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A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Academical Medical Center

To study the differentiation of hepatocytes along the biliary epithelial lineage in vivo, embryonic day 14 (E14) rat hepatocytes were isolated by differential centrifugation and transplanted as single-cell suspensions into the spleen of adult syngeneic rats. Hepatocytes and cholangiocytes were identified and their maturation characterized by the level of expression of alpha-fetoprotein (AFP), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase I (CPS); annexin IV, annexin V, cytokeratin 19 (CK-19), and cystic fibrosis transmembrane conductance regulator (CFTR); and electron microscopy. By correlating morphologic changes with the timing in the expression of these markers, we show that the organization of the transplanted E14 hepatocytes into lobular structures is accompanied by the formation and maturation of bile ducts around these developing lobules. Morphologic differentiation of the emerging bile ducts was accompanied by a gradual loss of hepatocyte markers and a gradual acquisition of cholangiocyte markers, with markers identifying a large-cholangiocyte phenotype appearing latest. Once fully differentiated, the intrasplenic liver lobules developed cholestatic features. The accompanying proliferation of bile ducts was due to cholangiocyte proliferation, but ductular transformation of hepatocytes was also observed. In conclusion, (1) bile duct formation at the interface between hepatocytes and connective tissue is an inherent component of liver development and (2) the susceptibility of developing hepatocytes to bile duct-inducing signals is highest in the fetal liver but that (3) this capacity is not irreversibly lost in otherwise mature hepatocytes.

Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineage.

The expression of glutamine synthetase (GS) in the mammalian liver is confined to the hepatocytes surrounding the central vein and can be induced in cultures of periportal hepatocytes by co-cultivation with the rat-liver epithelial cell line RL-ET-14. We exploited these observations to identify the regulatory regions of the GS gene and the responsible signal-transduction pathway that mediates this effect.

The RL-ET-14 cell line mediates expression of glutamine synthetase through the upstream enhancer/promoter region.

Glutamine synthetase (GS) is the only enzyme that can synthesize glutamine, but it also functions to detoxify glutamate and ammonia. Organs with high cellular concentrations of GS appear to function primarily to remove glutamate or ammonia, whereas those with a low cellular concentration appear to primarily produce glutamine. To validate this apparent dichotomy and to clarify its regulation, we determined the GS concentrations in 18 organs of the mouse. There was a >100-fold difference in GS mRNA, protein, and enzyme-activity levels among organs, whereas there was only a 20-fold difference in the GS protein:mRNA ratio, suggesting extensive transcriptional and posttranscriptional regulation. In contrast, only small differences in the GS enzyme activity : protein ratio were found, indicating that posttranslational regulation is of minor importance. The cellular concentration of GS was determined by relating the relative differences in cellular GS concentration, detected using image analysis of immunohistochemically stained tissue sections, to the biochemical data. There was a >1000-fold difference in cellular concentrations of GS between GS-positive cells in different organs, and cellular concentrations were up to 20x higher in subpopulations of cells within organs than in whole organs. GS activity was highest in pericentral hepatocytes (approximately 485 micromol.g(-1).min-(1), followed in descending order by epithelial cells in the epididymal head, Leydig cells in the testicular interstitium, epithelial cells of the uterine tube, acid-producing parietal cells in the stomach, epithelial cells of the S3 segment of the proximal convoluted tubule of the kidney, astrocytes of the central nervous tissue, and adipose tissue. GS activity in muscle amounted to only 0.4 micromol.g(-1).min(-1). Our findings confirmed the postulated dichotomy between cellular concentration and GS function.

Cellular concentrations of glutamine synthetase in murine organs.

In liver, most genes are expressed with a porto-central gradient. The transcription factor hepatic nuclear-factor4alpha (HNF4alpha) is associated with 12% of the genes in adult liver, but its involvement in zonation of gene expression has not been investigated. A putative HNF4alpha-response element in the upstream enhancer of glutamine synthetase (GS), an exclusively pericentral enzyme, was protected against DNase-I and interacted with a protein that is recognized by HNF4alpha-specific antiserum. Chromatin-immunoprecipitation assays of HNF4alpha-deficient (H4LivKO) and control (H4Flox) livers with HNF4alpha antiserum precipitated the GS upstream enhancer DNA only from H4Flox liver. Identical results were obtained with a histone-deacetylasel (HDAC1) antibody, but antibodies against HDAC3, SMRT and SHP did not precipitate the GS upstream enhancer. In H4Flox liver, GS, ornithine aminotransferase (OAT) and thyroid hormone-receptor beta1 (TRbeta1) were exclusively expressed in pericentral hepatocytes. In H4LivKO liver, this pericentral expression remained unaffected, but the genes were additionally expressed in the periportal hepatocytes, albeit at a lower level. The expression of the periportal enzyme phosphoenolpyruvate carboxykinase had declined in HNF4alpha-deficient hepatocytes. GS-negative cells, which were present as single, large hepatocytes or as groups of small cells near portal veins, did express HNF4alpha. Clusters of very small GS- and HNF4alpha-negative, and PCNA- and OV6-positive cells near portal veins were contiguous with streaks of brightly HNF4alpha-positive, OV6-, PCNA-, and PEPCK-dim cells.

Hepatic HNF4alpha deficiency induces periportal expression of glutamine synthetase and other pericentral enzymes.

STAT5 deficiency does not affect the expression of glutamine synthetase in mouse liver.

The perinatal changes in the pattern of expression of the thyroid hormone receptor (TR) isoforms TRalpha (1) TRalpha (2), TRbeta (1), and TRbeta (2) were investigated using in situ hybridization and immunohistochemistry, and RT-PCR and western blotting as visualization and quantification techniques respectively. In liver, lung, and kidney, TRalpha mRNA was expressed in the stromal and TRbeta mRNA in the parenchymal component of the tissues. When compared with liver, TRalpha mRNA concentrations were tenfold higher in lung, kidney, and intestine, and 100-fold higher in brain, with TRalpha (2) mRNA concentrations exceeding those of TRalpha (1) 5-to 10-fold. Tissue TRbeta (1) mRNA concentrations were similar in liver, lung, and brain, and 3- to 5-fold higher in kidney and intestine. None of the TRbeta (2) mRNA could be detected outside the pituitary. Tissue TRalpha (2) and TRbeta (1) protein levels reached adult levels at 5 days before birth, whereas TRalpha (1) protein peaked after birth. Because of the distinct time-course of thyroid hormone-binding receptors TRalpha (1) and TRbeta (1), we speculate that an initiating, TRbeta (1)-mediated signaling from the parenchyma is followed by a TRalpha (1)-mediated response in the stroma. When compared with organs with a complementary parenchymal-stromal expression pattern, organs with extensive cellular co-expression of TRalpha and TRbeta (brain and intestinal epithelium) were characterized by a very low TRalpha protein: mRNA ratio, implying a low translational efficiency of TR mRNA or a high turnover of TR protein. The data indicate that the TR-dependent regulatory cascades are controlled differently in organs with a complementary tissue expression pattern and in those with cellular co-expression of the TRalpha and TRbeta genes.

Expression of thyroid hormone receptors A and B in developing rat tissues; evidence for extensive posttranscriptional regulation.

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a beta-galactosidase-neomycine fusion gene. GS(+/LacZ) mice have no phenotype, but GS(LacZ/LacZ) mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS(LacZ/LacZ) embryos and GS(GFP/LacZ) ES cells survive in vitro in glutamine-containing medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity.

Glutamine synthetase is essential in early mouse embryogenesis.

Mechanisms underlying hepatic zonation are not completely elucidated. In vitro test systems may provide new insights into current hypotheses. In this study, zonally expressed proteins, i.e. glutamine synthetase (GS; pericentral) and carbamoylphosphate synthetase (CPS; periportal), were tested for their expression patterns in the bioartificial liver of the Academic Medical Center (AMC-BAL).

Expression of glutamine synthetase and carbamoylphosphate synthetase i in a bioartificial liver: markers for the development of zonation in vitro.

Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens.

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes.

The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/fl) mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in astrocytes. "GS-KO/A" mice were born without malformations, did not suffer from seizures, had a suckling reflex, and did drink immediately after birth, but then gradually failed to feed and died on postnatal day 3. Artificial feeding relieved hypoglycemia and prolonged life, identifying starvation as the immediate cause of death. Neuronal morphology and brain energy levels did not differ from controls. Within control brains, amino acid concentrations varied in a coordinate way by postnatal day 2, implying an integrated metabolic network had developed. GS deficiency caused a 14-fold decline in cortical glutamine and a sevenfold decline in cortical alanine concentration, but the rising glutamate levels were unaffected and glycine was twofold increased. Only these amino acids were uncoupled from the metabolic network. Cortical ammonia levels increased only 1.6-fold, probably reflecting reduced glutaminolysis in neurons and detoxification of ammonia to glycine. These findings identify the dramatic decrease in (cortical) glutamine concentration as the primary cause of brain dysfunction in GS-KO/A mice. The temporal dissociation between GS(fl) elimination and death, and the reciprocal changes in the cortical concentration of glutamine and alanine in GS-deficient and control neonates indicate that the phenotype of GS deficiency in the brain emerges coincidentally with the neonatal activation of the glutamine-glutamate and the associated alanine-lactate cycles.

Glutamine synthetase deficiency in murine astrocytes results in neonatal death.

Starvation elicits a complex adaptive response in an organism. No information on transcriptional regulation of metabolic adaptations is available. We, therefore, studied the gene expression profiles of brain, small intestine, kidney, liver, and skeletal muscle in mice that were subjected to 0-72 h of fasting. Functional-category enrichment, text mining, and network analyses were employed to scrutinize the overall adaptation, aiming to identify responsive pathways, processes, and networks, and their regulation. The observed transcriptomics response did not follow the accepted "carbohydrate-lipid-protein" succession of expenditure of energy substrates. Instead, these processes were activated simultaneously in different organs during the entire period. The most prominent changes occurred in lipid and steroid metabolism, especially in the liver and kidney. They were accompanied by suppression of the immune response and cell turnover, particularly in the small intestine, and by increased proteolysis in the muscle. The brain was extremely well protected from the sequels of starvation. 60% of the identified overconnected transcription factors were organ-specific, 6% were common for 4 organs, with nuclear receptors as protagonists, accounting for almost 40% of all transcriptional regulators during fasting. The common transcription factors were PPARα, HNF4α, GCRα, AR (androgen receptor), SREBP1 and -2, FOXOs, EGR1, c-JUN, c-MYC, SP1, YY1, and ETS1. Our data strongly suggest that the control of metabolism in four metabolically active organs is exerted by transcription factors that are activated by nutrient signals and serves, at least partly, to prevent irreversible brain damage.

Interorgan coordination of the murine adaptive response to fasting.

Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice.

Jacqueline L. M. Vermeulen

Tytgat Institute for Liver and Intestinal Research,
Department of Gasteroenterology and Hepatology,
Tytgat Institute for Liver and Intestinal Research, Department of Gasteroenterology and Hepatology

Academical Medical Center

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Jacqueline L. M. Vermeulen

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Academical Medical Center

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Tytgat Institute for Liver and Intestinal Research,
Department of Gasteroenterology and Hepatology,
Tytgat Institute for Liver and Intestinal Research, Department of Gasteroenterology and Hepatology