Galectin-9 and Interferon-Gamma Are Released by Natural Killer Cells upon Activation with Interferon-Alpha and Orchestrate the Suppression of Hepatitis C Virus Infection.

Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and can be activated by several cytokines, including interleukin-2 (IL-2), IL-15, and interferon-alpha (IFN-α). By exploiting the Huh7.5 hepatoma cell line infected with the HCV JFH1 genome, we provide novel insights into the antiviral effector functions of human primary NK cells after cytokine stimulation. NK cells activated with IFN-α (IFNα-NKs) had enhanced contact-dependent and -independent responses as compared with NK cells activated with IL-2/IL-15 (IL2/IL15-NKs) and could inhibit HCV replication both in vitro and in vivo. Importantly, IFN-α, but not IL-2/IL-15, protected NK cells from the functional inhibition exerted by HCV. By performing flow cytometry, multiplex cytokine profiling, and mass-spectrometry-based proteomics, we discovered that IFNα-NKs secreted high levels of galectin-9 and interferon-gamma (IFN-γ), and by conducting neutralization assays, we confirmed the major role of these molecules in HCV suppression. We speculated that galectin-9 might act extracellularly to inhibit HCV binding to host cells and downstream infection. In silico approaches predicted the binding of HCV envelope protein E2 to galectin-9 carbohydrate-recognition domains, and co-immunoprecipitation assays confirmed physical interaction. IFN-γ, on the other hand, triggered the intracellular expressions of two antiviral gate-keepers in target cells, namely, myxovirus-1 (MX1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Collectively, our data add more complexity to the antiviral innate response mediated by NK cells and highlight galectin-9 as a key molecule that might be exploited to neutralize productive viral infection.

Moving Towards Induced Pluripotent Stem Cell-based Therapies with Artificial Intelligence and Machine Learning.

The advent of induced pluripotent stem cell (iPSC) technology, which allows to transform one cell type into another, holds the promise to produce therapeutic cells and organs on demand. Realization of this objective is contingent on the ability to demonstrate quality and safety of the cellular product for its intended use. Bottlenecks and backlogs to the clinical use of iPSCs have been fully outlined and a need has emerged for safer and standardized protocols to trigger cell reprogramming and functional differentiation. Amidst great challenges, in particular associated with lengthy culture time and laborious cell characterization, a demand for faster and more accurate methods for the validation of cell identity and function at different stages of the iPSC manufacturing process has risen. Artificial intelligence-based methods are proving helpful for these complex tasks and might revolutionize the way iPSCs are managed to create surrogate cells and organs. Here, we briefly review recent progress in artificial intelligence approaches for evaluation of iPSCs and their derivatives in experimental studies.

Fat-associated lymphoid clusters as expandable niches for ectopic liver development.

BACKGROUND AND AIMS: Hepatocyte transplantation holds great promise as an alternative approach to whole-organ transplantation. Intraportal and intrasplenic cell infusions are primary hepatocyte transplantation delivery routes for this procedure. However, patients with severe liver diseases often have disrupted liver and spleen architectures, which introduce risks in the engraftment process. We previously demonstrated i.p. injection of hepatocytes as an alternative route of delivery that could benefit this subpopulation of patients, particularly if less invasive and low-risk procedures are required; and we have established that lymph nodes may serve as extrahepatic sites for hepatocyte engraftment. However, whether other niches in the abdominal cavity support the survival and proliferation of the transplanted hepatocytes remains unclear. APPROACH AND RESULTS: Here, we showed that hepatocytes transplanted by i.p. injection engraft and generate ectopic liver tissues in fat-associated lymphoid clusters (FALCs), which are adipose tissue-embedded, tertiary lymphoid structures localized throughout the peritoneal cavity. The FALC-engrafted hepatocytes formed functional ectopic livers that rescued tyrosinemic mice from liver failure. Consistently, analyses of ectopic and native liver transcriptomes revealed a selective ectopic compensatory gene expression of hepatic function-controlling genes in ectopic livers, implying a regulated functional integration between the two livers. The lack of FALCs in the abdominal cavity of immunodeficient tyrosinemic mice hindered ectopic liver development, whereas the restoration of FALC formation through bone marrow transplantation restored ectopic liver development in these mice. Accordingly, induced abdominal inflammation increased FALC numbers, which improved hepatocyte engraftment and accelerated the recovery of tyrosinemic mice from liver failure. CONCLUSIONS: Abdominal FALCs are essential extrahepatic sites for hepatocyte engraftment after i.p. transplantation and, as such, represent an easy-to-access and expandable niche for ectopic liver regeneration when adequate growth stimulus is present.

Zika Virus: A New Therapeutic Candidate for Glioblastoma Treatment.

Glioblastoma (GBM) is the most aggressive among the neurological tumors. At present, no chemotherapy or radiotherapy regimen is associated with a positive long-term outcome. In the majority of cases, the tumor recurs within 32-36 weeks of initial treatment. The recent discovery that Zika virus (ZIKV) has an oncolytic action against GBM has brought hope for the development of new therapeutic approaches. ZIKV is an arbovirus of the Flaviviridae family, and its infection during development has been associated with central nervous system (CNS) malformations, including microcephaly, through the targeting of neural stem/progenitor cells (NSCs/NPCs). This finding has led various groups to evaluate ZIKV's effects against glioblastoma stem cells (GSCs), supposedly responsible for GBM onset, progression, and therapy resistance. While preliminary data support ZIKV tropism toward GSCs, a more accurate study of ZIKV mechanisms of action is fundamental in order to launch ZIKV-based clinical trials for GBM patients.

Generation of Hepatobiliary Cell Lineages from Human Induced Pluripotent Stem Cells: Applications in Disease Modeling and Drug Screening.

The possibility to reproduce key tissue functions in vitro from induced pluripotent stem cells (iPSCs) is offering an incredible opportunity to gain better insight into biological mechanisms underlying development and disease, and a tool for the rapid screening of drug candidates. This review attempts to summarize recent strategies for specification of iPSCs towards hepatobiliary lineages -hepatocytes and cholangiocytes-and their use as platforms for disease modeling and drug testing. The application of different tissue-engineering methods to promote accurate and reliable readouts is discussed. Space is given to open questions, including to what extent these novel systems can be informative. Potential pathways for improvement are finally suggested.

Ex Vivo Cell Therapy by Ectopic Hepatocyte Transplantation Treats the Porcine Tyrosinemia Model of Acute Liver Failure.

The effectiveness of cell-based therapies to treat liver failure is often limited by the diseased liver environment. Here, we provide preclinical proof of concept for hepatocyte transplantation into lymph nodes as a cure for liver failure in a large-animal model with hereditary tyrosinemia type 1 (HT1), a metabolic liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) enzyme. Autologous porcine hepatocytes were transduced ex vivo with a lentiviral vector carrying the pig Fah gene and transplanted into mesenteric lymph nodes. Hepatocytes showed early (6 h) and durable (8 months) engraftment in lymph nodes, with reproduction of vascular and hepatic microarchitecture. Subsequently, hepatocytes migrated to and repopulated the native diseased liver. The corrected cells generated sufficient liver mass to clinically ameliorate the acute liver failure and HT1 disease as early as 97 days post-transplantation. Integration site analysis defined the corrected hepatocytes in the liver as a subpopulation of hepatocytes from lymph nodes, indicating that the lymph nodes served as a source for healthy hepatocytes to repopulate a diseased liver. Therefore, ectopic transplantation of healthy hepatocytes cures this pig model of liver failure and presents a promising approach for the development of cures for liver disease in patients.

Host Lymphotoxin-ß Receptor Signaling Is Crucial for Angiogenesis of Metanephric Tissue Transplanted into Lymphoid Sites.

The mouse lymph node (LN) can provide a niche to grow metanephric kidney to maturity. Here, we show that signaling through the lymphotoxin-ß receptor (LTßR) is critical for kidney organogenesis both in the LN and the omentum. By transplanting kidney rudiments either in the LNs of mice undergoing LTßR antagonist treatment or in the omenta of Ltbr knockout (Ltbr-/-) mice, the host LTßR signals were found to be crucial for obtaining a well-vascularized kidney graft. Indeed, defective LTßR signaling correlated with decreased expression of endothelial and angiogenic markers in kidney grafts as well as structural alterations. Because the number of glomerular endothelial cells expressing the LTßR target nuclear factor κB-inducing kinase (NIK) decreased in the absence of a functional LTßR, it was speculated that an LTßR/NIK axis mediated the angiogenetic signals required for successful ectopic kidney organogenesis, given the established role of NIK in neovascularization. However, the transplantation of kidney rudiments in omenta of Nik-/- mice revealed that NIK is dispensable for ectopic kidney vascular integration and maturation. Finally, defective LTßR signaling impaired compensatory glomerular adaptation to renal mass reduction, indicating that kidney regeneration approaches, besides whole kidney reconstruction, might benefit from the presence of LTßR signals.

Kidney-in-a-lymph node: A novel organogenesis assay to model human renal development and test nephron progenitor cell fates.

Stem cell-derived organoids are emerging as sophisticated models for studying development and disease and as potential sources for developing organ substitutes. Unfortunately, although organoids containing renal structures have been generated from mouse and human pluripotent stem cells, there are still critical unanswered questions that are difficult to attain via in vitro systems, including whether these nonvascularized organoids have a stable and physiologically relevant phenotype or whether a suitable transplantation site for long-term in vivo studies can be identified. Even orthotopic engraftment of organoid cultures in the adult does not provide an environment conducive to vascularization and functional differentiation. Previously, we showed that the lymph node offers an alternative transplantation site where mouse metanephroi can differentiate into mature renal structures with excretory, homeostatic, and endocrine functions. Here, we show that the lymph node lends itself well as a niche to also grow human primary kidney rudiments and can additionally be viewed as a platform to interrogate emerging renal organoid cultures. Our study has a wide-ranging impact for tissue engineering approaches to rebuild functional tissues in vivo including-but not limited to-the kidney.

Establishment and Characterization of 5-Fluorouracil-Resistant Human Colorectal Cancer Stem-Like Cells: Tumor Dynamics under Selection Pressure.

5-Fluorouracil (5-FU) remains the gold standard of first-line treatment for colorectal cancer (CRC). Although it may initially debulk the tumor mass, relapses frequently occur, indicating the existence of cancer cells that are therapy-resistant and are capable of refueling tumor growth. To identify mechanisms of drug resistance, CRC stem-like cells were subjected to long-term 5-FU selection using either intermittent treatment regimen with the IC50 drug dose or continuous treatment regimen with escalating drug doses. Parental cancer cells were cultivated in parallel. Real-time PCR arrays and bioinformatic tools were used to investigate gene expression changes. We found the first method selected for cancer cells with more aggressive features. We therefore transplanted these cancer cells or parental cells in mice, and again, found that not only did the 5-FU-selected cancer cells generate more aggressive tumors with respect to their parental counterpart, but they also showed a different gene expression pattern as compared to what we had observed in vitro, with ID1 the top upregulated gene. We propose ID1 as a stemness marker pervasively expressed in secondary lesions emerging after completion of chemotherapy.

Regenerating a kidney in a lymph node.

The ultimate treatment for end-stage renal disease (ESRD) is orthotopic transplantation. However, the demand for kidney transplantation far exceeds the number of available donor organs. While more than 100,000 Americans need a kidney, only 17,000 people receive a kidney transplant each year (National Kidney Foundation's estimations). In recent years, several regenerative medicine/tissue engineering approaches have been exploited to alleviate the kidney shortage crisis. Although these approaches have yielded promising results in experimental animal models, the kidney is a complex organ and translation into the clinical realm has been challenging to date. In this review, we will discuss cell therapy-based approaches for kidney regeneration and whole-kidney tissue engineering strategies, including our innovative approach to regenerate a functional kidney using the lymph node as an in vivo bioreactor.

Therapeutic potential of mTOR inhibitors for targeting cancer stem cells.

The mammalian target of rapamycin (mTOR) pathway is aberrantly activated in many cancer types. As the intricate network of regulatory mechanisms controlling mTOR activity is uncovered, more refined drugs are designed and tested in clinical trials. While first generation mTOR inhibitors have failed to show clinical efficacy due partly to the feedback relief of oncogenetic circuits, newly developed inhibitors show greater promise as anti-cancer agents. An effective drug must defeat the cancer stem cells (CSCs) while sparing the normal stem cells. Due to its opposing role on normal and malignant stem cells, mTOR lends itself very well as a therapeutic target. Indeed, a preferential inhibitory effect on CSCs has already been shown for some mTOR inhibitors. These results provide a compelling rationale for the clinical development of mTOR-targeted therapies.

Towards Organs on Demand: Breakthroughs and Challenges in Models of Organogenesis.

In recent years, functional three-dimensional (3D) tissue generation in vitro has been significantly advanced by tissue-engineering methods, achieving better reproduction of complex native organs compared to conventional culture systems. This review will discuss traditional 3D cell culture techniques as well as newly developed technology platforms. These recent techniques provide new possibilities in the creation of human body parts and provide more accurate predictions of tissue response to drug and chemical challenges. Given the rapid advancement in the human induced pluripotent stem cell (iPSC) field, these platforms also hold great promise in the development of patient-specific, transplantable tissues and organs on demand.

Pluripotent Stem Cells to Rebuild a Kidney: The Lymph Node as a Possible Developmental Niche.

Kidney disease poses a global challenge. Stem cell therapy may offer an alternative therapeutic approach to kidney transplantation, which is often hampered by the limited supply of donor organs. While specific surface antigen markers have yet to be identified for the analysis and purification of kidney stem/progenitor cells for research or clinical use, the reprogramming of somatic cells to pluripotent cells and their differentiation into the various kidney lineages might represent a valuable strategy to create a renewable cell source for regenerative purposes. In this review, we first provide an overview of kidney development and explore current knowledge about the role of extra- and intrarenal cells in kidney repair and organogenesis. We then discuss recent advances in the 1) differentiation of rodent and human embryonic stem cells (ESCs) into renal lineages; 2) generation of induced pluripotent stem cells (iPSCs) from renal or nonrenal (kidney patient-derived) adult cells; 3) differentiation of iPSCs into renal lineages; and 4) direct transcriptional reprogramming of adult renal cells into kidney progenitor cells. Finally, we describe the lymph node as a potential three-dimensional (3D) in vivo environment for kidney organogenesis from pluripotent stem cells.

The lymph node as a new site for kidney organogenesis.

The shortage of organs for kidney transplantation has created the need to develop new strategies to restore renal structure and function. Given our recent finding that the lymph node (LN) can serve as an in vivo factory to generate or sustain complex structures like liver, pancreas, and thymus, we investigated whether it could also support kidney organogenesis from mouse renal embryonic tissue (metanephroi). Here we provide the first evidence that metanephroi acquired a mature phenotype upon injection into LN, and host cells likely contributed to this process. Urine-like fluid-containing cysts were observed in several grafts 12 weeks post-transplantation, indicating metanephroi transplants' ability to excrete products filtered from the blood. Importantly, the kidney graft adapted to a loss of host renal mass, speeding its development. Thus, the LN might provide a unique tool for studying the mechanisms of renal maturation, cell proliferation, and fluid secretion during cyst development. Moreover, we provide evidence that inside the LN, short-term cultured embryonic kidney cells stimulated with the Wnt agonist R-Spondin 2 gave rise to a monomorphic neuron-like cell population expressing the neuronal 200-kDa neurofilament heavy marker. This finding indicates that the LN might be used to validate the differentiation potential of candidate stem cells in regenerative nephrology.

Maturation of embryonic tissues in a lymph node: a new approach for bioengineering complex organs.

Given our recent finding that the lymph node (LN) can serve as an in vivo factory to generate complex structures like liver, pancreas, and thymus, we investigated whether LN could also support early development and maturation from several mid-embryonic (E14.5/15.5) mouse tissues including brain, thymus, lung, stomach, and intestine. Here we observed brain maturation in LN by showing the emergence of astrocytes with well-developed branching processes. Thymus maturation in LN was monitored by changes in host immune cells. Finally, newly terminally differentiated mucus-producing cells were identified in ectopic tissues generated by transplantation of lung, stomach and intestine in LN. Thus, we speculate the LN offers a unique approach to study the intrinsic and extrinsic differentiation potential of cells and tissues during early development, and provides a new site for bioengineering complex body parts.

mTOR pathway in colorectal cancer: an update.

The mammalian target of rapamycin (mTOR) has emerged as a potential target for drug development, particularly due to the fact that it plays such a crucial role in cancer biology. In addition, next-generation mTOR inhibitors have become available, marking an exciting new phase in mTOR-based therapy. However, the verdict on their therapeutic effectiveness remains unclear. Here we review phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling as one of the primary mechanisms for sustaining tumor outgrowth and metastasis, recent advances in the development of mTOR inhibitors, and current studies addressing mTOR activation/inhibition in colorectal cancer (CRC). We will also discuss our recent comparative study of different mTOR inhibitors in a population of colon cancer stem cells (CSCs), and current major challenges for achieving individualized drug therapy using kinase inhibitors.

Selective targeting of human colon cancer stem-like cells by the mTOR inhibitor Torin-1.

Metastatic colorectal cancer (CRC) is incurable for most patients. Since mammalian target of rapamycin (mTOR) has been suggested as a crucial modulator of tumor biology, we aimed at evaluating the effectiveness of mTOR targeting for CRC therapy. To this purpose, we analyzed mTOR expression and the effect of mTOR inhibition in cancer stem-like cells isolated from three human metastatic CRCs (CoCSCs). CoCSCs exhibited a strong mTOR complex 2 (mTORC2) expression, and a rare expression of mTOR complex 1 (mTORC1). This latter correlated with differentiation, being expressed in CoCSC-derived xenografts. We indicate Serum/glucocorticoid-regulated kinase 1 (SGK1) as the possible main mTORC2 effector in CoCSCs, as highlighted by the negative effect on cancer properties following its knockdown. mTOR inhibitors affected CoCSCs differently, resulting in proliferation, autophagy as well as apoptosis induction. The apoptosis-inducing mTOR inhibitor Torin-1 hindered growth, motility, invasion, and survival of CoCSCs in vitro, and suppressed tumor growth in vivo with a concomitant reduction in vessel formation. Torin-1 also affected the expression of markers for cell proliferation, angio-/lympho-genesis, and stemness in vivo, including Ki67, DLL1, DLL4, Notch, Lgr5, and CD44. Importantly, Torin-1 did not affect the survival of normal colon stem cells in vivo, suggesting its selectivity towards cancer cells. Thus, we propose Torin-1 as a powerful drug candidate for metastatic CRC therapy.

Cancer Stem Cells: A Moving Target.

Even though the number of anti-cancer drugs entering clinical trials and approved by the FDA has increased in recent years, many cancer patients still experience poor survival outcome. The main explanation for such a dismal prognosis is that current therapies might leave behind a population of cancer cells with the capacity for long-term self-renewal, so-called cancer stem cells (CSCs), from which most tumors are believed to be derived and fueled. CSCs might favor local and distant recurrence even many years after initial treatment, thus representing a potential target for therapies aimed at improving clinical outcome. In this review, we will address the CSC hypothesis with a particular emphasis on its current paradigms and debates, and discuss several mechanisms of CSC resistance to conventional therapies.

Management of Liver Failure: From Transplantation to Cell-Based Therapy.

The severe shortage of deceased donor organs has driven a search for alternative methods of treating liver failure. In this context, cell-based regenerative medicine is emerging as a promising interdisciplinary field of tissue repair and restoration, able to contribute to improving health in a minimally invasive fashion. Several cell types have allowed long-term survival in experimental models of liver injury, but their therapeutic potential in humans should be regarded with deep caution, because few clinical trials are currently available and the number of patients enrolled so far is too small to assess benefits versus risks. This review summarizes the current literature on the physiological role of endogenous stem cells in liver regeneration and on the therapeutic benefits of exogenous stem cell administration with specific emphasis on the potential clinical uses of mesenchymal stem cells. Moreover, critical points that still need clarification, such as the exact identity of the stem-like cell population exerting the beneficial effects, as well as the limitations of stem cell-based therapies, are discussed.

Colon cancer stem cells: promise of targeted therapy.

First developed for hematologic disorders, the concept of cancer stem cells (CSCs) was expanded to solid tumors, including colorectal cancer (CRC). The traditional model of colon carcinogenesis includes several steps that occur via mutational activation of oncogenes and inactivation of tumor suppressor genes. Intestinal epithelial cells exist for a shorter amount of time than that required to accumulate tumor-inducing genetic changes, so researchers have investigated the concept that CRC arises from the long-lived stem cells, rather than from the differentiated epithelial cells. Colon CSCs were originally identified through the expression of the CD133 glycoprotein using an antibody directed to its epitope AC133. It is not clear if CD133 is a marker of colon CSCs-other cell surface markers, such as epithelial-specific antigen, CD44, CD166, Musashi-1, CD29, CD24, leucine-rich repeat-containing G-protein-coupled receptor 5, and aldehyde dehydrogenase 1, have been proposed. In addition to initiating and sustaining tumor growth, CSCs are believed to mediate cancer relapse after chemotherapy. How can we identify and analyze colon CSCs and what agents are being designed to kill this chemotherapy-refractory population?