Organoids: Avatars for Personalized Medicine.

Stem cells are the foundation of all mammalian life. Stem cells build and maintain our bodies throughout life. Two types of stem cells are discerned.1) Embryonic stem cells (ES cells) are briefly present in the early human or mouse embryo, a few days after fertilization. These ES cells can be grown indefinitely in the lab and have the potential to build each and every tissue in our body. Because of this 'pluripotency', ES cells hold great promise for therapeutic application in the field of regenerative medicine. It is also possible to take skin cells (or other cells) from adults and convert these in the lab into cells with ES properties, so called iPS cells. Many of the hurdles that ES cell technology have faced, do not exist for iPS cells.2) Adult stem cells. Every organ in our body is believed to harbor its own dedicated stem cells. These adult stem cells replace tissue that is lost due to wear and tear, trauma and disease. Adult stem cells are highly specialized and can only produce the tissue in which they reside; they are 'multipotent'. Examples are bone marrow stem cells that make all blood cells, skin stem cells and gut stem cells. Even the brain is now known to harbor its specialized stem cells. The adult stem cells allow us to live 80-90 years, but this comes at a cost: they are the cells that most easily transform into cancer cells.Both types of stem cells can be used to establish 'organoids', 3D structures established in a dish, that recapitulate many aspects of the organ they represent. Pluripotent stem cells can be taken through the developmental steps that establish organs during embryogenesis. This has worked particularly well for parts of the the central nervous system, the kidney and GI organs. We have shown that adult epithelial stem cells carrying the generic Lgr5 marker can be cultured under tissue-repair conditions and generate epithelial organoids directly from healthy and diseased organs such as the gut, the liver, the lung and the pancreas. Organoid technology opens a range of avenues for the study of development, physiology and disease, for drug development and for personalized medicine. In the long run, cultured mini-organs may replace transplant organs from donors and hold promise in gene therapy.

Mouse and human urothelial cancer organoids: A tool for bladder cancer research.

Bladder cancer is a common malignancy that has a relatively poor outcome. Lack of culture models for the bladder epithelium (urothelium) hampers the development of new therapeutics. Here we present a long-term culture system of the normal mouse urothelium and an efficient culture system of human bladder cancer cells. These so-called bladder (cancer) organoids consist of 3D structures of epithelial cells that recapitulate many aspects of the urothelium. Mouse bladder organoids can be cultured efficiently and genetically manipulated with ease, which was exemplified by creating genetic knockouts in the tumor suppressors Trp53 and Stag2. Human bladder cancer organoids can be derived efficiently from both resected tumors and biopsies and cultured and passaged for prolonged periods. We used this feature of human bladder organoids to create a living biobank consisting of bladder cancer organoids derived from 53 patients. Resulting organoids were characterized histologically and functionally. Organoid lines contained both basal and luminal bladder cancer subtypes based on immunohistochemistry and gene expression analysis. Common bladder cancer mutations like TP53 and FGFR3 were found in organoids in the biobank. Finally, we performed limited drug testing on organoids in the bladder cancer biobank.

Rectal Organoids Enable Personalized Treatment of Cystic Fibrosis.

In vitro drug tests using patient-derived stem cell cultures offer opportunities to individually select efficacious treatments. Here, we provide a study that demonstrates that in vitro drug responses in rectal organoids from individual patients with cystic fibrosis (CF) correlate with changes in two in vivo therapeutic endpoints. We measured individual in vitro efficaciousness using a functional assay in rectum-derived organoids based on forskolin-induced swelling and studied the correlation with in vivo effects. The in vitro organoid responses correlated with both change in pulmonary response and change in sweat chloride concentration. Receiver operating characteristic curves indicated good-to-excellent accuracy of the organoid-based test for defining clinical responses. This study indicates that an in vitro assay using stem cell cultures can prospectively select efficacious treatments for patients and suggests that biobanked stem cell resources can be used to tailor individual treatments in a cost-effective and patient-friendly manner.

FANTOM5 CAGE profiles of human and mouse samples.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells.

Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1 (SCA-1/LY6A). Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi) identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage-SCA-1+KIT+ (LSK) population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN). We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.

Programs for the persistence, vigilance and control of human CD8+ lung-resident memory T cells.

Tissue-resident memory T cells (TRM cells) in the airways mediate protection against respiratory infection. We characterized TRM cells expressing integrin αE (CD103) that reside within the epithelial barrier of human lungs. These cells had specialized profiles of chemokine receptors and adhesion molecules, consistent with their unique localization. Lung TRM cells were poised for rapid responsiveness by constitutive expression of deployment-ready mRNA encoding effector molecules, but they also expressed many inhibitory regulators, suggestive of programmed restraint. A distinct set of transcription factors was active in CD103+ TRM cells, including Notch. Genetic and pharmacological experiments with mice revealed that Notch activity was required for the maintenance of CD103+ TRM cells. We have thus identified specialized programs underlying the residence, persistence, vigilance and tight control of human lung TRM cells.

Identification of Different Classes of Luminal Progenitor Cells within Prostate Tumors.

Primary prostate cancer almost always has a luminal phenotype. However, little is known about the stem/progenitor properties of transformed cells within tumors. Using the aggressive Pten/Tp53-null mouse model of prostate cancer, we show that two classes of luminal progenitors exist within a tumor. Not only did tumors contain previously described multipotent progenitors, but also a major population of committed luminal progenitors. Luminal cells, sorted directly from tumors or grown as organoids, initiated tumors of adenocarcinoma or multilineage histological phenotypes, which is consistent with luminal and multipotent differentiation potentials, respectively. Moreover, using organoids we show that the ability of luminal-committed progenitors to self-renew is a tumor-specific property, absent in benign luminal cells. Finally, a significant fraction of luminal progenitors survived in vivo castration. In all, these data reveal two luminal tumor populations with different stem/progenitor cell capacities, providing insight into prostate cancer cells that initiate tumors and can influence treatment response.

Identification of multipotent luminal progenitor cells in human prostate organoid cultures.

The prostate gland consists of basal and luminal cells arranged as pseudostratified epithelium. In tissue recombination models, only basal cells reconstitute a complete prostate gland, yet murine lineage-tracing experiments show that luminal cells generate basal cells. It has remained challenging to address the molecular details of these transitions and whether they apply to humans, due to the lack of culture conditions that recapitulate prostate gland architecture. Here, we describe a 3D culture system that supports long-term expansion of primary mouse and human prostate organoids, composed of fully differentiated CK5+ basal and CK8+ luminal cells. Organoids are genetically stable, reconstitute prostate glands in recombination assays, and can be experimentally manipulated. Single human luminal and basal cells give rise to organoids, yet luminal-cell-derived organoids more closely resemble prostate glands. These data support a luminal multilineage progenitor cell model for prostate tissue and establish a robust, scalable system for mechanistic studies.

Retromer dependent recycling of the Wnt secretion factor Wls is dispensable for stem cell maintenance in the mammalian intestinal epithelium.

In C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian intestine, Wnt signaling is essential to maintain stem cells. This prompted us to ask if retromer mediated Wls recycling is also important for Wnt signaling and stem cell maintenance in this system. To answer this question, we generated a conditional Vps35 (fl) allele. As Vps35 is an essential subunit of the retromer complex, this genetic tool allowed us to inducibly interfere with retromer function in the intestinal epithelium. Using a pan-intestinal epithelial Cre line (Villin-CreERT2), we did not observe defects in crypt or villus morphology after deletion of Vps35 from the intestinal epithelium. Wnt secreted from the mesenchyme of the intestine may compensate for a reduction in epithelial Wnt secretion. To exclude the effect of the mesenchyme, we generated intestinal organoid cultures. Loss of Vps35 in intestinal organoids did not affect the overall morphology of the organoids. We were able to culture Vps35 (∆/∆) organoids for many passages without Wnt supplementation in the growth medium. However, Wls protein levels were reduced and we observed a subtle growth defect in the Vps35 (∆/∆) organoids. These results confirm the role of retromer in the retrograde trafficking of Wls in the intestine, but show that retromer mediated Wls recycling is not essential to maintain Wnt signaling or stem cell proliferation in the intestinal epithelium.

DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus.

BACKGROUND: DNA methylation is of pivotal importance during development. Previous genome-wide studies identified numerous differentially methylated regions upon differentiation of stem cells, many of them associated with transcriptional start sites. RESULTS: We present the first genome-wide, single-base-resolution view into DNA methylation dynamics during differentiation of a mammalian epithelial stem cell: the mouse small intestinal Lgr5+ stem cell. Very little change was observed at transcriptional start sites and our data suggest that differentiation-related genes are already primed for expression in the stem cell. Genome-wide, only 50 differentially methylated regions were identified. Almost all of these loci represent enhancers driving gene expression in the differentiated part of the small intestine. Finally, we show that binding of the transcription factor Tcf4 correlates with hypo-methylation and demonstrate that Tcf4 is one of the factors contributing to formation of differentially methylated regions. CONCLUSIONS: Our results reveal limited DNA methylation dynamics during small intestine stem cell differentiation and an impact of transcription factor binding on shaping the DNA methylation landscape during differentiation of stem cells in vivo.

Paneth cells: maestros of the small intestinal crypts.

Paneth cells are highly specialized epithelial cells of the small intestine, where they coordinate many physiological functions. First identified more than a century ago on the basis of their readily discernible secretory granules by routine histology, these cells are located at the base of the crypts of Lieberkühn, tiny invaginations that line the mucosal surface all along the small intestine. Investigations over the past several decades determined that these cells synthesize and secrete substantial quantities of antimicrobial peptides and proteins. More recent studies have determined that these antimicrobial molecules are key mediators of host-microbe interactions, including homeostatic balance with colonizing microbiota and innate immune protection from enteric pathogens. Perhaps more intriguing, Paneth cells secrete factors that help sustain and modulate the epithelial stem and progenitor cells that cohabitate in the crypts and rejuvenate the small intestinal epithelium. Dysfunction of Paneth cell biology contributes to the pathogenesis of chronic inflammatory bowel disease.

The R-spondin protein family.

The four vertebrate R-spondin proteins are secreted agonists of the canonical Wnt/ß-catenin signaling pathway. These proteins are approximately 35 kDa, and are characterized by two amino-terminal furin-like repeats, which are necessary and sufficient for Wnt signal potentiation, and a thrombospondin domain situated more towards the carboxyl terminus that can bind matrix glycosaminoglycans and/or proteoglycans. Although R-spondins are unable to initiate Wnt signaling, they can potently enhance responses to low-dose Wnt proteins. In humans, rare disruptions of the gene encoding R-spondin1 cause a syndrome of XX sex reversal (phenotypic male), palmoplantar keratosis (a thickening of the palms and soles caused by excess keratin formation) and predisposition to squamous cell carcinoma of the skin. Mutations in the gene encoding R-spondin4 cause anonychia (absence or hypoplasia of nails on fingers and toes). Recently, leucine-rich repeat-containing G-protein-coupled receptor (Lgr)4, Lgr5 and Lgr6, three closely related orphans of the leucine-rich repeat family of G-protein-coupled receptors, have been identified as receptors for R-spondins. Lgr5 and Lgr6 are markers for adult stem cells. Because R-spondins are potent stimulators of adult stem cell proliferation in vivo and in vitro, these findings might guide the therapeutic use of R-spondins in regenerative medicine.

The SRY-HMG box gene, SOX4, is a target of gene amplification at chromosome 6p in lung cancer.

The search for oncogenes is becoming increasingly important in cancer genetics because they are suitable targets for therapeutic intervention. To identify novel oncogenes, activated by gene amplification, we analyzed cDNA microarrays by high-resolution comparative genome hybridization and compared DNA copy number and mRNA expression levels in lung cancer cell lines. We identified several amplicons (5p13, 6p22-21, 11q13, 17q21 and 19q13) that had a concomitant increase in gene expression. These regions were also found to be amplified in lung primary tumours. We mapped the boundaries and measured expression levels of genes within the chromosome 6p amplicon. The Sry-HMG box gene SOX4 (sex-determining region Y box 4), which encodes a transcription factor involved in embryonic cell differentiation, was overexpressed by a factor of 10 in cells with amplification relative to normal cells. SOX4 expression was also stronger in a fraction of lung primary tumours and lung cancer cell lines and was associated with the presence of gene amplification. We also found variants of SOX4 in lung primary tumours and cancer cell lines, including a somatic mutation that introduced a premature stop codon (S395X) at the serine-rich C-terminal domain. Although none of the variants increased the transactivation ability of SOX4, overexpression of the wildtype and of the non-truncated variants in NIH3T3 cells significantly increased the transforming ability of the weakly oncogenic RHOA-Q63L. In conclusion, our results show that, in lung cancer, SOX4 is overexpressed due to gene amplification and provide evidence of oncogenic properties of SOX4.

Identification of primary MAFB target genes in multiple myeloma.

OBJECTIVE: In multiple myeloma (MM), seven primary recurrent translocations involving the immunoglobulin heavy chain locus have been identified. One of the partner loci maps to 20q12 and involves the MAFB gene resulting in its ectopic expression. We attempt here to identify MAFB target genes in MM. MATERIALS AND METHODS: We used an inducible system to upregulate MAFB in MM cell lines not carrying the t(14;20). Microarray expression analysis was used to detect gene expression changes upon MAFB expression. These genes were further evaluated comparatively with gene expression profiles obtained from MM or plasma cell leukemia tumors carrying an activated MAFB gene. Functional implications of these upregulated genes were studied by testing their promoter activity in reporter assays. C-MAF was included comparatively as well. RESULTS: The inducible cell lines identified a total of 284 modulated transcripts. After further evaluation using ex vivo data 14 common upregulated genes were found, common to the C-MAF pathway as well. The promoter activity of some of these secondary genes proved a functional relationship with MAFB. In connection with one of these secondary genes (NOTCH2), even tertiary upregulated genes were found. Functional studies indicated that inducible MAFB expression conferred antiapoptotic effects. CONCLUSION: We identified 14 upregulated genes, and their downstream consequences in the combined MAFB/C-MAF pathway. Eleven of these genes are novel in the C-MAF pathway as well. These direct target genes may be responsible for the oncogenic transformation of MAF expressing myeloma cells.

Suppression of tubulin polymerization by the LKB1-microtubule-associated protein/microtubule affinity-regulating kinase signaling.

LKB1, a tumor suppressor gene mutated in the Peutz-Jeghers syndrome, encodes a serine/threonine protein kinase. Recent biochemical studies have shown that LKB1 activates 14 AMP-activated protein kinase-related kinases including MARKs (microtubule-associated protein/microtubule affinity-regulating kinases) that regulate microtubule dynamics. Here we show in vitro that LKB1 phosphorylates and activates MARK2, which in turn phosphorylates microtubule-associated protein Tau at the KXGS motif and suppresses tubulin polymerization. In cells, forced expression of LKB1 suppresses microtubule regrowth, whereas LKB1 knockdown accelerates it. We further show that the phosphorylation of Tau by the LKB1-MARK signaling triggers proteasome-mediated degradation of Tau. These results indicate that LKB1 is involved in the regulation of microtubule dynamics through the activation of MARKs.

Adenomatous polyposis coli-deficient zebrafish are susceptible to digestive tract neoplasia.

Truncation of the tumour suppressor adenomatous polyposis coli (APC) constitutively activates the Wnt/beta-catenin signalling pathway. This event constitutes the primary transforming event in sporadic colorectal cancer in humans. Moreover, humans or mice carrying germline truncating mutations in APC develop large numbers of intestinal adenomas. Here, we report that zebrafish that are heterozygous for a truncating APC mutation spontaneously develop intestinal, hepatic and pancreatic neoplasias that are highly proliferative, accumulate beta-catenin and express Wnt target genes. Treatment with the chemical carcinogen 7,12-dimethylbenz[a]anthracene accelerates the induction of these lesions. These observations establish apc-mutant zebrafish as a bona fide model for the study of digestive tract cancer.

The HMG box transcription factor Sox4 contributes to the development of the endocrine pancreas.

To investigate the role of the Sry/hydroxymethylglutaryl box (Sox) transcription factors in the development of the pancreas, we determined the expression pattern of Sox factors in the developing mouse pancreas. By RT-PCR, we detected the presence of multiple Sox family members in both the developing pancreas and mature islets and then focused on two factors, Sox2 and Sox4. The expression field of Sox2, which plays a role in the maintenance of some stem cell populations, included the developing duodenum, but Sox2 was specifically excluded from the pancreatic buds. In contrast, Sox4 was detected broadly in the early pancreatic buds and eventually became restricted to the nuclei of all islet cells in the adult mouse. Mice homozygous for a null mutation of the sox4 gene showed normal pancreatic bud formation and endocrine cell differentiation up to embryonic day 12.5. Beyond that date, cultured pancreatic explants lacking sox4 failed to form normal islets. Instead, a markedly reduced number of endocrine cells were found scattered through the explant. We show here that several Sox transcription factors are expressed in the developing pancreas and in the islet, and that one of these factors, Sox4, is required for the normal development of pancreatic islets.

WNT signalling and haematopoiesis: a WNT-WNT situation.

The evolutionarily conserved WNT-signalling pathway has pivotal roles during the development of many organ systems, and dysregulated WNT signalling is a key factor in the initiation of various tumours. Recent studies have implicated a role for WNT signal transduction at several stages of lymphocyte development and in the self-renewal of haematopoietic stem cells. Here, we outline new insights into the WNT-signalling pathway, review its role in the self-renewal of haematopoietic stem cells and in the development of T and B cells, and discuss controversies and future developments with regard to WNT signalling in the thymus.