Future medicine: from molecular pathways to the collective intelligence of the body.

The remarkable anatomical homeostasis exhibited by complex living organisms suggests that they are inherently reprogrammable information-processing systems that offer numerous interfaces to their physiological and anatomical problem-solving capacities. We briefly review data suggesting that the multiscale competency of living forms affords a new path for biomedicine that exploits the innate collective intelligence of tissues and organs. The concept of tissue-level allostatic goal-directedness is already bearing fruit in clinical practice. We sketch a roadmap towards 'somatic psychiatry' by using advances in bioelectricity and behavioral neuroscience to design methods that induce self-repair of structure and function. Relaxing the assumption that cellular control mechanisms are static, exploiting powerful concepts from cybernetics, behavioral science, and developmental biology may spark definitive solutions to current biomedical challenges.

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.

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.

Development of Ectopic Livers by Hepatocyte Transplantation Into Swine Lymph Nodes.

Orthotopic liver transplantation continues to be the only effective therapy for patients with end-stage liver disease. Unfortunately, many of these patients are not considered transplant candidates, lacking effective therapeutic options that would address both the irreversible progression of their hepatic failure and the control of their portal hypertension. In this prospective study, a swine model was exploited to induce subacute liver failure. Autologous hepatocytes, isolated from the left hepatic lobe, were transplanted into the mesenteric lymph nodes (LNs) by direct cell injection. At 30-60 days after transplantation, hepatocyte engraftment in LNs was successfully identified in all transplanted animals with the degree of ectopic liver mass detected being proportional to the induced native liver injury. These ectopic livers developed within the LNs showed remarkable histologic features of swine hepatic lobules, including the formation of sinusoids and bile ducts. On the basis of our previous tyrosinemic mouse model and the present pig models of induced subacute liver failure, the generation of auxiliary liver tissue using the LNs as hepatocyte engraftment sites represents a potential therapeutic approach to supplement declining hepatic function in the treatment of liver disease.

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.

Adult hepatocytes direct liver organogenesis through non-parenchymal cell recruitment in the kidney.

BACKGROUND & AIMS: Since the first account of the myth of Prometheus, the amazing regenerative capacity of the liver has fascinated researchers because of its enormous medical potential. Liver regeneration is promoted by multiple types of liver cells, including hepatocytes and liver non-parenchymal cells (NPCs), through complex intercellular signaling. However, the mechanism of liver organogenesis, especially the role of adult hepatocytes at ectopic sites, remains unknown. In this study, we demonstrate that hepatocytes alone spurred liver organogenesis to form an organ-sized complex 3D liver that exhibited native liver architecture and functions in the kidneys of mice. METHODS: Isolated hepatocytes were transplanted under the kidney capsule of monocrotaline (MCT) and partial hepatectomy (PHx)-treated mice. To determine the origin of NPCs in neo-livers, hepatocytes were transplanted into MCT/PHx-treated green fluorescent protein transgenic mice or wild-type mice transplanted with bone marrow cells isolated from green fluorescent protein-mice. RESULTS: Hepatocytes engrafted at the subrenal space of mice underwent continuous growth in response to a chronic hepatic injury in the native liver. More than 1.5 years later, whole organ-sized liver tissues with greater mass than those of the injured native liver had formed. Most remarkably, we revealed that at least three types of NPCs with similar phenotypic features to the liver NPCs were recruited from the host tissues including bone marrow. The neo-livers in the kidney exhibited liver-specific functions and architectures, including sinusoidal vascular systems, zonal heterogeneity, and emergence of bile duct cells. Furthermore, the neo-livers successfully rescued the mice with lethal liver injury. CONCLUSION: Our data clearly show that adult hepatocytes play a leading role as organizer cells in liver organogenesis at ectopic sites via NPC recruitment. LAY SUMMARY: The role of adult hepatocytes at ectopic locations has not been clarified. In this study, we demonstrated that engrafted hepatocytes in the kidney proliferated, recruited non-parenchymal cells from host tissues including bone marrow, and finally created an organ-sized, complex liver system that exhibited liver-specific architectures and functions. Our results revealed previously undescribed functions of hepatocytes to direct liver organogenesis through non-parenchymal cell recruitment and organize multiple cell types into a complex 3D liver at ectopic sites. Transcript profiling: Microarray data are deposited in GEO (GEO accession: GSE99141).

Transplantation of hepatocytes from genetically engineered pigs into baboons.

BACKGROUND: Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes. METHODS: Baboons (n=3) were recipients of GTKO/hCD46 pig hepatocytes. All three baboons received anti-thymocyte globulin (ATG) induction and tapering methylprednisolone. Baboon 1 received maintenance immunosuppressive therapy with tacrolimus and rapamycin. Baboons 2 and 3 received an anti-CD40mAb/rapamycin-based regimen that prevents sensitization to pig solid organ grafts. The baboons were euthanized 4 or 5 weeks after hepatocyte transplantation. The baboon immune response was monitored by the measurement of anti-non-Gal IgM and IgG antibodies (by flow cytometry) and CFSE-mixed lymphocyte reaction. Monitoring for hepatocyte survival and function was by (i) real-time PCR detection of porcine DNA, (ii) real-time PCR for porcine gene expression, and (iii) pig serum albumin levels (by ELISA). The sites of hepatocyte injection were examined microscopically. RESULTS: Detection of porcine DNA and porcine gene expression was minimal at all sites of hepatocyte injection. Serum levels of porcine albumen were very low-500-1000-fold lower than in baboons with orthotopic pig liver grafts, and approximately 5000-fold lower than in healthy pigs. No hepatocytes or infiltrating immune cells were seen at any of the injection sites. Two baboons (Baboons 1 and 3) demonstrated a significant increase in anti-pig IgM and an even greater increase in IgG, indicating sensitization to pig antigens. DISCUSSION AND CONCLUSIONS: As a result of this disappointing experience, the following points need to be considered. (i) Were the isolated pig hepatocytes functionally viable? (ii) Are pig hepatocytes more immunogenic than pig hearts, kidneys, artery patch grafts, or islets? (iii) Does injection of pig cells (antigens) into the spleen and/or lymph nodes stimulate a greater immune response than when pig tissues are grafted at other sites? (iv) Did the presence of the recipient's intact liver prevent survival and proliferation of pig hepatocytes? (v) Is pig CD47-primate SIRP-α compatibility essential? In conclusion, the transplantation of genetically engineered pig hepatocytes into multiple sites in immunosuppressed baboons was associated with very early graft failure. Considerable further study is required before clinical trials should be undertaken.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66- is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance.

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.

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.

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.

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.

Tissue-Specific Effects of Esophageal Extracellular Matrix.

Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate repair or remodeling of numerous tissues, including the esophagus. The theoretically ideal scaffold for tissue repair is the ECM derived from the particular tissue to be treated, that is, site-specific or homologous ECM. The preference or potential advantage for the use of site-specific ECM remains unknown in the esophageal location. The objective of the present study was to characterize the in vitro cellular response and in vivo host response to a homologous esophageal ECM (eECM) versus nonhomologous ECMs derived from small intestinal submucosa and urinary bladder. The in vitro response of esophageal stem cells was characterized by migration, proliferation, and three-dimensional (3D) organoid formation assays. The in vivo remodeling response was evaluated in a rat model of esophageal mucosal resection. Results of the study showed that the eECM retains favorable tissue-specific characteristics that enhance the migration of esophageal stem cells and supports the formation of 3D organoids to a greater extent than heterologous ECMs. Implantation of eECM facilitates the remodeling of esophageal mucosa following mucosal resection, but no distinct advantage versus heterologous ECM could be identified.

Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation.

Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically.

Distinct human stem cell populations in small and large intestine.

The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.

Identification of a candidate stem cell in human gallbladder.

There are currently no reports of identification of stem cells in human gallbladder. The differences between human gallbladder and intrahepatic bile duct (IHBD) cells have also not been explored. The goals of this study were to evaluate if human fetal gallbladder contains a candidate stem cell population and if fetal gallbladder cells are distinct from fetal IHBD cells. We found that EpCAM+CD44+CD13+ cells represent the cell population most enriched for clonal self-renewal from primary gallbladder. Primary EpCAM+CD44+CD13+ cells gave rise to EpCAM+CD44+CD13+ and EpCAM+CD44+CD13- cells in vitro, and gallbladder cells expanded in vitro exhibited short-term engraftment in vivo. Last, we found that CD13, CD227, CD66, CD26 and CD49b were differentially expressed between gallbladder and IHBD cells cultured in vitro indicating clear phenotypic differences between the two cell populations. Microarray analyses of expanded cultures confirmed that both cell types have unique transcriptional profiles with predicted functional differences in lipid, carbohydrate, nucleic acid and drug metabolism. In conclusion, we have isolated a distinct clonogenic population of epithelial cells from primary human fetal gallbladder with stem cell characteristics and found it to be unique compared to IHBD 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.