Renal stem cell reprogramming: Prospects in regenerative medicine

Stem cell therapy is a promising future enterprise for renal replacement in patients with acute and chronic kidney disease, conditions which affect millions worldwide and currently require patients to undergo lifelong medical treatments through dialysis and/or organ transplant. Reprogramming differentiated renal cells harvested from the patient back into a pluripotent state would decrease the risk of tissue rejection and provide a virtually unlimited supply of cells for regenerative medicine treatments, making it an exciting area of current research in nephrology. Among the major hurdles that need to be overcome before stem cell therapy for the kidney can be applied in a clinical setting are ensuring the fidelity and relative safety of the reprogrammed cells, as well as achieving feasible efficiency in the reprogramming processes that are utilized. Further, improved knowledge about the genetic control of renal lineage development is vital to identifying predictable and efficient reprogramming approaches, such as the expression of key modulators or the regulation of geneactivity through small molecule mimetics. Here, we discuss several recent advances in induced pluripotent stem cell technologies. We also explore strategies that have been successful in renal progenitor generation, and explore what these methods might mean for the development of cell-based regenerative therapies for kidney disease.     

Renal stem cells: fact or science fiction?

The kidney is widely regarded as an organ without regenerative abilities. However, in recent years this dogma has been challenged on the basis of observations of kidney recovery following acute injury, and the identification of renal populations that demonstrate stem cell characteristics in various species. It is currently speculated that the human kidney can regenerate in some contexts, but the mechanisms of renal regeneration remain poorly understood. Numerous controversies surround the potency, behaviour and origins of the cell types that are proposed to perform kidney regeneration. The present review explores the current understanding of renal stem cells and kidney regeneration events, and examines the future challenges in using these insights to create new clinical treatments for kidney disease.     

Renal organogenesis

The increasing prevalence of chronic kidney disease in the absence of new treatment modalities has become a strong driver for innovation in nephrology. An increasing understanding of stem cell biology has kindled the prospects of regenerative options for kidney disease. However, the kidney itself is not a regenerative organ, as all the nephrons are formed during embryonic development. Here, we will investigate advances in the molecular genetics of renal organogenesis, including what this can tell us about lineage relationships, and discuss how this may serve to inform us about both the normal processes of renal repair and options for regenerative therapies.     

Renal organogenesis: What can it tell us about renal repair and regeneration?

The increasing prevalence of chronic kidney disease in the absence of new treatment modalities has become a strong driver for innovation in nephrology. An increasing understanding of stem cell biology has kindled the prospects of regenerative options for kidney disease. However, the kidney itself is not a regenerative organ, as all the nephrons are formed during embryonic development. Here, we will investigate advances in the molecular genetics of renal organogenesis, including what this can tell us about lineage relationships, and discuss how this may serve to inform us about both the normal processes of renal repair and options for regenerative therapies.     

Targeting the redox system for cardiovascular regeneration in aging

Cardiovascular aging presents a formidable challenge, as the aging process can lead to reduced cardiac function and heightened susceptibility to cardiovascular diseases. Consequently, there is an escalating, unmet medical need for innovative and effective cardiovascular regeneration strategies aimed at restoring and rejuvenating aging cardiovascular tissues. Altered redox homeostasis and the accumulation of oxidative damage play a pivotal role in detrimental changes to stem cell function and cellular senescence, hampering regenerative capacity in aged cardiovascular system. A mounting body of evidence underscores the significance of targeting redox machinery to restore stem cell self-renewal and enhance their differentiation potential into youthful cardiovascular lineages. Hence, the redox machinery holds promise as a target for optimizing cardiovascular regenerative therapies. In this context, we delve into the current understanding of redox homeostasis in regulating stem cell function and reprogramming processes that impact the regenerative potential of the cardiovascular system. Furthermore, we offer insights into the recent translational and clinical implications of redox-targeting compounds aimed at enhancing current regenerative therapies for aging cardiovascular tissues.     

慢性肾功能衰竭的治疗现状及研究前景

CRF是威胁人类健康及生命的常见病之一,近年来平均每年以约8%的速度在增长。依靠慢性肾功能衰竭肾脏母体及机体的再生潜能在脱细胞基质支架上修复重建肾脏结构与功能,这将是慢性肾功能衰竭治疗的一种全新的途径。而去细胞基质在组织工程、干细胞及再生医学的大量应用为解决组织器官的修复和重建等难题带来了希望。本文就目前CRF的治疗现状及、肾脏组织工程研究前景进行简要综述。     

Kidney Specific Protein-Positive Cells Derived from Embryonic Stem Cells Reproduce Tubular Structures In Vitro and Differentiate into Renal Tubular Cells

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.     

In vitro regeneration of kidney from pluripotent stem cells

Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.     

Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors

The adult brain is extremely vulnerable to various insults. The recent discovery of neural progenitors in adult mammals, however, raises the possibility of repairing damaged tissue by recruiting their latent regenerative potential. Here we show that activation of endogenous progenitors leads to massive regeneration of hippocampal pyramidal neurons after ischemic brain injury. Endogenous progenitors proliferate in response to ischemia and subsequently migrate into the hippocampus to regenerate new neurons. Intraventricular infusion of growth factors markedly augments these responses, thereby increasing the number of newborn neurons. Our studies suggest that regenerated neurons are integrated into the existing brain circuitry and contribute to ameliorating neurological deficits. These results expand the possibility of novel neuronal cell regeneration therapies for stroke and other neurological diseases.     

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.     

Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay

Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.     

Insights into kidney stem cell development and regeneration using zebrafish

Kidney disease is an escalating global health problem,for which the formulation of therapeutic approaches using stem cells has received increasing research attention.The complexity of kidney anatomy and function,which includes the diversity of renal cell types,poses formidable challenges in the identification of methods to generate replacement structures.Recent work using the zebrafish has revealed their high capacity to regenerate the integral working units of the kidney,known as nephrons,following acute injury.Here,we discuss these findings and explore the ways that zebrafish can be further utilized to gain a deeper molecular appreciation of renal stem cell biology,which may uncover important clues for regenerative medicine.     

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.     

Selecting the optimal cell for kidney regeneration: fetal, adult or reprogrammed stem cells

Chronic kidney disease (CKD) is a progressive loss in renal function over a period of months or years. End-stage renal disease (ESRD) or stage 5 CKD ensues when renal function deteriorates to under 15% of the normal range. ESRD requires either dialysis or, preferentially, a kidney organ allograft, which is severely limited due to organ shortage for transplantation. To combat this situation, one needs to either increase supply of organs or decrease their demand. Two strategies therefore exist: for those that have completely lost their kidney function (ESRD), we will need to supply new kidneys. Taking into account the kidneys' extremely complex structure, this may prove to be impossible in the near future. In contrast, for those patients that are in the slow progression route from CKD to ESRD but still have functional kidneys, we might be able to halt progression by introducing stem cell therapy to diseased kidneys to rejuvenate or regenerate individual cell types. Multiple cell compartments that fall into three categories are likely to be worthy targets for cell repair: vessels, stroma (interstitium) and nephron epithelia. Different stem/progenitor cells can be linked to regeneration of specific cell types; hematopoietic progenitors and hemangioblastic cell types have specific effects on the vascular niche (vasculogenesis and angiogenesis). Multipotent stromal cells (MSC), whether derived from the bone marrow or isolated from the kidney's non-tubular compartment, may, in turn, heal nephron epithelia via paracrine mechanisms. Nevertheless, as we now know that all of the above lack nephrogenic potential, we should continue our quest to derive genuine nephron (epithelial) progenitors from differentiated pluripotent stem cells, from fetal and adult kidneys and from directly reprogrammed somatic cells.     

Disagreements in the therapeutic use of mesenchymal stem cell-derived secretome

In a recent article, the authors provide a detailed summary of the characteristics and biological functions of mesenchymal stem cells (MSCs), as well as a discussion on the potential mechanisms of action of MSC-based therapies. They describe the morphology, biogenesis, and current isolation techniques of exosomes, one of the most important fractions of the MSC-derived secretome. They also summarize the characteristics of MSC-derived exosomes and highlight their functions and therapeutic potential for tissue/organ regeneration and for kidney, liver, cardiovascular, neurological, and musculoskeletal diseases, as well as cutaneous wound healing. Despite the fact that MSCs are regarded as an important pillar of regenerative medicine, their regenerative potential has been demonstrated to be limited in a number of pathological conditions. The negative effects of MSC-based cell therapy have heightened interest in the therapeutic use of MSC-derived secretome. On the other hand, MSC-derived exosomes and microvesicles possess the potential to have a significant impact on disease development, including cancer. MSCs can interact with tumor cells and promote mutual exchange and induction of cellular markers by exchanging secretome. Furthermore, enzymes secreted into and activated within exosomes can result in tumor cells acquiring new properties. As a result, therapeutic applications of MSC-derived secretomes must be approached with extreme caution.     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

Stem cells and neonatal brain injury

Recent advances in regenerative medicine and in our understanding of neurogenesis may lead to new ways of recovering neuronal function lost or damaged during the perinatal period; such injuries are not amenable to conventional therapies. We review recent experimental studies based on immature rodental models of neonatal brain injury, especially hypoxic-ischemic encephalopathy. The developing brain is revealed to have considerable potential with respect to proliferation and migration to the injured site. However, the generation of fully differentiated neurons is extremely limited after brain injuries. Aggressive efforts to adjust the environment of the damaged brain in which tissue regeneration is occurring or more cautious stem cell transplantation will be required for the successful treatment of developmental brain injury. This work was supported by a Research Grant for Cardiovascular Diseases (18C-1) from the Ministry of Health, Labour and Welfare.     

Chemical modulation of cell fates: in situ regeneration

Chemical modulation of cell fates has been widely used to promote tissue and organ regeneration. Small molecules can target the self-renewal, expansion, differentiation, and survival of endogenous stem cells for enhancing their regenerative power or induce dedifferentiation or transdifferentiation of mature cells into proliferative progenitors or specialized cell types needed for regeneration. Here, we discuss current progress and potential using small molecules to promote in vivo regenerative processes by regulating the cell fate. Current studies of small molecules in regeneration will provide insights into developing safe and efficient chemical approaches for in situ tissue repair and regeneration.     

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.