embryo can still be used as a host embryo for ES cell injection. Our results show that eight-cell stage embryos can develop into blastocysts and yield pups after cold storage for 24 and 48 h. After ES cell injection, stored eight-cell stage embryos can support ES cells developing to F0 pups. In summary, cool storage can preserve the developmental ability of eight-cell stage embryos for at least 48 h, allowing transportation of the embryos at refrigeration temperature between different labs and their subsequent use as host embryos for ES cell injection.

Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.

Gene expression profiles of germ-free and conventional piglets from the same litter.

Germ-free (GF) pigs have clear microbiological backgrounds, and are extensively used as large animal models in the biomedical sciences. However, investigations of the transcriptomic differences between GF and cesarean-derived conventional (CV) piglets are limited. To improve our understanding of GF pigs, and to increase the utility of pigs as an alternative non-rodent model, we used RNA sequencing to profile gene expression in five tissues (the oral mucosae, jejunum, colon, liver, and spleen) of four male GF piglets and four male CV piglets from the same litter. We identified 14 genes that were differentially expressed in all five tissues. Seven of these common differentially expressed genes (DEGs) were interferon-inducible genes, and all 14 were consistently downregulated in the GF piglets as compared to the CV piglets. Compared to the other tissues tested, the expression of transcription factors (TFs) in the colon was most affected by the absence of a microbiota. The expression patterns of immune-related genes were downregulated in the GF piglets as compared to the CV piglets, indicating that the intestinal microbiota influenced gene expression in other tissues besides the gut. Gene Ontology (GO) analysis indicated that, in pigs, the intestinal microbiota affected the expression of genes related to immune system function and development.

Extracellular matrix component expression in human pluripotent stem cell-derived retinal organoids recapitulates retinogenesis in vivo and reveals an important role for IMPG1 and CD44 in the development of photoreceptors and interphotoreceptor matrix.

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis. STATEMENT OF SIGNIFICANCE: The expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch's membrane; whereas Brevican, Versican, IMPG1 & IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation.