Developmental engineering the kidney: Leveraging principles of morphogenesis for renal regeneration

Multiple methodological approaches are currently under active development for application in tissue engineering and regenerative medicine of tubular and solid organs. Most recently, developmental engineering (TE/RM), or the leveraging of embryonic and morphological paradigms to recapitulate aspects of organ development, has been proposed as a strategy for the sequential, iterative de novo assembly of tissues and organs as discrete developmental modules ex vivo, prior to implantation in vivo. In this article, we focus on the kidney to highlight in detail how principles of developmental biology are impacting approaches to TE of this complex solid organ. Ultimately, such methodologies may facilitate the establishment of clinically relevant therapeutic strategies for regeneration of renal structure and function, greatly impacting treatment regimens for chronic kidney disease. Birth Defects Research (Part C) 96:30–38, 2012. © 2012 Wiley Periodicals, Inc.     

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.     

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.     

斑马鱼在再生医学研究中的应用及进展

组织器官的再生现象一直以来吸引着众多生物学家们的关注。再生能力在不同物种间差异很大, 与人及高等脊椎动物相比, 低等脊椎动物(如：斑马鱼)有着较高的再生能力。斑马鱼的鳍、心脏、视网膜、视神经、脊髓、肝脏及感觉毛细胞等都具有很强的再生能力。因此, 从斑马鱼再生过程的研究中将获得大量有用的信息, 促进对人类再生能力缺陷的认识, 进而推动再生医学的发展。文章就斑马鱼在心脏、神经系统、肝脏、鳍再生医学研究中的进展及应用做一综述。     

Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death.     

Inducing cellular dedifferentiation: a potential method for enhancing endogenous regeneration in mammals

Salamanders have the remarkable ability to regenerate lost body parts and injured organs. This regenerative ability requires fully-differentiated cells in the vicinity of the injury to dedifferentiate, proliferate, and then redifferentiate to form the specialized cells that comprise the regenerated structure or organ. The dedifferentiation stage plays a crucial role in the regenerative response and distinguishes the salamander from other vertebrates with more limited regenerative abilities. Recently, several investigators have shown that certain mammalian cell types can be induced to dedifferentiate to progenitor cells when stimulated with the appropriate signals. This discovery opens the possibility that researchers might one day enhance the endogenous regenerative capacity of mammals by inducing cellular dedifferentiation in vivo.     

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases¹. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease². Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells³. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.     

Improving cardiac regeneration after injury: Are we a step closer?

During regeneration, lost functional tissue can, in general, be replaced by different mechanisms, including proliferation of terminally differentiated cells or through differentiation of resident stem cells. It is a well-accepted dogma that the mammalian heart cannot efficiently regenerate upon injury as a consequence of insufficient oxygen supply. This is in sharp contrast to the hearts of adult zebrafish or newts that are able to replace lost ventricular tissue. Novel data indicate that the young murine heart also has the ability to regenerate within the first week after birth using mechanisms apparently quite similar to those observed in fish. This now provides us with a good starting point to identify the molecular mechanisms that led to the loss of the regenerative capacity of the adult mammalian heart. These future studies will also indicate whether it will be possible to reawaken the regenerative capability of cardiomyocytes in the human heart by treatment with selected pharmaceuticals.     

Analysing regenerative potential in zebrafish models of congenital muscular dystrophy

The congenital muscular dystrophies (CMDs) are a clinically and genetically heterogeneous group of muscle disorders. Clinically hypotonia is present from birth, with progressive muscle weakness and wasting through development. For the most part, CMDs can mechanistically be attributed to failure of basement membrane protein laminin-α2 sufficiently binding with correctly glycosylated α-dystroglycan. The majority of CMDs therefore arise as the result of either a deficiency of laminin-α2 (MDC1A) or hypoglycosylation of α-dystroglycan (dystroglycanopathy). Here we consider whether by filling a regenerative medicine niche, the zebrafish model can address the present challenge of delivering novel therapeutic solutions for CMD. In the first instance the readiness and appropriateness of the zebrafish as a model organism for pioneering regenerative medicine therapies in CMD is analysed, in particular for MDC1A and the dystroglycanopathies. Despite the recent rapid progress made in gene editing technology, these approaches have yet to yield any novel zebrafish models of CMD. Currently the most genetically relevant zebrafish models to the field of CMD, have all been created by N-ethyl-N-nitrosourea (ENU) mutagenesis. Once genetically relevant models have been established the zebrafish has several important facets for investigating the mechanistic cause of CMD, including rapid ex vivo development, optical transparency up to the larval stages of development and relative ease in creating transgenic reporter lines. Together, these tools are well suited for use in live-imaging studies such as in vivo modelling of muscle fibre detachment. Secondly, the zebrafish's contribution to progress in effective treatment of CMD was analysed. Two approaches were identified in which zebrafish could potentially contribute to effective therapies. The first hinges on the augmentation of functional redundancy within the system, such as upregulating alternative laminin chains in the candyfloss fish, a model of MDC1A. Secondly high-throughput small molecule screens not only provide effective therapies, but also an alternative strategy for investigating CMD in zebrafish. In this instance insight into disease mechanism is derived in reverse. Zebrafish models are therefore clearly of critical importance in the advancement of regenerative medicine strategies in CMD.This article is part of a Directed Issue entitled: Regenerative Medicine: The challenge of translation.     

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction¹. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life^2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues^4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury^6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex^2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish^8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.     

Using zebrafish as the model organism to understand organ regeneration

The limited regenerative capacity of several organs, such as central nervous system(CNS), heart and limb in mammals makes related major diseases quite difficult to recover. Therefore, dissection of the cellular and molecular mechanisms underlying organ regeneration is of great scientific and clinical interests. Tremendous progression has already been made after extensive investigations using several model organisms for decades. Unfortunately, distance to the final achievement of the goal still remains. Recently, zebrafish became a popular model organism for the deep understanding of regeneration based on its powerful regenerative capacity, in particular the organs that are limitedly regenerated in mammals. Additionally, zebrafish are endowed with other advantages good for the study of organ regeneration. This review summarizes the recent progress in the study of zebrafish organ regeneration, in particular regeneration of fin, heart, CNS, and liver as the representatives. We also discuss reasons of the reduced regenerative capacity in higher vertebrate, the roles of inflammation during regeneration, and the difference between organogenesis and regeneration.     

Nerve signaling regulates basal keratinocyte proliferation in the blastema apical epithelial cap in the axolotl (Ambystoma mexicanum)

The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration.     

Cellular and molecular processes of regeneration, with special emphasis on fish fins

The phenomenon of 'epimorphic regeneration', a complete reformation of lost tissues and organs from adult differentiated cells, has been fascinating many biologists for many years. While most vertebrate species including humans do not have a remarkable ability for regeneration, the lower vertebrates such as urodeles and fish have exceptionally high regeneration abilities. In particular, the teleost fish has a high ability to regenerate a variety of tissues and organs including scales, muscles, spinal cord and heart among vertebrate species. Hence, an understanding of the regeneration mechanism in teleosts will provide an essential knowledge base for rational approaches to tissue and organ regeneration in mammals. In the last decade, small teleost fish such as the zebrafish and medaka have emerged as powerful animal models in which a variety of developmental, genetic and molecular approaches are applicable. In addition, rapid progress in the development of genome resources such as expressed sequence tags and genome sequences has accelerated the speed of the molecular analysis of regeneration. This review summarizes the current status of our understanding of the cellular and molecular basis of regeneration, particularly that regarding fish fins.     

Regenerating cardiac cells: insights from the bench and the clinic

A major challenge in cardiovascular regenerative medicine is the development of novel therapeutic strategies to restore the function of cardiac muscle in the failing heart. The heart has historically been regarded as a terminally differentiated organ that does not have the potential to regenerate. This concept has been updated by the discovery of cardiac stem and progenitor cells that reside in the adult mammalian heart. Whereas diverse types of adult cardiac stem or progenitor cells have been described, we still do not know whether these cells share a common origin. A better understanding of the physiology of cardiac stem and progenitor cells should advance the successful use of regenerative medicine as a viable therapy for heart disease. In this review, we summarize current knowledge of the various adult cardiac stem and progenitor cell types that have been discovered. We also review clinical trials presently being undertaken with adult stem cells to repair the injured myocardium in patients with coronary artery disease.     

Surface functionalization of nanobiomaterials for application in stem cell culture,tissue engineering,and regenerative medicine

Stem cell‐based approaches offer great application potential in tissue engineering and regenerative medicine owing to their ability of sensing the microenvironment and respond accordingly (dynamic behavior). Recently, the combination of nanobiomaterials with stem cells has paved a great way for further exploration. Nanobiomaterials with engineered surfaces could mimic the native microenvironment to which the seeded stem cells could adhere and migrate. Surface functionalized nanobiomaterial‐based scaffolds could then be used to regulate or control the cellular functions to culture stem cells and regenerate damaged tissues or organs. Therefore, controlling the interactions between nanobiomaterials and stem cells is a critical factor. However, surface functionalization or modification techniques has provided an alternative approach for tailoring the nanobiomaterials surface in accordance to the physiological surrounding of a living cells; thereby, enhancing the structural and functional properties of the engineered tissues and organs. Currently, there are a variety of methods and technologies available to modify the surface of biomaterials according to the specific cell or tissue properties to be regenerated. This review highlights the trends in surface modification techniques for nanobiomaterials and the biological relevance in stem cell‐based tissue engineering and regenerative medicine. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:554–567, 2016     

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine¹. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans¹. The zebrafish has become a powerful animal model for the study of vertebrate cell biology². There has been extensive documentation and analysis of embryonic development in the zebrafish³. Only recently have scientists sought to document adult anatomy and surgical dissection techniques⁴, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer⁵. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish^6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate⁸. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species^9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals. Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)^11,12, expression profiling^13,14, and transplantation^11,15. We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.     

Appendage regeneration in anamniotes utilizes genes active during larval-metamorphic stages that have been lost or altered in amniotes: The case for studying lizard tail regeneration

This review elaborates the idea that organ regeneration derives from specific evolutionary histories of vertebrates. Regenerative ability depends on genomic regulation of genes specific to the life-cycles that have differentially evolved in anamniotes and amniotes. In aquatic environments, where fish and amphibians live, one or multiple metamorphic transitions occur before the adult stage is reached. Each transition involves the destruction and remodeling of larval organs that are replaced with adult organs. After organ injury or loss in adult anamniotes, regeneration uses similar genes and developmental process than those operating during larval growth and metamorphosis. Therefore, the broad presence of regenerative capability across anamniotes is possible because generating new organs is included in their life history at metamorphic stages. Soft hyaluronate-rich regenerative blastemas grow in submersed or in hydrated environments, that is, essential conditions for regeneration, like during development. In adult anamniotes, the ability to regenerate different organs decreases in comparison to larval stages and becomes limited during aging. Comparisons of genes activated during metamorphosis and regeneration in anamniotes identify key genes unique to these processes, and include thyroid, wnt and non-coding RNAs developmental pathways. In the terrestrial environment, some genes or developmental pathways for metamorphic transitions were lost during amniote evolution, determining loss of regeneration. Among amniotes, the formation of soft and hydrated blastemas only occurs in lizards, a morphogenetic process that evolved favoring their survival through tail autotomy, leading to a massive although imperfect regeneration of the tail. Deciphering genes activity during lizard tail regeneration would address future attempts to recreate in other amniotes regenerative blastemas that grow into variably completed organs.     

Orchestrating cell/material interactions for tissue engineering of surgical implants

Research groups are currently recognising a critical clinical need for innovative approaches to organ failure and agenesis. Allografting, autologous reconstruction and prosthetics are hampered with severe limitations. Pertinently, readily available 'laboratory-grown' organs and implants are becoming a reality. Tissue engineering constructs vary in their design complexity depending on the specific structural and functional demands. Expeditious methods on integrating autologous stem cells onto nanoarchitectured 3D nanocomposites, are being transferred from lab to patients with a number of successful first-in-man experiences. Despite the need for a complete understanding of cell/material interactions tissue engineering is offering a plethora of exciting possibilities in regenerative medicine.     

Organ repair and regeneration: An overview

A number of organs have the intrinsic ability to regenerate, a distinctive feature that varies among organisms. Organ regeneration is a process not fully yet understood. However, when its underlying mechanisms are unraveled, it holds tremendous therapeutic potential for humans. In this review, we chose to summarize the repair and regenerative potential of the following organs and organ systems: thymus, adrenal gland, thyroid gland, intestine, lungs, heart, liver, blood vessels, germ cells, nervous system, eye tissues, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone, and cartilage. For each organ, a review of the following is presented: (a) factors, pathways, and cells that are involved in the organ's intrinsic regenerative ability, (b) contribution of exogenous cells – such as progenitor cells, embryonic stem cells, induced pluripotent stem cells, and bone marrow‐, adipose‐ and umbilical cord blood‐derived stem cells – in repairing and regenerating organs in the absence of an innate intrinsic regenerative capability, (c) and the progress made in engineering bio‐artificial scaffolds, tissues, and organs. Organ regeneration is a promising therapy that can alleviate humans from diseases that have not been yet cured. It is also superior to already existing treatments that utilize exogenous sources to substitute for the organ's lost structure and/or function(s). (Part C) 96:1–29, 2012. © 2012 Wiley Periodicals, Inc.