Will cell therapies provide the solution for the shortage of transplantable organs? Purpose of review The potential to regenerate ischemically damaged kidneys while being perfused ex-vivo offers the best near-term solution to increasing kidney allografts for transplantation. Recent findings There are a number of stem-cell sources including: stromal mesenchymal cells (MSC), induced adult pluripotent stem cells, fetal stem cells from placenta, membranes, amniotic fluid and umbilical cord and hematopoietic cells. MSC are increasingly the stem cell of choice and studies are primarily focused on novel induction immunosuppression to prevent rejection. Stem-cell therapies applied in vivo may be of limited benefit because the nonintegrating cells do not remain in the kidney and are not detectable in the body after several days. MSC therapies for transplantation have demonstrated early safety and feasibility. However, efficacy has not been clearly established. A more feasible application of a stem-cell therapy in transplantation is the administration of MSC to treat damaged renal allografts directly while being perfused ex vivo. Initial feasibility has been established demonstrating MSC-treatment results in statistically significant reduction of inflammatory responses, increased ATP and growth factor synthesis and mitosis. Summary The ability to regenerate renal tissue ex-vivo sufficiently to result in immediate function could revolutionize transplantation by solving the chronic organ shortage. Correspondence to Lauren Brasile, PhD, BREONICS Inc., 44 Dalliba Avenue, Watervliet, NY 12189, USA. Tel: +1 518 459 2112; e-mail: lbrasile@citlink.net Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

The ins and outs of engineering functional tissues and organs: evaluating the in-vitro and in-situ processes Purpose of review For many disorders that result in loss of organ function, the only curative treatment is organ transplantation. However, this approach is severely limited by the shortage of donor organs. Tissue engineering has emerged as an alternative solution to this issue. This review discusses the concept of tissue engineering from a technical viewpoint and summarizes the state of the art as well as the current shortcomings, with the aim of identifying the key lessons that we can learn to further advance the engineering of functional tissues and organs. Recent findings A plethora of tissue-engineering strategies have been recently developed. Notably, these strategies put different emphases on the in-vitro and in-situ processes (i.e. preimplantation and postimplantation) that take place during tissue formation. Biophysical and biomechanical interactions between the cells and the scaffold/biomaterial play a crucial role in all steps and have started to be exploited to steer tissue regeneration. Summary Recent works have demonstrated the need to better understand the in-vitro and in-situ processes during tissue formation, in order to regenerate complex, functional organs with desired cellular organization and tissue architecture. A concerted effort from both fundamental and tissue-specific research has the potential to accelerate progress in the field. Correspondence to Nicholas A. Kurniawan, PhD, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. Tel: +31 40 247 2347; e-mail: kurniawan@tue.nl Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Infection in xenotransplantation: opportunities and challenges Purpose of review Posttransplantation infections are common. It is anticipated that infection will be no less common in xenotransplantation recipients. Prolonged xenograft survivals have resulted from advances in immunosuppressive strategies and development of swine that decrease host immune responses via genetic manipulation, notably CRISPR/cas9 manipulation. As prospects for clinical trials improve, consideration of the unique infectious risks posed by xenotransplantation reemerge. Recent findings Organisms likely to cause infection in human recipients of porcine xenografts are unknown in advance of clinical trials. Microbiological screening of swine intended as xenograft donors can be more intensive than is currently feasible for human allograft donors. Monitoring infection in recipients will also be more intensive. Key opportunities in infectious diseases of xenotransplantation include major technological advances in evaluation of the microbiome by unbiased metagenomic sequencing, assessments of some risks posed by porcine endogenous retroviruses (PERVs) including antiretroviral susceptibilities, availability of swine with deletion of genomic PERVs, and recognition of the rapidly changing epidemiology of infection in swine worldwide. Summary Unknown infectious risks in xenotransplantation requires application of advanced microbiological techniques to discern and prevent infection in graft recipients. Clinical trials will provide an opportunity to advance the safety of all of organ transplantation. Correspondence to Jay A. Fishman, MD, Transplantation Infectious Disease and Compromised Host Program, Infectious Disease Division and MGH Transplant Center, Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, WH510A, Boston, MA 02114, USA. Tel: +1 617 643 4809; e-mail: Fishman.jay@mgh.harvard.edu Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

The role of industry in advancing xenotransplantation Purpose of review Xenotransplantation offers the opportunity to alleviate the imbalance between the demand of patients with end stage organ failure and the supply of organs available for transplantation but remains aspirational. This review highlights how collaboration between academia and industry are essential for success. Recent findings The science of xenotransplantation has accelerated in recent years with key discoveries in genetic engineering, enabling disruption of genes facilitating rejection, and transgenic expression of desired human genes. Combined with similar progress directed toward induction of transplant tolerance, the stage has been set for meaningful progress. These advances are reviewed in detail elsewhere in this volume and argue that the breakthroughs needed to deliver substantial cross-species organ survival have largely been achieved, heralding a liminal stage of human xenotransplantation. However, xenotransplantation as a meaningful therapy for medically refractory end organ failure will not be realized through scientific innovation alone. The advent of broadly available, therapeutic xenogeneic tissues requires extensive development and regulatory expertise; the biotechnology/pharmaceutical industry can provide extensive resources and expertise in those essential areas. Summary Successful delivery of xenotransplantation as an available therapy for curing end stage organ failure is best accomplished through partnership and collaboration between academia and industry. Correspondence to Marc I. Lorber, MD, Sr. Vice President and Chief Medical Officer, Lung Biotechnology, PBC, United Therapeutics Corporation, 1040 Spring Street, Silver Spring, MD 20910, USA. Tel: +1 301 608 9292; e-mail: mlorber@lungbiotechnology.com Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

The potential role of 3D-bioprinting in xenotransplantation Purpose of review To review the impact of a new technology, 3D-bioprinting, in xenotransplantation research. Recent findings Genetically engineered pigs, beginning with human (h) CD55-transgenic and Gal-knockout pigs, have improved the outcomes of xenotransplantation research. Today, there are more than 30 different genetically engineered pigs either expressing human gene(s) or lacking pig gene(s). CRIPSR/cas9 technology has facilitated the production of multigene pigs (up to nine genes in a single pig), which lack multiple pig xenoantigens, and express human transgenes, such as hCD46, hCD55, hThrombomodulin, hCD39, etc. Although recent studies in nonhuman primates (NHPs) have demonstrated prolonged survival after life-supporting pig kidney, heart, and islet xenotransplantation, researchers have difficulty determining the best genetic combination to test in NHPs because of a potential greater than 100 000 genetic combinations. 3D-bioprinting of genetically engineered pig cells: is superior to 2D in-vitro testing, enables organ-specific testing, helps to understand differences in immunogenicity between organs, and is faster and cheaper than testing in NHPs. Moreover, 3D-bioprinted cells can be continuously perfused in a bioreactor, controlling for all variables, except the studied variable. Summary 3D-bioprinting can help in the study of the impact of specific genes (human or pig) in xenotransplantation in a rapid, inexpensive, and reliable way. Correspondence to Burcin Ekser, MD, PhD, Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine, 550N University Blvd, UH 4601, Indianapolis, IN 46202, USA. Tel: +1 317 948 3835; fax: +1 317 968 1254; e-mail: bekser@iupui.edu Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Immune responses towards bioengineered tissues and strategies to control them Purpose of review Research into development of artificial tissues and bioengineered organs to replace physiological functions of injured counterparts has highlighted a previously underestimated challenge for its clinical translatability: the immune response against biomaterials. Herein, we will provide an update and review current knowledge regarding this important barrier to regenerative medicine. Recent findings Although a clear understanding of the immune reactivity against biomaterials remains elusive, accumulating evidence indicates that innate immune cells, primarily neutrophils and macrophages, play a key role in the initial phases of the immune response. More recently, data have shown that in later phases, T and B cells are also involved. The use of physicochemical modifications of biomaterials and cell-based strategies to modulate the host inflammatory response is being actively investigated for effective biomaterial integration. Summary The immune response towards biomaterials and bioengineered organs plays a crucial role in determining their utility as transplantable grafts. Expanding our understanding of these responses is necessary for developing protolerogenic strategies and delivering on the ultimate promise of regenerative medicine. Correspondence to Paolo Cravedi, MD, PhD, Department of Medicine, Icahn School of Medicine at Mount Sinai, 1 Levy Place, New York, NY 10029, USA. Tel: +1 212 241 3349; fax: +1 212 987 0389; e-mail: paolo.cravedi@mssm.edu Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Chimeric xenotransplantation Purpose of review Organ transplantation is an effective treatment for selected patients with end-stage organ disease or specific cancer types. Its main limitations are the chronic lack of grafts and the lifetime need for immunosuppression. The advent of autologous organs generated into xenogeneic species has the potential to solve these issues. Recent findings The current review discusses about the recent discoveries in the filed of organ generation by interspecific pre and postimplantation embryo complementation. Moreover, it describes the recent progress in postnatal xenogeneic liver repopulation and the transplantation of chimeric tissues and organs. Summary Thanks to the groundbreaking discoveries of the last few years, these strategies are becoming more and more real, yet with still a number of key steps to overcome. Correspondence to Christian Toso, MD, PhD, Hepato-Pancreato-Biliary Centre, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14, Switzerland. Tel: +41 22 3727693; e-mail: christian.toso@hcuge.ch Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Regulatory barriers to xenotransplantation Purpose of review There is a grave discordance between supply and demand for patients with failing organs largely due to an insufficient donor pool for transplantation. Xenotransplantation has been proposed as a solution to bridge this gap. Recent findings Recent success over the last decade in nonhuman primate models, due to emerging gene-editing technologies combined with novel immunosuppression regimens, has produced promising results in pancreatic islet cell, heart, lung, kidney and liver xenotransplantations. Summary As the prospect of xenotransplantation is realized, safety and ethical considerations have come to the forefront of discussion. The WHO and World Health Assembly have encouraged member states to form regulatory bodies to govern human xenotransplantation studies with the highest standards. Here, we summarize the current regulatory landscape governing preclinical advances toward the first human clinical trials. Correspondence to Muhammad M. Mohiuddin, MD, Cardiac Xenotransplantation Program, University of Maryland School of Medicine, 10 S. Pine Street, MSTF 434B, Baltimore, MD 21201, USA. Tel: +1 410 706 6081; e-mail: mmohiuddin@som.umaryland.edu Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Organoids for replacement therapy: expectations, limitations and reality Purpose of review To discuss existing expectations from organoids and how they can affect biomedical research and society, and to analyse the current limitations and how they can potentially be overcome. Recent findings Recent success with engineering human organoids has created great enthusiasm and expectations, especially for their potential as tissue substitutes. The most feasible applications for organoid technologies at the moment are: drug testing, disease modelling and studying of human development. Summary Being able to engineer transplantable tissues in a dish would fundamentally change the way we conduct biomedical research and clinical practice, and impact important aspects of science and society – from animal experimentation to personalized medicine, bioethics, transplantation and gene therapy. However, whether organoids will soon be able to fulfil these expectations is still unclear, because of significant existing limitations. By answering a set of questions, here I will examine the expectations on the future of organoids and how they can affect the field and the society, I will analyse the most important limitations that still prevent the production of transplantable human tissues in a dish, and discuss possible solution strategies. Correspondence to Christodoulos Xinaris, Laboratory of Organ Regeneration, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Centro Anna Maria Astori Science and Technology Park Kilometro Rosso, Via Stezzano, 87 24126 Bergamo, Italy. Tel: +00 39 035 42131; fax: +00 39 035 319331; e-mail: christodoulos.xinaris@marionegri.it Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

The pathology of solid organ xenotransplantation Purpose of review The use of genetically modified pigs has resulted in prolonged xenograft organ survival, overcoming the initial barriers that lead to hyperacute rejection and immediate loss of the graft. The purpose of the present review is to revisit the xenogeneic response and the pathologic changes in the xenograft organ in the context of recent publications of large animal studies that highlight existing challenges. Recent findings Transgenic modifications that have included complement regulatory proteins and coagulation regulatory proteins have prolonged xenograft survival in pig to nonhuman primate kidneys, livers, and hearts. Modifications of immunosuppressive regimens such as the addition of mTOR inhibition and costimulatory blockade have also led to better outcomes. Antibody-mediated rejection and thrombotic microangiopathy persist as primary challenges to the field and require further systematic exploration. Summary The efforts to overcome the natural antibody response to xenoantigens are largely sufficient. There is great opportunity for designing immunosuppression protocols and for detecting early coagulopathies, complement activation, and donor-specific antibody response. With graft survival prolongation, there is also a greater need to understand mechanisms and to enhance diagnostic tools for pathologic evaluation. Correspondence to Ivy A. Rosales, Immunopathology Research Laboratory, Massachusetts General Hospital, Boston, MA 02114, USA. Tel: +1-617-7260233; mobile: +1-617-4606211; e-mail: irosales@mgh.harvard.edu Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.