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.

Supplementary Procedures, Figures 1-4 from BCL-2 and Mutant NRAS Interact Physically and Functionally in a Mouse Model of Progressive Myelodysplasia

Immunologic aging is defined as a process in which the immune system undergoes tremendous decline in T cell numbers and function, thus affecting one’s ability to effectively mount an immune response. The degeneration of the thymus, the primary immune organ responsible for T cell development, is central to immunologic aging. Various studies have assessed thymic regeneration as an effective way to offset immune function decline. In this study, we determined the effect of mouse adult thymus transplants in the lymph node of athymic nude mice to rejuvenate thymus function. We transplanted thymuses of increasing ages and assessed engrafted ectopic thymuses by histology as well as for blood T cell numbers and function. We observed that transplanting aged thymuses, up to 8 months old, in the lymph node regenerated thymus function and corresponding T cell activation with some thymic rejuvenation when compared to the expected native thymus in control animals. However, transplanting 11- and 14-month old thymus in the lymph node had decreased-to-limited thymic function with no thymic rejuvenation detected. These observations provide important insights into the plasticity and regenerative capabilities of the thymus during age-associated involution.

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.

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.

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.

The effectiveness of cell-based therapies to treat liver failure is limited by the diseased liver environment. Herein we provide preclinical proof-of-concept for the treatment of liver failure through hepatocyte transplantation into lymph nodes in a large-animal model of hereditary tyrosinemia type 1 (HT1), a metabolic liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) enzyme. FAH-deficient pigs received autologous hepatocyte transplantation into mesenteric lymph nodes after ex vivo transduction with a lentiviral vector carrying the pig Fah gene. Hepatocytes showed early (6 hour) and durable (8 month) 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 enough liver mass to clinically ameliorate disease as early as 97 days post-transplantation, with complete normalization of tyrosine levels and liver function tests. Integration site analysis defined the corrected hepatocytes in the liver as a subpopulation of hepatocytes in the lymph nodes, indicating that the lymph nodes served as a source for healthy hepatocytes to repopulate a diseased liver. Ectopic transplantation of hepatocytes cures the pig model of HT1 and presents a promising approach to the treatment of liver disease in patients with pre-existing liver damage and fibrosis. One Sentence Summary Transplantation of corrected hepatocytes in mesenteric lymph nodes can cure fatal metabolic liver disease by providing organized liver tissue and by repopulating the diseased liver in the pig tyrosinemia model.

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.

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.

The mouse lymph node (LN) can provide a niche to grow metanephric kidney to maturity. Here, we show that signaling through the lymphotoxin-β receptor (LTβR) is critical for kidney organogenesis both in the LN and the omentum. By transplanting kidney rudiments either in the LNs of mice undergoing LTβR antagonist treatment or in the omenta of Ltbr knockout (Ltbr-/-) mice, the host LTβR signals were found to be crucial for obtaining a well-vascularized kidney graft. Indeed, defective LTβR 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 LTβR target nuclear factor κB-inducing kinase (NIK) decreased in the absence of a functional LTβR, it was speculated that an LTβR/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 LTβR signaling impaired compensatory glomerular adaptation to renal mass reduction, indicating that kidney regeneration approaches, besides whole kidney reconstruction, might benefit from the presence of LTβR signals.

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/PHxtreated 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). (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Chimeric liver transplantation reveals interspecific graft remodellingJournal of HepatologyVol. 69Issue 5PreviewThe use of chimeric animals with organs compatible with specific patients in need of transplantation has the potential to solve the chronic lack of organ donors. The idea of using animals as incubators of human tissue is becoming more and more realistic, especially with the recent observation that human induced pluripotent stem cells (hiPSCs) can lead to chimeras after injection into pig blastocysts.1 At this stage, the experiment was terminated before birth, and the contribution of human cells to the final chimeric pig embryos has remained low, but future studies are looking at replacing entire organs, as has been the case for various rodent combinations. Full-Text PDF ‘History of medicine tells us that procedures that were inconceivable yesterday, barely achievable today often become the routine of tomorrow’.[1]Starzl T.E. Iwatsuki S. Van Thiel D.H. et al.Evolution of liver transplantation.Hepatology. 1982; 2: 614-636Crossref PubMed Scopus (613) Google Scholar When Dr. Thomas E. Starzl made this remarkable statement in 1982, he knew the medical, surgical, immunological, and even political challenges to performing a liver transplantation. In the 1980s, liver transplantation was a barely achievable procedure. In 2018, we are living in Dr. Starzl’s tomorrow, where the liver transplantation is a daily routine, not only in Europe[2]Bramhall S.R. Minford E. Gunson B. Buckels J.A. Liver transplantation in the UK.World J Gastroenterol. 2001; 7: 602-611Crossref PubMed Scopus (36) Google Scholar or the United States.[3]Jadlowiec C.C. Taner T. Liver transplantation: current status and challenges.World J Gastroenterol. 2016; 22: 4438-4445Crossref PubMed Scopus (150) Google Scholar but around the world.4Lee S.G. Moon D.B. Hwang S. et al.Liver transplantation in Korea: past, present, and future.Transplant Proc. 2015; 47: 705-708Crossref PubMed Scopus (41) Google Scholar, 5Egawa H. Tanabe K. Fukushima N. Date H. Sugitani A. Haga H. Current status of organ transplantation in Japan.Am J Transplant. 2012; 12: 523-530Crossref PubMed Scopus (55) Google Scholar. So, what is the future for liver transplantation? Despite the success achieved in liver transplantation with a one-year patient survival >90% and five-year patient survival >80%,[6]UNOS. United Network for Organ Sharing. ; Accessed on July 31, 2018.Google Scholar two major problems are still limiting the field. The most important of which is the shortage of organs, despite the utilization of marginal donors, donation after circulatory (cardiac) death, and living donors.[7]Ekser B. Cooper D.K.C. Tector A.J. The need for xenotransplantation as a source of organs and cells for clinical transplantation.Int J Surg. 2015; 23: 199-204Crossref PubMed Scopus (72) Google Scholar When a transplantable liver is available, the second most important challenge is to avoid rejection and side effects of immunosuppressive drugs. Solving both issues would dramatically improve patient access to liver transplantation, as well as quality of life after transplantation.[8]Onghena L. Develtere W. Poppe C. et al.Quality of life after liver transplantation: state of the art.World J Hepatol. 2016; 8: 749-756Crossref PubMed Scopus (37) Google Scholar For decades, physicians, surgeons, and researchers have been trying to overcome both problems. The use of “humanized” animal organs could solve the most important problem of organ shortage[9]Cooper D.K. Ekser B. Ramsoondar J. Phelps C. Ayares D. The role of genetically engineered pigs in xenotransplantation research.J Pathol. 2016; 238: 288-299Crossref PubMed Scopus (148) Google Scholar but ironically, it creates an additional problem, the presence of xenogeneic cells in these humanized organs that may render the prospect of interspecies chimeric liver a daunting possibility for clinical applications. With recent advances in genetic engineering,[9]Cooper D.K. Ekser B. Ramsoondar J. Phelps C. Ayares D. The role of genetically engineered pigs in xenotransplantation research.J Pathol. 2016; 238: 288-299Crossref PubMed Scopus (148) Google Scholar stem cell technology,[10]Francipane M.G. Lagasse E. Pluripotent stem cells to rebuild a kidney: the lymph node as a possible developmental niche.Cell Transplant. 2016; 25: 1007-1023Crossref PubMed Scopus (7) Google Scholar tissue engineering, research in complex animal models, and 3D-bioprinting [11]Smith L.J. Li P. Holland M.R. Ekser B. FABRICA: a bioreactor platform for printing, perfusing, observing, & stimulating 3D tissues.Sci Rep. 2018; 8: 7561Crossref PubMed Scopus (31) Google Scholar, the hope of seeing a xenotransplanted organ (e.g. genetically engineered pig-to-human), an engineered liver tissue, or a 3D-bioprinted liver is closer to reality than ever.12Ekser B. Ezzelarab M. Hara H. et al.Clinical xenotransplantation: the next medical revolution?.Lancet. 2012; 379: 672-683Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 13Komori J. Boone L. DeWard A. Hoppo T. Lagasse E. The mouse lymph node as an ectopic transplantation site for multiple tissues.Nat Biotechnol. 2012; 30: 976-983Crossref PubMed Scopus (81) Google Scholar In the current issue of Journal of Hepatology, Oldani and colleagues[14]Oldani G. Peloso A. Vijgen S. et al.Chimeric liver transplantation reveals interspecific graft remodelling.J Hepatol. 2018; 69: 1025-1036Abstract Full Text Full Text PDF Scopus (5) Google Scholar transplanted chimeric livers with a high level of xenogeneic cells, a critical aspect in the field of interspecies chimeric organs that had not been tested previously. In their report, the authors observed a surprising outcome with the chimeric livers revealing interspecific graft remodeling after transplantation in a small animal model. Although the validation of their findings needs to be confirmed in a large animal model, their perfectly designed and executed study increases the odds that both challenges cited above could be overcome in the future. Oldani et al.[14]Oldani G. Peloso A. Vijgen S. et al.Chimeric liver transplantation reveals interspecific graft remodelling.J Hepatol. 2018; 69: 1025-1036Abstract Full Text Full Text PDF Scopus (5) Google Scholar created chimeric livers by transplanting Lewis rat hepatocytes into genetically engineered mice (FRG mice, C57Bl/6Fah−/−Rag2−/−IL2rg−/−). After generating chimeric mouse liver containing rat hepatocytes, they transplanted liver grafts into newly weaned Lewis rats with or without immunosuppression. In this challenging model, they first optimized the surgical technique, by (i) using dynamic cuffs for vena porta anastomosis, so they could avoid narrowing in the anastomosis, while the rat recipient and transplanted chimeric mouse liver grow. They (ii) avoided arterialization of mouse liver in rats, so excessive flow would not destroy the mouse liver in a rat body (although vessel calipers were kept the same, mouse liver grafts had 30–40% less parenchyma compared to native rat liver).[15]Oldani G. Lacotte S. Orci L.A. et al.Efficient nonarterialized mouse liver transplantation using 3-dimensional-printed instruments.Liver Transpl. 2016; 22: 1688-1696Crossref PubMed Scopus (10) Google Scholar They also recognized the importance of the choice of gender (female rats were chosen because of the possible absence of neoangiogenesis in male rats due to fast grow), so recipients could survive and grow normally. All non-immunosuppressed rats experienced acute rejection and died, as expected. More importantly, the authors showed that high immunosuppression is still needed for chimeric grafts, although less than fully xenogeneic grafts. Interestingly and to a certain extend surprisingly, the authors observed remodeling of the chimeric livers.[14]Oldani G. Peloso A. Vijgen S. et al.Chimeric liver transplantation reveals interspecific graft remodelling.J Hepatol. 2018; 69: 1025-1036Abstract Full Text Full Text PDF Scopus (5) Google Scholar Chimeric mice livers in rat body required up to two months before rat hepatocytes fully colonized the mouse liver, in addition transdifferentiation of the rat hepatocytes into cholangiocytes replaced many of the mouse cholangiocytes, and rat portal endothelial cells were replaced by the host rat endothelial cells. Particularly for cholangiocytes, this study confirms recent data by Willenbring’s group demonstrating transdifferentiation of hepatocytes into cholangiocytes that failed to form during development and would suggest de novo formation of a biliary system is possible in chimeric livers.[16]Schaub J.R. Huppert K.A. Kurial S.N. et al.De novo formation of the biliary system by TGFB-mediated hepatocyte transdifferentiation.Nature. 2018; 557: 247-251Crossref PubMed Scopus (142) Google Scholar The evidence of how liver parenchymal regeneration was carried out by the resident (host) cells only is summarized (Fig. 1). In fact, functionality studies confirmed that mouse albumin was detectable in rat plasma and became undetectable 56 days after transplantation. The authors show that there was a full replacement of remaining xenogeneic mouse hepatocytes, fumarylacetoacetate hydrolase− (FAH−), by autologous rat hepatocytes (FAH+) starting at 56 days post-transplantation. The proposed research can be, as of now, only performed in an animal model with the help of the FAH gene, with the selective potential of the FAH positive hepatocytes in an FAH-knockout liver environment.[17]Grompe M. Fah knockout animals as models for therapeutic liver repopulation.Adv Exp Med Biol. 2017; 959: 215-230Crossref PubMed Scopus (23) Google Scholar If Oldani et al.’s[14]Oldani G. Peloso A. Vijgen S. et al.Chimeric liver transplantation reveals interspecific graft remodelling.J Hepatol. 2018; 69: 1025-1036Abstract Full Text Full Text PDF Scopus (5) Google Scholar findings are confirmed in large animal models, chimeric genetically engineered animal organs created by patient-specific induced pluripotent stem cells (example of a personalized medicine), will have the potential to solve organ shortage with even better outcomes than allotransplantation. However, genetically engineered pig liver clinical xenotransplantation could be the most promising alternative for organ shortage in the near future.7Ekser B. Cooper D.K.C. Tector A.J. The need for xenotransplantation as a source of organs and cells for clinical transplantation.Int J Surg. 2015; 23: 199-204Crossref PubMed Scopus (72) Google Scholar, 12Ekser B. Ezzelarab M. Hara H. et al.Clinical xenotransplantation: the next medical revolution?.Lancet. 2012; 379: 672-683Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 18Cooper D.K. Dou K.F. Tao K.S. Yang Z.X. Tector A.J. Ekser B. Pig liver xenotransplantation: a review of progress toward the clinic.Transplantation. 2016; 100: 2039-2047Crossref PubMed Scopus (36) Google Scholar A limitation of the study by Oldani et al., as pointed out by the authors, is that the study was performed using phylogenetically close species (rat and mouse). Albumin levels are similar in mice and rats. However, this is not the case in pig-to-nonhuman primate or possible pig-to-human xenotransplantation.[19]Ekser B. Bianchi J. Ball S. et al.Comparison of hematologic, biochemical, and coagulation parameters in α1,3-galactosyltransferase gene-knockout pigs, wild-type pigs, and four primate species.Xenotransplantation. 2012; 19: 342-354Crossref PubMed Scopus (38) Google Scholar Finally, the high levels of immunosuppression still needed to retain the chimeric rodent graft suggest that more than human hepatocyte might be needed in humanized liver for a clinical application, possibly involving human endothelial cells. In conclusion, it is exciting to see a mouse liver repopulated with rat hepatocytes and transplanted into rats with long-term survival under sub-optimal immunosuppression. Moreover, findings regarding chimeric liver grafts, which undergo recipient-oriented remodeling of hepatocytes, cholangiocytes, and endothelial cells are important for scientifically proven discovery. Whether this approach will be confirmed in a large animal model or not, the current research by Oldani et al. will definitely help to further liver-related research in small animal models. The authors received no financial support for the production of this manuscript. B.E.’s research, in part, is supported by the Sponsored Research Agreement with Lung Biotechnology LLC, United States, the Indiana University-Purdue University (IUPUI), United States, Research Support Funds Grants from the Office of the Vice Chancellor for Research, and the Board of Directors of the Indiana University Health Values Fund for Research Award and the Indiana Clinical and Translational Sciences Institute funded, in part by Grant # UL1TR001108 from the NIH, United States, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. E.L.’s research is supported by the NIH/NIDDK, United States, grants #DK114282 and DK113261, and the Commonwealth of PA, United States. The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details. Both authors wrote and approved the final version of the manuscript. Download .pdf (.09 MB) Help with pdf files Supplementary data 1

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 5weeks 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 low500-1000-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 ConclusionsAs 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- 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.

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.

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.