Kidney repair using stem cells

143/jnonline – Thorough CriTiCal appraisalEPHROL JN (2010;:02)143-14623© 2010 Società Italiana di Nefrologia - ISSN 1121-8428Kidney repair using stem cells:myth or reality as a therapeutic option?hirotsugu iwatani, Enyu imaiDepartment of Nephrology, Osaka University Graduate School of Medicine, Suita, Osaka - JapanI ntroductIonThe kidney has been considered a highly terminally differen-tiated organ of the body, and indeed its proliferative potential is much lower than that of epithelial cells of the intestine. The-se characteristics of kidney are borne out by the incidence of malignant tumors, as the kidney is less likely to be the origin of a tumor than is the gastrointestinal tract. The lower prolife-rative fate of the kidney may be due to gene programming of its constituent cells. More precisely, partial and programmed gene inactivation of genomic DNA that is controlled in time and space, may be involved in the regulation of kidney fate. For these reasons, kidney has been thought of as a most un-likely organ for regeneration. Moreover, regeneration of cells alone is not sufficient for organ regeneration, especially for regeneration of the kidney, because it is composed of manycell types that function as a tissue unit and not as individual cells, making up a highly organized and elaborately structu-red organ with a sophisticated architecture. Restoration of organ architecture, which requires the placement of specific cell types adjacent to each other in a proper spatial fashion, is extremely important for kidney regeneration.However, the kidney has some regenerative potential. It can recover from acute kidney injury such as acute tubular ne-crosis. As for chronic kidney disease, if diabetic nephropathy patients can maintain long-term optimal blood glucose levels by pancreas transplantation, then the amelioration of the le-sions of diabetic nephropathy has been observed (1). The-refore, although the capacity of the kidney for proliferation is lower than that of other organs, renal repair is both theoreti-cally and actually possible. What remains to be determined is how to elicit the regenerative potential of damaged kidney and how to use this potential to the best advantage.Stem/progenitor cells and the proper microenvironment are essential for the repair or regeneration of damaged kidney. A suitable microenvironment includes the presence of lo-cal cytokines as well as of extracellular matrix which can function as a scaffold and give rise to cytokine or growth factors around the stem/progenitor cell.b one mArrow -derIved stem cellsThere have been many attempts to establish the existence of extrarenal kidney stem/progenitor cells. In the early 2000s, pluripotent bone marrow–derived stem cells were thought to contribute to kidney repair. Bone marrow includes several cell types such as hematopoietic stem cells (HSCs), mesen-chymal stromal cells (MSCs), which is often referred to as mesenchymal stem cells, and endothelial progenitor cells.Bone marrow–derived stem cells appeared to have a capaci-ty for transdifferentiation and to be able to replace damaged renal tissue by replacing tubular epithelial cells (2), mesangial cells (3, 4), endothelial cells (5) and even podocytes (6, 7). The experimental method used to analyze their ability to mediate kidney repair was the transplantation of bone marrow–derived stem cells. In many cases, cells of donor origin were tracedIwatani and Imai: Myth or reality as a therapeutic option?by labeling the cells with green fluorescent protein or, if the donor was male, Y chromosome–positive cells were identified by in situ hybridization. Engraftment of bone marrow–derived stem cells to kidney component cells is often confirmed by double staining the cells of donor origin and kidney-specific markers. Investigation of the DNA karyotype of engrafted cells confirmed the existence of double the content of the normal genomic DNA. Therefore, cell fusion events appear to represent a part of the previously reported transdifferentiation cases. The strict meaning of transdifferentiation is conversion from one cell lineage to another, different lineage with con-version of cellular functions and markers and maintenance of a normal karyotype. Although, cell phenotype is transmitted via the transplant method, it appears that cell fusion as well as transdifferentiation occurs under these circumstances. In studies of this period, the concept of transdifferentiation was loosely defined, and clear transdifferentiation was not demonstrated in many of the studies. These studies should therefore be interpreted with caution.m esenchymAl stromAl cells Transplantation of bone marrow–derived pluripotent MSCs, but not HSCs, has been shown to result in engraftment to tubular epithelial cells in an experimental kidney injury model and did induce recovery of lost renal function in re-cipients (8). Although bone marrow contains several cell types including HSC, MSC and endothelial progenitor cells, engraftment of bone marrow–derived cells to kidney com-ponent cells is most likely to have been due to the MSC population (8, 9). Therefore, MSC are more promising as a source of stem/progenitor cells than crude bone marrow–derived cells. Furthermore, it has recently been reported that kidney-derived MSCs contribute to vasculogenesis, angiogenesis and endothelial repair (10). This finding sup-ports the possibility that MSCs residing in kidney can par-ticipate in kidney repair or regeneration.t herApeutIc mechAnIsm of cell trAnsplAntAtIonRecent studies argue against direct transdifferentiation of bone marrow–derived stem cells or MSCs into kidney. The engraftment frequency of bone marrow–derived stem cells was reported to be relatively small, ranging from 3% to 22% (11). This finding means that the majority of the regenerating cells were derived from resident kidney cells and not from extrarenal cells such as bone marrow–derived cells. Fur-thermore, the observed improvement in renal function did not appear to be attained via the engrafted cells but through other mechanisms. Studies of a renal ischemia/reperfusion model indicated that endogenous kidney cells rather than bone marrow–derived cells contributed to the repair of tubu-lar epithelial cells (12). Although the percentage of engraft-ment of donor cells to kidney component cells is very small, functional recovery of the injured kidney is apparent when MSCs are transplanted. This may be because various cyto-kines excreted by MSCs may make an appropriate microen-vironment in autocrine or paracrine fashion and finally work to promote kidney repair not as a cell basis. Indeed, MSCs have the ability to secrete various cytokines such as vascu-lar endothelial growth factor (VEGF), hepatocyte growth fac-tor (HGF) and insulin-like growth factor 1 (IGF-1) (13). Con-sistent with a potential cytokine-producing role for MSCs, it has been reported that injection of bone marrow–derived stromal cells into the peritoneal cavity reduced the severity of cisplatin-induced acute renal failure without any integra-tion of the donor cells into tubular cells. Furthermore, the conditioned media obtained from culture of these stromal cells induced migration and proliferation of kidney-derived epithelial cells and significantly diminished cisplatin-induced proximal tubule cell death (14). The findings of this study the-refore provide new evidence that it is humoral factors pro-duced from bone marrow–derived MSCs, and not the bone marrow–derived MSC themselves, which are important for kidney repair or recovery. The combined reports suggest that what is important for kidney repair or recovery is not the local engraftment of MSCs to kidney, but secretion of humo-ral factors by MSCs that modulate the injured kidney.w hAt Are the humorAl fActors?If this is the case, then what are the humoral factors that are important for mediating kidney repair or regeneration? Many factors have been proposed as candidate factors to fulfill this role. One report indicated that administration of MSCs to a rat model of ischemia-reperfusion-induced acu-te renal failure improved renal function, whereas admini-stration of syngeneic fibroblasts did not. This study further indicated that the growth factors VEGF, HGF and IGF-1 were more highly expressed in MSCs than in fibroblasts (13). Some of these factors are known to modulate kidney function or repair. For example, VEGF attenuates glomeru-lar inflammation and accelerates glomerular capillary repair (15). HGF, an angiogenic growth factor, prevents epithelial cell death and enhances regeneration and remodeling of injured or fibrotic renal tissue (16). The described effects of these growth factors on kidney regeneration are effects of the administration of individual growth factors. However, if a mixture of several of these growth factors were to be admi-144145EPHROL JN (2010;:02)143-14623nistered, or if the timing of their administration was altered, their effects would be more complicated. Recently, other than soluble factors, a new mechanism of communication among cells was proven: microvesicles. Microvesicles de-rived from MSCs activate the proliferation of surviving renal tubular cells after injury by transferring mRNA (17). Thus, MSCs can repair the damaged tubules by secreting micro-vesicles which function in a paracrine fashion.r enAl stem /progenItor cellsA second question is, what are the cellular targets of these humoral factors? Of course, these targets would be renal stem/progenitor cells. Many researchers have taken on the challenge of searching for resident tissue stem/progenitor cells in the kidney. Using the characteristics that stem cells must be label-retaining cells because of slow-cycling, renal stem cells have been reported to exist in renal papilla (18) or tubular epithelial cells (19). Bowman’s capsule (20) and the S3 segment of the proximal tubules (21) have also been re-ported to contain renal stem/progenitor cells. CD133+ CD24+ cells within the population of parietal epithelial cells are re-ported to engraft to tubular cells. Analysis of side population (SP) phenotypes has been adopted as an approach to search for renal stem cells that have no markers. Under our expe-rimental conditions, kidney-derived SP cells did not convert to kidney component cells (22). It has also been reported that kidney-derived SP cells can differentiate into multiple lineages in vitro and that injection of these cells ameliorates lost renal function without significant engraftment to kid-ney component cells (23). These data indicate that humoral factors may account for the observed improvement in renal function, and SP cells are essentially heterogeneous. It still remains elusive as to whether kidney-derived SP cells are stem cells of the kidney.e xtrArenAl source of stem cellsIf an extrarenal source of stem cells is required for kidney repair, the target cells will be embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. The ES cells, which are derived from the inner cell mass of the blastocyst, are pluri-potent. However, transplantation of nondifferentiated ES cel-ls induces teratomas. Therefore, it is essential to differentiate ES cells to some degree before transplantation. Cultured ES cells can be induced to express markers of the intermediate mesoderm from which kidneys arise, by the application of retinoic acid, activin-A and Bmp7. Moreover, when injected into a developing metanephros, ES cells are incorporated into tubular epithelial cells with almost 100% efficiency (24).ES cells are therefore one of the best cell sources for kidney repair and represent a powerful tool for this purpose. Howe-ver, there are ethical and legal problems in the creation and manipulation of human ES cells. An alternative to ES cells are the recently developed iPS cells (25). Retroviral transfer of just 4 genes (Oct3/4, Sox2, Klf4 and c-Myc ) into cultured somatic cells converts the cells into pluripotent ES-like cel-ls. The 4 genes in question are transcription factors related to pluripotency. It was later shown that iPS cells can be cre-ated without addition of the oncogene c-Myc (26). Recently, human iPS cells have been successfully induced from skin fibroblasts (27). Since iPS cells, similar to ES cells, are pluri-potent and, furthermore, can be created from adult somatic cells, it is possible to prepare patient-specific pluripotent cells without manipulating germ cells. This means that there is no ethical problem with the use of iPS cells, and therefore iPS cells are a very promising source of cells for kidney re-pair or regeneration. However, even if a cell source for kid-ney repair is available, the induction or differentiation of this cell into kidney remains a difficult problem. One major pro-blem is that the risk of forming teratomas from transplanted undifferentiated iPS cells is just as high as it is for ES cells (25). Nevertheless, successful differentiation of iPS cells into cells of the cardiovascular and hematopoietic lineages has been reported (28-30).In conclusion, iPS cells are one of the most promising sources of stem cells for kidney repair and regeneration. Ho-wever, the method of induction of stem/progenitor cells to differentiated kidney component cells remains to be clarified. Therefore, the most important future issue is identification of tissue stem/progenitor cells in the kidney and recruitment of these cells in response to injury. A detailed observation and elucidation of the microenvironment of kidney cells after injury would then be the second step toward kidney repair using stem cells. Highly innovative techniques or approaches that are capable of grasping and reorganizing multifactorial changes in the milieu of time and space around the stem cells would be essential to ultimately solve this issue.Financial support: No financial support.Conflict of interest statement: None declared.Address for correspondence:Hirotsugu Iwatani, MD, PhD Department of NephrologyOsaka University Graduate School of Medicine Box B62-2 Yamadaoka, Suita Osaka 565-0871, Japan hiro@kid.med.osaka-u.ac.jpIwatani and Imai: Myth or reality as a therapeutic option?r eferencesFioretto P, Steffes MW, Sutherland DE, Goetz FC, Mauer M.1.Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med. 1998;339:69-75.Poulsom R, Forbes SJ, Hodivala-Dilke K, et al. Bone marrow 2.contributes to renal parenchymal turnover and regeneration.J Pathol. 2001;195:229-235.Imasawa T, Utsunomiya Y, Kawamura T, et al. 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