Single-Cell Sequencing and Organoids: A Powerful Combination for Modelling Organ Development and Diseases.

The development and function of a particular organ and the pathogenesis of various diseases remain intimately linked to the features of each cell type in the organ. Conventional messenger RNA- or protein-based methodologies often fail to elucidate the contribution of rare cell types, including some subpopulations of stem cells, short-lived progenitors and circulating tumour cells, thus hampering their applications in studies regarding organ development and diseases. The scRNA-seq technique represents a new approach for determining gene expression variability at the single-cell level. Organoids are new preclinical models that recapitulate complete or partial features of their original organ and are thought to be superior to cell models in mimicking the sophisticated spatiotemporal processes of the development and regeneration and diseases. In this review, we highlight recent advances in the field of scRNA-seq, organoids and their current applications and summarize the advantages of using a combination of scRNA-seq and organoid technology to model diseases and organ development.

Modeling liver cancer and therapy responsiveness using organoids derived from primary mouse liver tumors.

The current understanding of cancer biology and development of effective treatments for cancer remain far from satisfactory. This in turn heavily relies on the availability of easy and robust model systems that resemble the architecture/physiology of the tumors in patients to facilitate research. Cancer research in vitro has mainly been based on the use of immortalized 2D cancer cell lines that deviate in many aspects from the original primary tumors. The recent development of the organoid technology allowing generation of organ-buds in 3D culture from adult stem cells has endowed the possibility of establishing stable culture from primary tumors. Although culturing organoids from liver tumors is thought to be difficult, we now convincingly demonstrate the establishment of organoids from mouse primary liver tumors. We have succeeded in culturing 91 lines from 129 liver tissue/tumors. These organoids can be grown in long-term cultures in vitro. About 20% of these organoids form tumors in immunodeficient mice upon (serial) transplantation, confirming their tumorigenic and self-renewal properties. Interestingly, single cells from the tumor organoids have high efficiency of organoid initiation, and a single organoid derived from a cancer cell is able to initiate a tumor in mice, indicating the enrichment of tumor-initiating cells in the tumor organoids. Furthermore, these organoids recapitulate, to some extent, the heterogeneity of liver cancer in patients, with respect to phenotype, cancer cell composition and treatment response. These model systems shall provide enormous opportunities to advance our research on liver cancer (stem cell) biology, drug development and personalized medicine.

The RNA genome of hepatitis E virus robustly triggers an antiviral interferon response.

The outcomes of hepatitis E virus (HEV) infection are diverse, ranging from asymptomatic carrier, self-limiting acute infection, and fulminant hepatitis to persistent infection. This is closely associated with the immunological status of the host. This study aimed to understand the innate cellular immunity as the first-line defense mechanism in response to HEV infection. Phosphorylation of signal transducer and activator of transcription 1, a hallmark of the activation of antiviral interferon (IFN) response, was observed in the liver tissues of the majority of HEV-infected patients but not in the liver of uninfected individuals. In cultured cell lines and primary liver organoids, we found that HEV RNA genome potently induced IFN production and antiviral response. This mechanism is conserved among different HEV strains, including genotypes 1, 3, and 7 as tested. Interestingly, single-stranded HEV RNA is sufficient to trigger the antiviral response, without the requirement of viral RNA synthesis and the generation of an RNA replicative form or replicative intermediate. Surprisingly, the m7 G cap and poly A tail are not required, although both are key features of the HEV genome. Mechanistically, this antiviral response occurs in a retinoic acid-inducible gene-I-independent, melanoma differentiation-associated protein 5-independent, mitochondrial antiviral signaling protein-independent, and ß-catenin-independent but IRF3-dependent and IRF7-dependent manner. Furthermore, the integrity of the Janus kinase-signal transducer and activator of transcription pathway is essentially required. CONCLUSION: HEV infection elicits an active IFN-related antiviral response in vitro and in patients, triggered by the viral RNA and mediated by IFN regulatory factors 3 and 7 and the Janus kinase-signal transducer and activator of transcription cascade; these findings have revealed new insights into HEV-host interactions and provided the basis for understanding the pathogenesis and outcome of HEV infection. (Hepatology 2018;67:2096-2112).

The Eukaryotic Translation Initiation Factor 4F Complex Restricts Rotavirus Infection via Regulating the Expression of IRF1 and IRF7.

The eIF4F complex is a translation initiation factor that closely regulates translation in response to a multitude of environmental conditions including viral infection. How translation initiation factors regulate rotavirus infection remains poorly understood. In this study, the knockdown of the components of the eIF4F complex using shRNA and CRISPR/Cas9 were performed, respectively. We have demonstrated that loss-of-function of the three components of eIF4F, including eIF4A, eIF4E and eIF4G, remarkably promotes the levels of rotavirus genomic RNA and viral protein VP4. Consistently, knockdown of the negative regulator of eIF4F and programmed cell death protein 4 (PDCD4) inhibits the expression of viral mRNA and the VP4 protein. Mechanically, we confirmed that the silence of the eIF4F complex suppressed the protein level of IRF1 and IRF7 that exert potent antiviral effects against rotavirus infection. Thus, these results demonstrate that the eIF4F complex is an essential host factor restricting rotavirus replication, revealing new targets for the development of new antiviral strategies against rotavirus infection.

Nitazoxanide Inhibits Human Norovirus Replication and Synergizes with Ribavirin by Activation of Cellular Antiviral Response.

Norovirus is the main cause of viral gastroenteritis worldwide. Although norovirus gastroenteritis is self-limiting in immunocompetent individuals, chronic infections with debilitating and life-threatening complications occur in immunocompromised patients. Nitazoxanide (NTZ) has been used empirically in the clinic and has demonstrated effectiveness against norovirus gastroenteritis. In this study, we aimed at uncovering the antiviral potential and mechanisms of action of NTZ and its active metabolite, tizoxanide (TIZ), using a human norovirus (HuNV) replicon. NTZ and TIZ, collectively referred to as thiazolides (TZD), potently inhibited replication of HuNV and a norovirus surrogate, feline calicivirus. Mechanistic studies revealed that TZD activated cellular antiviral response and stimulated the expression of a subset of interferon-stimulated genes (ISGs), particularly interferon regulatory factor 1 (IRF-1), not only in a Huh7 cell-based HuNV replicon, but also in naive Huh7 and Caco-2 cells and novel human intestinal organoids. Overexpression of exogenous IRF-1 inhibited HuNV replication, whereas knockdown of IRF-1 largely attenuated the antiviral activity of TZD, suggesting that IRF-1 mediated TZD inhibition of HuNV. By using a Janus kinase (JAK) inhibitor, CP-690550, and a STAT1 knockout approach, we found that TZD induced antiviral response independently of the classical JAK-signal transducers and activators of transcription (JAK-STAT) pathway. Furthermore, TZD and ribavirin synergized to inhibit HuNV replication and completely depleted the replicons from host cells after long-term treatment. In summary, our results demonstrated that TZD combated HuNV replication through activation of cellular antiviral response, in particular by inducing a prominent antiviral effector, IRF-1. NTZ monotherapy or combination with ribavirin represent promising options for treating norovirus gastroenteritis, especially in immunocompromised patients.

Dynamics of Proliferative and Quiescent Stem Cells in Liver Homeostasis and Injury.

BACKGROUND & AIMS: Adult liver stem cells are usually maintained in a quiescent/slow-cycling state. However, a proliferative population, marked by leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), was recently identified as an important liver stem cell population. We aimed to investigate the dynamics and functions of proliferative and quiescent stem cells in healthy and injured livers. METHODS: We studied LGR5-positive stem cells using diphtheria toxin receptor and green fluorescent protein (GFP) knock-in mice. In these mice, LGR5-positive cells specifically coexpress diphtheria toxin receptor and the GFP reporter. Lineage-tracing experiments were performed in mice in which LGR5-positive stem cells and their daughter cells expressed a yellow fluorescent protein/mTmG reporter. Slow-cycling stem cells were investigated using GFP-based, Tet-on controlled transgenic mice. We studied the dynamics of both stem cell populations during liver homeostasis and injury induced by carbon tetrachloride. Stem cells were isolated from mouse liver and organoid formation assays were performed. We analyzed hepatocyte and cholangiocyte lineage differentiation in cultured organoids. RESULTS: We did not detect LGR5-expressing stem cells in livers of mice at any stage of a lifespan, but only following liver injury induced by carbon tetrachloride. In the liver stem cell niche, where the proliferating LGR5+ cells are located, we identified a quiescent/slow-cycling cell population, called label-retaining cells (LRCs). These cells were present in the homeostatic liver, capable of retaining the GFP label over 1 year, and expressed a panel of progenitor/stem cell markers. Isolated single LRCs were capable of forming organoids that could be carried in culture, expanded for months, and differentiated into hepatocyte and cholangiocyte lineages in vitro, demonstrating their bona fide stem cell properties. More interestingly, LRCs responded to liver injury and gave rise to LGR5-expressing stem cells, as well as other potential progenitor/stem cell populations, including SOX9- and CD44-positive cells. CONCLUSIONS: Proliferative LGR5 cells are an intermediate stem cell population in the liver that emerge only during tissue injury. In contrast, LRCs are quiescent stem cells that are present in homeostatic liver, respond to tissue injury, and can give rise to LGR5 stem cells, as well as SOX9- and CD44-positive cells.

RIG-I is a key antiviral interferon-stimulated gene against hepatitis E virus regardless of interferon production.

Interferons (IFNs) are broad antiviral cytokines that exert their function by inducing the transcription of hundreds of IFN-stimulated genes (ISGs). However, little is known about the antiviral potential of these cellular effectors on hepatitis E virus (HEV) infection, the leading cause of acute hepatitis globally. In this study, we profiled the antiviral potential of a panel of important human ISGs on HEV replication in cell culture models by overexpression of an individual ISG. The mechanism of action of the key anti-HEV ISG was further studied. We identified retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5, and IFN regulatory factor 1 (IRF1) as the key anti-HEV ISGs. We found that basal expression of RIG-I restricts HEV infection. Pharmacological activation of the RIG-I pathway by its natural ligand 5'-triphosphate RNA potently inhibits HEV replication. Overexpression of RIG-I activates the transcription of a wide range of ISGs. RIG-I also mediates but does not overlap with IFN-α-initiated ISG transcription. Although it is classically recognized that RIG-I exerts antiviral activity through the induction of IFN production by IRF3 and IRF7, we reveal an IFN-independent antiviral mechanism of RIG-I in combating HEV infection. We found that activation of RIG-I stimulates an antiviral response independent of IRF3 and IRF7 and regardless of IFN production. However, it is partially through activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) cascade of IFN signaling. RIG-I activated two distinct categories of ISGs, one JAK-STAT-dependent and the other JAK-STAT-independent, which coordinately contribute to the anti-HEV activity. CONCLUSION: We identified RIG-I as an important anti-HEV ISG that can be pharmacologically activated; activation of RIG-I stimulates the cellular innate immunity against HEV regardless of IFN production but partially through the JAK-STAT cascade of IFN signaling. (Hepatology 2017;65:1823-1839).

Hepatitis E Virus Infects Neurons and Brains.

Hepatitis E virus (HEV), as a hepatotropic virus, is supposed to exclusively infect the liver and only cause hepatitis. However, a broad range of extrahepatic manifestations (in particular, idiopathic neurological disorders) have been recently reported in association with its infection. In this study, we have demonstrated that various human neural cell lines (embryonic stem cell-derived neural lineage cells) induced pluripotent stem cell-derived human neurons and primary mouse neurons are highly susceptible to HEV infection. Treatment with interferon-α or ribavirin, the off-label antiviral drugs for chronic hepatitis E, exerted potent antiviral activities against HEV infection in neural cells. More importantly, in mice and monkey peripherally inoculated with HEV particles, viral RNA and protein were detected in brain tissues. Finally, patients with HEV-associated neurological disorders shed the virus into cerebrospinal fluid, indicating a direct infection of their nervous system. Thus, HEV is neurotropic in vitro, and in mice, monkeys, and possibly humans. These results challenge the dogma of HEV as a pure hepatotropic virus and suggest that HEV infection should be considered in the differential diagnosis of idiopathic neurological disorders.

Inhibition of Calcineurin or IMP Dehydrogenase Exerts Moderate to Potent Antiviral Activity against Norovirus Replication.

Norovirus is a major cause of acute gastroenteritis worldwide and has emerged as an important issue of chronic infection in transplantation patients. Since no approved antiviral is available, we evaluated the effects of different immunosuppressants and ribavirin on norovirus and explored their mechanisms of action by using a human norovirus (HuNV) replicon-harboring model and a surrogate murine norovirus (MNV) infectious model. The roles of the corresponding drug targets were investigated by gain- or loss-of-function approaches. We found that the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506) moderately inhibited HuNV replication. Gene silencing of their cellular targets, cyclophilin A, FKBP12, and calcineurin, significantly inhibited HuNV replication. A low concentration, therapeutically speaking, of mycophenolic acid (MPA), an uncompetitive IMP dehydrogenase (IMPDH) inhibitor, potently and rapidly inhibited norovirus replication and ultimately cleared HuNV replicons without inducible resistance following long-term drug exposure. Knockdown of the MPA cellular targets IMPDH1 and IMPDH2 suppressed HuNV replication. Consistent with the nucleotide-synthesizing function of IMPDH, exogenous guanosine counteracted the antinorovirus effects of MPA. Furthermore, the competitive IMPDH inhibitor ribavirin efficiently inhibited norovirus and resulted in an additive effect when combined with immunosuppressants. The results from this study demonstrate that calcineurin phosphatase activity and IMPDH guanine synthase activity are crucial in sustaining norovirus infection; thus, they can be therapeutically targeted. Our results suggest that MPA shall be preferentially considered immunosuppressive medication for transplantation patients at risk of norovirus infection, whereas ribavirin represents as a potential antiviral for both immunocompromised and immunocompetent patients with norovirus gastroenteritis.

IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

IFN regulatory factor 1 (IRF1) is one of the most important IFN-stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi-mediated gene-silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK-STAT cascade. Furthermore, induction of ISGs and the anti-HEV effect of IRF1 overlapped that of IFNα, but was potentiated by ribavirin. We demonstrated that IRF1 effectively inhibits HEV replication through the activation of the JAK-STAT pathway, and the subsequent transcription of antiviral ISGs, but independent of IFN production.-Xu, L., Zhou, X., Wang, W., Wang, Y., Yin, Y., van der Laan, L. J. W., Sprengers, D., Metselaar, H. J., Peppelenbosch, M. P., Pan, Q. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

Nucleoside analogue 2'-C-methylcytidine inhibits hepatitis E virus replication but antagonizes ribavirin.

Hepatitis E virus (HEV) infection has emerged as a global health issue, but no approved medication is available. The nucleoside analogue 2'-C-methylcytidine (2CMC), a viral polymerase inhibitor, has been shown to inhibit infection with a variety of viruses, including hepatitis C virus (HCV). Here, we report that 2CMC significantly inhibits the replication of HEV in a subgenomic replication model and in a system using a full-length infectious virus. Importantly, long-term treatment with 2CMC did not result in a loss of antiviral potency, indicating a high barrier to drug resistance development. However, the combination of 2CMC with ribavirin, an off-label treatment for HEV, exerts antagonistic effects. Our results indicate that 2CMC serves as a potential antiviral drug against HEV infection.

Metformin Hydrochloride Significantly Inhibits Rotavirus Infection in Caco2 Cell Line, Intestinal Organoids, and Mice.

Rotavirus is one of the main pathogens that causes severe diarrhea in children under the age of 5, primarily infecting the enterocytes of the small intestine. Currently, there are no specific drugs available for oral rehydration and antiviral therapy targeting rotavirus. However, metformin hydrochloride, a drug known for its antiviral properties, shows promise as it accumulates in the small intestine and modulates the intestinal microbiota. Therefore, we formulated a hypothesis that metformin hydrochloride could inhibit rotavirus replication in the intestine. To validate the anti-rotavirus effect of metformin hydrochloride, we conducted infection experiments using different models, ranging from in vitro cells and organoids to small intestines in vivo. The findings indicate that a concentration of 0.5 mM metformin hydrochloride significantly inhibits the expression of rotavirus mRNA and protein in Caco-2 cells, small intestinal organoids, and suckling mice models. Rotavirus infections lead to noticeable pathological changes, but treatment with metformin has been observed to mitigate the lesions caused by rotavirus infection in the treated group. Our study establishes that metformin hydrochloride can inhibit rotavirus replication, while also affirming the reliability of organoids as a virus model for in vitro research.

Dietary High Dose of Iron Aggravates the Intestinal Injury but Promotes Intestinal Regeneration by Regulating Intestinal Stem Cells Activity in Adult Mice With Dextran Sodium Sulfate-Induced Colitis.

The effects of excessive dietary iron intake on the body have been an important topic. The purpose of this study was to investigate the effects of high-dose iron on intestinal damage and regeneration in dextran sodium sulfate (DSS)-induced colitis model mice. A total of 72 8-week-old adult C57BL/6 mice were randomly divided into two dietary treatment groups: the basal diet supplemented with 45 (control) and 450 mg/kg iron (high-iron) from ferrous sulfate. The mice were fed different diets for 2 weeks, and then 2.5% DSS was orally administered to all mice for 7 days. Samples of different tissues were collected on days 0, 3, and 7 post administration (DPA). High-iron treatment significantly decreased the relative weight of the large intestine at 7 DPA but not at 0 DPA or 3 DPA. High dietary iron increased the jejunal villus width at 0 DPA, decreased the villus width and the crypt depth of the jejunum at 3 DPA, and decreased the number of colonic crypts at 7 DPA. Meanwhile, high dietary iron decreased the number of goblet cells in the jejunal villi and the Paneth cells in the jejunal crypts at 0 DPA, increased the number of goblet cells per crypt of the colon at 3 DPA, and the number of Paneth cells in the jejunal crypts, the goblet cells in the colon, the Ki67-positive proliferating cells in the colon, and the Sex-determining region Y-box transcription factor 9+ (SOX9) cells in the jejunum crypts and colon at 7 DPA. The organoid formation rate was increased by high-iron treatments at 3 DPA and 7 DPA. High dietary iron treatment decreased the mRNA level of jejunal jagged canonical Notch ligand 2 (Jag-2) at 0 DPA and bone morphogenetic protein 4 (Bmp4) and neural precursor cell-expressed developmentally downregulated 8 (Nedd8) in the jejunum and colon at 7 DPA, whereas it increased the mRNA expression of the serum/glucocorticoid-regulated kinase 1 (Sgk1) in the colon at 3 DPA. The results suggested that a high dose of iron aggravated intestinal injury but promoted intestinal repair by regulating intestinal epithelial cell renewal and intestinal stem cell activity in adult mice with colitis.

N-Acetyl-D-glucosamine improves the intestinal development and nutrient absorption of weaned piglets via regulating the activity of intestinal stem cells.

Early weaning in piglets can cause a series of negative effects. This causes serious losses to the livestock industry. N-Acetyl-D-glucosamine (D-GlcNAc) plays an important role in regulating the homeostasis of the intestine. This study aimed to investigate the effects of D-GlcNAc on the growth performance and intestinal function of weaned piglets. Twenty-four weaned piglets ([Yorkshire × Landrace] × Duroc, 6.58 ± 0.15 kg, n = 8) at 21 d old were fed 3 diets supplemented with 0 (control), 1 and 3 g/kg D-GlcNAc. The intestinal organoid model was used to verify the regulatory mechanism of D-GlcNAc on intestinal epithelial cells. On the whole, supplementation of D-GlcNAc in the piglet diet has no significant effect on the growth performance and diarrhoea of weaned piglets (P > 0.05). The apparent digestibility of nutrients and mRNA abundance of nutrient transporters in the 1 g/kg D-GlcNAc group were increased significantly (P < 0.05). D-GlcNAc did not affect villus height (VH) and crypt depth (CD) but resulted in a numerically shorter VH and shallower CD, which lead to an increase in ileal VH:CD ratio (P < 0.05). Cell shedding rates in the ileum villi increased (P < 0.05). The relative length and weight of the small intestine of weaned piglets increased (P < 0.05). In vitro studies found that the budding rates of organoids treated with 0.1 mmol/L D-GlcNAc increased on the d 3 and 5 (P < 0.05). The average budding numbers per budding organoid treated with 0.1 and 10 mmol/L D-GlcNAc increased on d 3 (P < 0.05). D-GlcNAc upregulated leucine rich repeat containing G protein-coupled receptor 5 (Lgr5 + ) and Chromogranin A mRNA abundance in organoids (P < 0.05). Mucin 2 (Muc2) expression increased when treated with 1 and 10 mmol/L D-GlcNAc (P < 0.05). In conclusion, dietary D-GlcNAc cannot improve the growth performance of weaned piglets. However, it can promote the growth and development of the intestinal tract and improve the digestion and absorption capacity of the intestine, which is achieved by affecting the activity of intestinal stem cells.

Changes in progenitors and differentiated epithelial cells of neonatal piglets.

This study aimed to assess the changes of small intestinal morphology, progenitors, differentiated epithelial cells, and potential mechanisms in neonatal piglets. Hematoxylin and eosin staining of samples from 36 piglets suggested that dramatic changes were observed in the jejunum crypts depth and crypt fission index of neonatal piglets (P < 0.001). The number of intestinal stem cells (ISC) tended to increase (P < 0.10), and a decreased number of enteroendocrine cells appeared in the jejunal crypt on d 7 (P < 0.05). Furthermore, the mRNA expression of jejunal chromogranin A (ChgA) was down-regulated in d 7 piglets (P < 0.05). There was an up-regulation of the adult ISC marker gene of SPARC related modular calcium binding 2 (Smoc2), and Wnt/ß-catenin target genes on d 7 (P < 0.05). These results were further verified in vitro enteroid culture experiments. A mass of hollow spheroids was cultured from the fetal intestine of 0-d-old piglets (P < 0.001), whereas substantial organoids with budding and branching structures were cultured from the intestine of 7-d-old piglets (P < 0.001). The difference was reflected by the organoid budding efficiency, crypt domains per organoid, and the surface area of the organoid. Furthermore, spheroids on d 0 had more Ki67-positive cells and enteroendocrine cells (P < 0.05) and showed a decreasing trend in the ISC and goblet cells (P < 0.10). Moreover, the mRNA expression of spheroids differed markedly from that of organoids, with low expression of intestinal differentiation gene (Lysozyme; P < 0.05), epithelial-specific markers (Villin, E-cadherin; P < 0.05), and adult ISC markers (leucine-rich repeat-containing G protein-coupled receptor 5 [Lgr5], Smoc2; P < 0.001), and up-regulation of fetal marker (connexin 43 [Cnx43]; P < 0.05). The mRNA expression of relevant genes was up-regulated, and involved in Wnt/ß-catenin, epidermal growth factor (EGF), Notch, and bone morphogenetic protein (BMP) signaling on d 7 organoids (P < 0.05). Spheroids displayed low differentiated phenotype and high proliferation, while organoids exhibited strong differentiation potential. These results indicated that the conversion from the fetal progenitors (spheroids) to adult ISC (normal organoids) might largely be responsible for the fast development of intestinal epithelial cells in neonatal piglets.

Dietary Copper Improves Intestinal Morphology via Modulating Intestinal Stem Cell Activity in Pigs.

Copper (Cu) is an essential micronutrient for animals. Many studies have been conducted on the effects of dietary Cu on growth performance, intestinal morphology, and function of piglets. However, the underlying mechanism remains to be explored. Intestinal stem cells (ISC) drive the development and constant renewal of intestinal epithelium. Therefore, we hypothesized that dietary Cu affects piglets' intestinal development via modulating ISC activity. A total of eighty-five 21-day-old piglets were randomly assigned to five groups, where 25, 50, 75, 100, and 125 mg CuSO4/kg on a dry matter basis were supplemented to the basal diet at phase 1 (day 0 to 21). Increasing the dietary Cu concentration decreased (p < 0.05) villus width but increased (p < 0.001) the number of Ki67-positive cells. At phase 2 (day 22 to 163), the other 45 pigs were offered the same diets. Villus height in the 125 mg/kg Cu group was greater (p < 0.001) than in the other groups. Moreover, the effects of Cu on ISC activity in vitro were tested to explore the underlying mechanism. Compared to the control group, 10 µmol/L CuSO4·5H2O increased (p < 0.001) the organoid budding efficiency, crypt depth, and crypts per organoid. Dietary Cu improved the intestinal morphology of finishing pigs via promoting cell proliferation and modulating ISC activity.

Dietary vitamin A affects growth performance, intestinal development, and functions in weaned piglets by affecting intestinal stem cells.

Vitamin A (VA) is an important nutrient for weaning piglets. It plays a significant role in the normal formation, development, and maintenance of epithelial cells. Previous studies have shown that VA supplements could improve the host's intestinal barrier function. Therefore, we hypothesized that VA supplements can affect intestinal function in weaned piglets by regulating intestinal stem cells. Thirty-two 21-d-old weaned [(Yorkshire × Landrace) × Duroc] piglets with an average weight of 8.34 ± 0.13 kg were randomly divided into 4 treatment groups, with 1) 2 mg/kg (control), 2) 4 mg/kg, 3) 8 mg/kg, and 4) 16 mg/kg doses of VA, respectively. The experiment lasted for 14 d. Weaned piglets were given ad libitum access to food and water during the test. The ADG (linear, P = 0.020) and G:F (linear, P = 0.005) of the piglets were found to increase significantly from days 8 to 14. The Lgr5+ gene expression (P = 0.012) in the jejunum mucosa of the 16 mg/kg VA group was increased. The jejunum villus height (P = 0.027) and villi surface area (P = 0.035) were significantly increased in the 4 mg/kg VA treatment group. The crypt depth increased significantly in the 4 and 8 mg/kg VA treatment groups (quadratic, P = 0.043), and the ratios of villus height to crypt depth significantly increased in the 16 mg/kg VA group (quadratic, P = 0.015). The maltase (P = 0.032), sucrose (P = 0.041), and alkaline phosphatase activity (linear, P = 0.024) were significantly increased when further supplemented with 4 mg/kg VA. Slc2a2 mRNA abundance was significantly increased in the 2 mg/kg VA group (linear, P = 0.024). Moreover, the budding rates, buddings number per organoid, and Chromogranin A and Muc2 expression of piglet intestinal organoids were significantly reduced (P < 0.05) by VA and its metabolites (retinoic acid). Compared with the control group, the expression of Spp1 and Trop2 increased. These results indicated that VA may increase the stemness of intestinal stem cell in vitro. This study suggested that VA could affect growth performance and intestinal function by regulating intestinal stem cells in the jejunum of weaned piglets.

Rotavirus Infection and Cytopathogenesis in Human Biliary Organoids Potentially Recapitulate Biliary Atresia Development.

Biliary atresia (BA) is a neonatal liver disease characterized by progressive fibroinflammatory obliteration of both intrahepatic and extrahepatic bile ducts. The etiologies of BA remain largely unknown, but rotavirus infection has been implicated at least for a subset of patients, and this causal relation has been well demonstrated in mouse models. In this study, we aim to further consolidate this evidence in human biliary organoids. We obtained seven batches of human biliary organoids cultured from fetal liver, adult liver, and bile duct tissues. We found that these organoids are highly susceptible and support the full life cycle of rotavirus infection in three-dimensional culture. The robust infection triggers active virus-host interactions, including interferon-based host defense mechanisms and injury responses. We have observed direct cytopathogenesis in organoids upon rotavirus infection, which may partially recapitulate the development of BA. Importantly, we have demonstrated the efficacy of mycophenolic acid and interferon alpha but not ribavirin in inhibiting rotavirus in biliary organoids. Furthermore, neutralizing antibody targeting rotavirus VP7 protein effectively inhibits infection in organoids. Thus, we have substantiated the causal evidence of rotavirus inducing BA in humans and provided potential strategies to combat the disease.IMPORTANCE There is substantial evidence indicating the possible involvement of rotavirus in biliary atresia (BA) development, at least in a subset of patients, but concrete proof remains lacking. In a mouse model, it has been well demonstrated that rotavirus can infect the biliary epithelium to cause biliary inflammation and obstruction, representing the pathogenesis of BA in humans. By using recently developed organoids technology, we now have demonstrated that human biliary organoids are susceptible to rotavirus infection, and this provokes active virus-host interactions and causes severe cytopathogenesis. Thus, our model recapitulates some essential aspects of BA development. Furthermore, we have demonstrated that antiviral drugs and neutralizing antibodies are capable of counteracting the infection and BA-like morphological changes, suggesting their potential for mitigating BA in patients.

Drug screening identifies gemcitabine inhibiting rotavirus through alteration of pyrimidine nucleotide synthesis pathway.

Although rotavirus infection is usually acute and self-limiting, it can cause chronic infection with severe diseases in immunocompromised patients, including organ transplantation recipients and cancer patients irrespective of pediatric or adult patients. Since no approved medication against rotavirus infection is available, this study screened a library of safe-in-man broad-spectrum antivirals. We identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of rotavirus infection. We confirmed this effect in 2D cell cultures and 3D cultured human intestinal organoids with both laboratory-adapted rotavirus strains and five clinical isolates. Supplementation of UTP or uridine largely abolished the anti-rotavirus activity of gemcitabine, suggesting its function through inhibition of pyrimidine biosynthesis pathway. Our results support repositioning of gemcitabine for treating rotavirus infection, especially for infected cancer patients.