Non-stem cell lineages as an alternative origin of intestinal tumorigenesis in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, has been shown to suppress intestinal stemness. Here, we used Paneth cells as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation in mice. Upon inflammation, Paneth cell-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in patients with inflammatory bowel disease, but also of a larger fraction of human sporadic colon cancers. The latter is possibly because of the inflammatory consequences of western-style dietary habits, a major colon cancer risk factor. Machine learning methods designed to predict the cell-of-origin of cancer from patient-derived tumor samples confirmed that, in a substantial fraction of sporadic cases, the origins of colon cancer reside in secretory lineages and not in stem cells.

Tropomyosin1 isoforms underlie epithelial to mesenchymal plasticity, metastatic dissemination, and resistance to chemotherapy in high-grade serous ovarian cancer.

Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions.

The origin of intestinal cancer in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, was shown to suppress intestinal stemness. Here, we employed Paneth cells (PCs) as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation. Upon inflammation, PC-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in inflammatory bowel disease (IBD) patients but also of a larger fraction of sporadic colon cancers. The latter is likely due to the inflammatory consequences of Western-style dietary habits, the major colon cancer risk factor. Computational methods designed to predict the cell-of-origin of cancer confirmed that, in a substantial fraction of sporadic colon cancers the cells-of-origin are secretory lineages and not stem cells.

3D-Informed Targeting of the Trop-2 Signal-Activation Site Drives Selective Cancer Vulnerability.

Next-generation Trop-2-targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti-Trop-2 mAbs recognize a single immunodominant epitope. Hence, we removed it by deletion mutagenesis. Cancer-specific, glycosylation-prone mAbs were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation site-mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2-expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art complementarity determining region grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd < 10-12 mol/L, and uncleaved/wtTrop-2 in normal cells with Kd 3.16×10-8 mol/L, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2-expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2-targeted therapy.

A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine.

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

Paneth cells as the origin of intestinal cancer in the context of inflammation.

Paneth cells (PCs), responsible for the secretion of antimicrobial peptides in the small intestine and for niche support to Lgr5+ crypt-base columnar stem cells (CBCs), have been shown to respond to inflammation by dedifferentiating into stem-like cells in order to sustain a regenerative response1,2. Therefore, PCs may represent the cells-of-origin of intestinal cancer in the context of inflammation. To test this hypothesis, we targeted Apc, Kras, and Tp53 mutations in Paneth cells by Cre-Lox technology and modelled inflammation by dextran sodium sulfate (DSS) administration. PC-specific loss of Apc resulted in multiple small intestinal tumors, whereas Kras or Tp53 mutations did not. Compound Apc and Kras mutations in PCs resulted in a striking increase in tumor multiplicity even in the absence of the inflammatory insult. By combining scRNAseq with lineage tracing to capture the conversion of PCs into bona fide tumor cells, we show that they progress through a "revival stem cell" (RSC) state characterized by high Clusterin (Clu) expression and Yap1 signaling, reminiscent of what has been previously observed upon irradiation of the mouse digestive tract3. Accordingly, comparison of PC- and Lgr5-derived murine intestinal tumors revealed differences related to Wnt signaling and inflammatory pathways which match the dichotomy of CBCs and injury-induced RSCs4 between human sporadic colon cancers and those arising in the context of inflammatory bowel diseases. Last, we show that western-style dietary habits, known to trigger a low-grade inflammation throughout the intestinal tract, underlie the analogous dedifferentiation of Paneth cells and their acquisition of stem-like features. Taken together, our results show that intestinal cancer arises in the context of inflammation through the dedifferentiation of committed secretory lineages such as Paneth cells and the activation of the revival stem cell state. As such, a true quiescent stem cell identity may be hidden in fully committed and postmitotic lineages which, upon inflammation, support the regenerative response by re-entering the cell cycle and dedifferentiating into RSCs. The chronic nature of the tissue insult in inflammatory bowel diseases and even in the context of western-style dietary habits is likely to result in the expansion of cell targets for tumor initiation and progression.

Alternative splicing downstream of EMT enhances phenotypic plasticity and malignant behavior in colon cancer.

Phenotypic plasticity allows carcinoma cells to transiently acquire the quasi-mesenchymal features necessary to detach from the primary mass and proceed along the invasion-metastasis cascade. A broad spectrum of epigenetic mechanisms is likely to cause the epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) transitions necessary to allow local dissemination and distant metastasis. Here, we report on the role played by alternative splicing (AS) in eliciting phenotypic plasticity in epithelial malignancies with focus on colon cancer. By taking advantage of the coexistence of subpopulations of fully epithelial (EpCAMhi) and quasi-mesenchymal and highly metastatic (EpCAMlo) cells in conventional human cancer cell lines, we here show that the differential expression of ESRP1 and other RNA-binding proteins (RBPs) downstream of the EMT master regulator ZEB1 alters the AS pattern of a broad spectrum of targets including CD44 and NUMB, thus resulting in the generation of specific isoforms functionally associated with increased invasion and metastasis. Additional functional and clinical validation studies indicate that both the newly identified RBPs and the CD44s and NUMB2/4 splicing isoforms promote local invasion and distant metastasis and are associated with poor survival in colon cancer. The systematic elucidation of the spectrum of EMT-related RBPs and AS targets in epithelial cancers, apart from the insights in the mechanisms underlying phenotypic plasticity, will lead to the identification of novel and tumor-specific therapeutic targets.

Effectiveness and Quality of Life with Paliperidone Palmitate 3-Monthly in Comparison with Other Long-Acting Drugs.

Purpose: Antipsychotic long-acting injections (AP-LAIs) are indicated for patients affected by schizophrenia especially those with poor treatment adherence. Patients and Methods: To compare paliperidone palmitate 3-monthly (PP3M), paliperidone palmitate one-monthly (PP1M) and haloperidol decanoate (HAL-D) treatment, we enrolled 90 patients with schizophrenia treated in Mental Health Center with one of the three AP-LAIs for at least six months and followed them for another 6 months. At 6 and 12 months of treatment we administered Clinical Global Impression-Severity, Global Assessment of Functioning and World Health Organization Quality of Life-26 items (WHOQOL-BREF). At 1-year treatment, we evaluated relapses (psychiatric hospitalizations and urgent consultations), side effects and drop-outs. Results: We did not highlight any statistically significant difference among the three treatments in relapses and scale scores. Weight increase was significantly higher in PP1M and PP3M groups. Twelve patients (13.3%) discontinued AP-LAI. At 1-year AP-LAI treatment, 69% of patients rated quality of life as "good" or "very good" and 71% declared themselves to be "satisfied" or "very satisfied". Conclusion: HAL-D, PP1M and PP3M 1-year treatments were similarly effective in preventing relapses and improving quality of life and health satisfaction. All discontinuations in the new 3-monthly antipsychotic treatment were caused by patient refusal to continue it.

In desmoid-type fibromatosis cells sorafenib induces ferroptosis and apoptosis, which are enhanced by autophagy inhibition.

INTRODUCTION: Desmoid-type fibromatosis (DTF) is a rare, soft tissue tumour. Sorafenib, a multikinase inhibitor, has demonstrated antitumour efficacy in DTF patients. Little is known about the underlying molecular mechanisms, which are crucial to know to further optimize systemic treatments. Here we investigated the molecular effects of sorafenib exposure on DTF and stromal cells, with an emphasis on cell death mechanisms. MATERIAL AND METHODS: DTF primary cell cultures, with known CTNNB1 status, and primary stromal cell cultures, derived from DTF tissue, were exposed to clinically relevant concentrations of sorafenib in the presence or absence of inhibitors of ferroptosis, apoptosis and autophagy. Cell viability was determined after 24 and 48 h using MTT assays. Annexin V/PI staining, lipid peroxidation analysis and immunoblotting were performed to assess apoptosis, ferroptosis and autophagy. RESULTS: Exposure to sorafenib caused a significant, concentration- and time-dependent decrease in cell viability in all primary DTF and stromal cell cultures. Inhibitors of ferroptosis and apoptosis protected against sorafenib-mediated cytotoxicity implicating that both cell death mechanisms are activated. Annexin V/PI stainings and lipid peroxidation analyses confirmed induction of apoptosis and ferroptosis, respectively. Autophagy inhibition enhanced the cytotoxic effect of sorafenib and led to a stronger induction of apoptosis and ferroptosis. CONCLUSION: This study identified ferroptosis and apoptosis as mechanisms for the sorafenib induced cell death in DTF cells as well as stromal cells. Furthermore, autophagy inhibition enhanced the cytotoxic effects of sorafenib. Knowledge of the mechanisms by which sorafenib affects DTF at a cellular level may help to optimize its clinical efficacy and mitigate toxic effects.

Trop-2, Na+/K+ ATPase, CD9, PKCα, cofilin assemble a membrane signaling super-complex that drives colorectal cancer growth and invasion.

Trop-2 is a transmembrane signal transducer that is overexpressed in most human cancers, and drives malignant progression. To gain knowledge on the higher-order molecular mechanisms that drive Trop-2 signaling, we applied next-generation sequencing, proteomics, and high-resolution microscopy to models and primary cases of human colorectal cancer (CRC). We had previously shown that Trop-2 induces a Ca2+ signal. We reveal here that Trop-2 binds the cell membrane Na+/K+-ATPase, and that clustering of Trop-2 induces an intracellular Ca2+ rise followed by membrane translocation of PKCα, which in turn phosphorylates the Trop-2 cytoplasmic tail. This feed-forward signaling is promoted by the binding of Trop-2 to the PKCα membrane-anchor CD9. CRISPR-based inactivation of CD9 in CRC cells shows that CD9 is required by Trop-2 for recruiting PKCα and cofilin-1 to the cell membrane. This induces malignant progression through proteolytic cleavage of E-cadherin, remodeling of the ß-actin cytoskeleton, and activation of Akt and ERK. The interaction between Trop-2 and CD9 was validated in vivo in murine models of CRC growth and invasion. Overexpression of the components of this Trop-2-driven super-complex significantly worsened disease-free and overall survival of CRC patients, supporting a pivotal relevance in CRC malignant progression. Our findings demonstrate a previously unsuspected layer of cancer growth regulation, which is dormant in normal tissues, and is activated by Trop-2 in cancer cells.

Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer.

Phenotypic plasticity represents the most relevant hallmark of the carcinoma cell as it bestows it with the capacity of transiently altering its morphological and functional features while en route to the metastatic site. However, the study of phenotypic plasticity is hindered by the rarity of these events within primary lesions and by the lack of experimental models. Here, we identified a subpopulation of phenotypic plastic colon cancer cells: EpCAMlo cells are motile, invasive, chemo-resistant, and highly metastatic. EpCAMlo bulk and single-cell RNAseq analysis indicated (1) enhanced Wnt/ß-catenin signaling, (2) a broad spectrum of degrees of epithelial to mesenchymal transition (EMT) activation including hybrid E/M states (partial EMT) with highly plastic features, and (3) high correlation with the CMS4 subtype, accounting for colon cancer cases with poor prognosis and a pronounced stromal component. Of note, a signature of genes specifically expressed in EpCAMlo cancer cells is highly predictive of overall survival in tumors other than CMS4, thus highlighting the relevance of quasi-mesenchymal tumor cells across the spectrum of colon cancers. Enhanced Wnt and the downstream EMT activation represent key events in eliciting phenotypic plasticity along the invasive front of primary colon carcinomas. Distinct sets of epithelial and mesenchymal genes define transcriptional trajectories through which state transitions arise. pEMT cells, often earmarked by the extracellular matrix glycoprotein SPARC together with nuclear ZEB1 and ß-catenin along the invasive front of primary colon carcinomas, are predicted to represent the origin of these (de)differentiation routes through biologically distinct cellular states and to underlie the phenotypic plasticity of colon cancer cells.

Circulating angiogenic cells in glioblastoma: toward defining crucial functional differences in CAC-induced neoplastic versus reactive neovascularization.

BACKGROUND: In order to identify suitable therapeutic targets for glioma anti-angiogenic therapy, the process of neovascularization mediated by circulating angiogenic cells (CACs) needs to be scrutinized. METHODS: In the present study, we compared the expression of neovascularization-related genes by 3 circulating CAC subsets (hematopoietic progenitor cells [HPCs], CD34+, and KDR+ cells; internal controls: peripheral blood mononuclear cells and circulating endothelial cells) of treatment-naïve patients with glioblastoma (GBM) to those of patients undergoing reactive neovascularization (myocardial infarction (MI). CACs from umbilical cord (representing developmental neovascularization) and healthy subjects served as controls. Fluorescent-activated cell sorting was used to isolate CACs, RT-PCR to determine the expression levels of a panel of 48 neovascularization-related genes, and Luminex assays to measure plasma levels of 21 CAC-related circulating molecules. RESULTS: We found essential differences in gene expression between GBM and MI CACs. GBM CACs had a higher expression of proangiogenic factors (especially, KITL, CXCL12, and JAG1), growth factor and chemotactic receptors (IGF1R, TGFBR2, CXCR4, and CCR2), adhesion receptor monomers (ITGA5 and ITGA6), and matricellular factor POSTN. In addition, we found major differences in the levels of neovascularization-related plasma factors. A strong positive correlation between plasma MMP9 levels and expression of CXCR4 in the CAC subset of HPCs was found in GBM patients. CONCLUSIONS: Our findings indicate that CAC-mediated neovascularization in GBM is characterized by more efficient CAC homing to target tissue and a more potent proangiogenic response than in physiologic tissue repair in MI. Our findings can aid in selecting targets for therapeutic strategies acting against GBM-specific CACs.

Preliminary results of a multidisciplinary Italian study adopting a Psycho-Neuro-Endocrine-Immunological (PNEI) approach to the study of colorectal adenomas.

Background and aim of the work Colorectal mucosal precancerous lesions, metabolic syndrome (MetS) and psychiatric disorders may share a common low-grade local and systemic inflammation. Aim is to report on preliminary data concerning a research adopting a psycho-neuro-endocrine-immune (PNEI) approach to study outpatients undergoing colonoscopy. Methods A sample of patients undergoing colonoscopy was cross-sectionally investigated. Data on colorectal adenomas, MetS, early atherosclerosis, anxious-depressive symptoms, personality traits, and inflammatory markers were statistically analyzed. Results Sixty-two patients were recruited (female 50%, mean age: 60.8±9.4 years). The prevalence of adenomas and MetS was respectively of 45.2% and 41.9%. Anxiety and depressive symptoms were detected in 16 (32.7%) and 9 (18.4%) subjects, respectively. The presence of adenomas positively correlated with male sex (p=0.01), age (p<0.01), IL-6 (p=0.03), hsCRP (p=0.04), and MetS (p=0.03); it was also associated with hsCRP concentration (aOR=3.81, p=0.03). Conclusions Proinflammatory atherogenic status, psychological traits, increased mucosal inflammation, and metabolic parameters may share a common a pathogenic mechanism, worth studying.

Ectopic activation of WNT signaling in human embryonal carcinoma cells and its effects in short- and long-term in vitro culture.

Human embryonal carcinoma (EC) cells comprise the pluripotent stem cells of malignant non-seminomatous germ cell tumors (GCTs) and represent the malignant counterpart of embryonic stem cells (ESCs). WNT/ß-catenin signaling has been implicated in regulating adult and embryonic stem cells although its role in EC cells is less investigated. Here, we studied WNT signaling in a panel of representative pluripotent and nullipotent human EC cell lines. We found that EC cell lines show distinct levels of intrinsic WNT signaling and respond differently to ectopic WNT activation. Short-term activation of WNT signaling induced a differentiation-response in the pluripotent EC cells (NT2 and NCCIT) whereas the nullipotent EC cells (TERA1 and 2102Ep) were refractory and maintained high levels of OCT4 and SSEA4 expression. Long-term activation of WNT signaling in NCCIT and, to a lesser extent, TERA1 cells led to (re)gain of OCT4 expression and a switch from SSEA4 to SSEA1 surface antigens ultimately resulting in OCT4+/SSEA4-/SSEA1+ profile. Cisplatin treatment indicated that the OCT4+/SSEA4-/SSEA1+ NCCIT cells became more resistant to chemotherapy treatment. Our findings are of particular interest for the GCT and ES cell biology and shed light on the role of WNT signaling in human EC cells.

Circulating Proangiogenic Cells and Proteins in Patients with Glioma and Acute Myocardial Infarction: Differences in Neovascularization between Neoplasia and Tissue Regeneration.

Although extensive angiogenesis takes place in glial tumors, antiangiogenic therapies have remained without the expected success. In the peripheral circulation of glioma patients, increased numbers of endothelial precursor cells (EPCs) are present, potentially offering targets for antiangiogenic therapy. However, for an antiangiogenic therapy to be successful, the therapy should specifically target glioma-related EPC subsets and secreted factors only. Here, we compared the EPC subsets and plasma factors in the peripheral circulation of patients with gliomas to acute myocardial infarctions. We investigated the five most important EPC subsets and 21 angiogenesis-related plasma factors in peripheral blood samples of 29 patients with glioma, 14 patients with myocardial infarction, and 20 healthy people as controls, by FACS and Luminex assay. In GBM patients, all EPC subsets were elevated as compared to healthy subjects. In addition, HPC and KDR+ cell fractions were higher than in MI, while CD133+ and KDR+CD133+ cell fractions were lower. There were differences in relative EPC fractions between the groups: KDR+ cells were the largest fraction in GBM, while CD133+ cells were the largest fraction in MI. An increase in glioma malignancy grade coincided with an increase in the KDR+ fraction, while the CD133+ cell fraction decreased relatively. Most plasma angiogenic factors were higher in GBM than in MI patients. In both MI and GBM, the ratio of CD133+ HPCs correlated significantly with elevated levels of MMP9. In the GBM patients, MMP9 correlated strongly with levels of all HPCs. In conclusion, the data demonstrate that EPC traffic in patients with glioma, representing neoplasia, is different from that in myocardial infarction, representing tissue regeneration. Glioma patients may benefit from therapies aimed at lowering KDR+ cells and HPCs.

Paneth Cells Respond to Inflammation and Contribute to Tissue Regeneration by Acquiring Stem-like Features through SCF/c-Kit Signaling.

IBD syndromes such as Crohn's disease and ulcerative colitis result from the inflammation of specific intestinal segments. Although many studies have reported on the regenerative response of intestinal progenitor and stem cells to tissue injury, very little is known about the response of differentiated lineages to inflammatory cues. Here, we show that acute inflammation of the mouse small intestine is followed by a dramatic loss of Lgr5+ stem cells. Instead, Paneth cells re-enter the cell cycle, lose their secretory expression signature, and acquire stem-like properties, thus contributing to the tissue regenerative response to inflammation. Stem cell factor secretion upon inflammation triggers signaling through the c-Kit receptor and a cascade of downstream events culminating in GSK3ß inhibition and Wnt activation in Paneth cells. Hence, the plasticity of the intestinal epithelium in response to inflammation goes well beyond stem and progenitor cells and extends to the fully differentiated and post-mitotic Paneth cells.

Metabolic Syndrome, Anxiety and Depression in a Sample of Italian Primary Care Patients.

This cross-sectional study aimed at measuring the correlation and association of anxiety, depression and comorbid anxiety-depression symptoms with metabolic syndrome (MetS) in a sample of Italian primary care patients who attended their General Practitioner clinics over a 1-month period in 2013. The Hospital Anxiety and Depression Scale (HADS) was used to assess anxiety and depressive symptoms. The sample was made up of 129 patients (57% women; mean age, 61 ± 12 years). The prevalence of MetS varied from 40% (Adult Treatment Panel III-Revised criteria) to 48% (International Diabetes Federation criteria). The prevalence of symptoms of anxiety, depression and comorbid anxiety and depression was, respectively, 26%, 2%, and 15%. MetS (defined according to Adult Treatment Panel III-Revised criteria) was associated with comorbid anxiety-depressive symptoms (odds ratio [OR] = 3.84, 95% confidence interval [CI] = 1.26-11.71), but not with anxiety or depressive symptoms only. Out of the individual components of MetS, enlarged waist circumference was associated with anxiety symptoms (OR = 4.22, 95% CI = 1.56-11.44).

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberkühn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12. When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells. Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as "organoid reconstitution assay" (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol.

The characterization of circulating endothelial progenitor cells (EPCs) is fundamental to any study related to angiogenesis. Unfortunately, current literature lacks consistency in the definition of EPC subsets due to variations in isolation strategies and inconsistencies in the use of lineage markers. Here we address critical points in the identification of hematopoietic progenitor cells (HPCs), circulating endothelial cells (CECs), and culture-generated outgrowth endothelial cells (OECs) from blood samples of healthy adults (AB) and umbilical cord (UCB). Peripheral blood mononuclear cells (PBMCs) were enriched using a Ficoll-based gradient followed by an optimized staining and gating strategy to enrich for the target cells. Sorted EPC populations were subjected to RT-PCR for tracing the expression of markers beyond the limits of cell surface-based immunophenotyping. Using CD34, CD133 and c-kit staining, combined with FSC and SSC, we succeeded in the accurate and reproducible identification of four HPC subgroups and found significant differences in the respective populations in AB vs. UCB. Co-expression analysis of endothelial markers on HPCs revealed a complex pattern characterized by various subpopulations. CECs were identified by using CD34, KDR, CD45, and additional endothelial markers, and were subdivided according to their apoptotic state and expression of c-kit. Comparison of UCB-CECs vs. AB-CECs revealed significant differences in CD34 and KDR levels. OECs were grown from PBMC-fractions We found that viable c-kit+ CECs are a candidate circulating precursor for CECs. RT-PCR to angiogenic factors and receptors revealed that all EPC subsets expressed angiogenesis-related molecules. Taken together, the improvements in immunophenotyping and gating strategies resulted in accurate identification and comparison of better defined cell populations in a single procedure.

Bifurcation trees of Stark-Wannier ladders for accelerated Bose-Einstein condensates in an optical lattice.

In this paper we show that in the semiclassical regime of periodic potential large enough, the Stark-Wannier ladders become a dense energy spectrum because of a cascade of bifurcations while increasing the ratio between the effective nonlinearity strength and the tilt of the external field; this fact is associated to a transition from regular to quantum chaotic dynamics. The sequence of bifurcation points is explicitly given.