骨髓干细胞转分化为肾祖细胞并参与修复急性肾脏损伤的研究

目的 观察骨髓干细胞是否可以向肾祖细胞转分化,成为肾脏祖细胞库的肾外来源;验证粒细胞集落刺激因子(G-CSF)是否可以促进骨髓干细胞向肾脏祖细胞的转分化,提高肾脏修复的效能.方法 6周龄全身表达绿色荧光蛋白(GFP)的C57BL/6J转基因小鼠提供骨髓,6~8周龄同种无荧光标记的C57BL/6J小鼠40只作为骨髓受体.骨髓移植前,受体小鼠接受致死剂量的γ放射线137Cs照射,骨髓重建情况经流式细胞仪检测确认.骨髓重建完毕后所有小鼠均接受单侧肾脏缺血再灌注损伤.干细胞动员效果及向肾脏归巢情况经流式细胞仪检测鉴定.损伤4、8周后取肾脏标本行免疫荧光组织化学染色,观察骨髓来源的肾脏祖细胞数以及骨髓细胞在微血管形成中的作用.损伤4周后通过组织切片免疫荧光组织化学方法观察并计数微血管细胞数.结果 G-CSF动员1 d后,分别为CD29、CD34、Sca-1、c-Kit、Flk-1阳性的干细胞占外周血非红系细胞的比例均高于对照组(P<0.05).损伤4周后,G-CSF动员组的肾脏中,骨髓来源并且分别表达Sca-1/GFP、CD29/GFP的干细胞的比例均高于对照组(P<0.05);在损伤4周及8周后,肾脏切片免疫荧光组织化学显示G-CSF动员肾脏中骨髓来源的肾祖细胞即Sca-1/GFP双阳性的细胞数量高于对照组.损伤4周后,动员组肾脏中表达CD31的微血管密度高于对照组(P<0.05).损伤4周后肾脏组织中存在CD105/GFP及α-SMA/GFP双阳性的细胞.结论 ①骨髓干细胞可以转分化为器官特异性干细胞-肾脏祖细胞;②G-CSF可以加速这一转分化的过程,并使损伤肾脏得到更好的修复.     

The regenerative potential of stem cells in acute renal failure

Adult stem cells have been characterized in several tissues as a subpopulation of cells able to maintain. generate, and replace terminally differentiated cells in response to physiological cell turnover or tissue injury. Little is known regarding the presence of stem cells in the adult kidney but it is documented that under certain conditions, such as the recovery from acute injury, the kidney can regenerate itself by increasing the proliferation of some resident cells. The origin of these cells is largely undefined; they are often considered to derive from resident renal stem or progenitor cells. Whether these immature cells are a subpopulation preserved from the early stage of nephrogenesis is still a matter of investigation and represents an attractive possibility. Moreover, the contribution of bone marrow-derived stem cells to renal cell turnover and regeneration has been suggested. In mice and humans, there is evidence that extrarenal cells of bone marrow origin take part in tubular epithelium regeneration. Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. Recent studies have demonstrated that hematopoietic stem cells were mobilized following ischemia/reperfusion and engrafted the kidney to differentiate into tubular epithelium in the areas of damage. The evidence that mesenchymal stem cells, by virtue of their renoprotective property, restore renal tubular structure and also ameliorate renal function during experimental acute renal failure provides opportunities for therapeutic intervention.     

Isolation and characterization of nontubular sca-1+lin- multipotent stem/progenitor cells from adult mouse kidney

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Recently, stage-specific embryonic kidney progenitor tissue was used to regenerate nephrons. Through fluorescence-activated cell sorting, microarray analysis, in vitro differentiation assays, mixed lymphocyte reaction, and a model of ischemic kidney injury, this study sought to identify and characterize multipotent organ stem/progenitor cells in the adult kidney. Herein is reported the existence of nontubular cells that express stem cell antigen-1 (Sca-1). This population of small cells includes a CD45-negative fraction that lacks hematopoietic stem cell and lineage markers and resides in the renal interstitial space. In addition, these cells are enriched for beta1-integrin, are cytokeratin negative, and show minimal expression of surface markers that typically are found on bone marrow-derived mesenchymal stem cells. Global gene profiling reveals enrichment for many genes downstream of developmental signaling molecules and self-renewal pathways, such as TGF-beta/bone morphogenic protein, Wnt, or fibroblast growth factor, as well as for those that are involved in specification of mesodermal lineages (myocyte enhancer factor 2A, YY1-associated factor 2, and filamin-beta). In vitro, they are plastic adherent and slowly proliferating and result in inhibition of alloreactive CD8(+) T cells, indicative of an immune-privileged behavior. Furthermore, clonal-derived lines can be differentiated into myogenic, osteogenic, adipogenic, and neural lineages. Finally, when injected directly into the renal parenchyma, shortly after ischemic/reperfusion injury, renal Sca-1(+)Lin(-) cells, derived from ROSA26 reporter mice, adopt a tubular phenotype and potentially could contribute to kidney repair. These data define a unique phenotype for adult kidney-derived cells, which have potential as stem cells and may contribute to the regeneration of injured kidneys.     

干细胞在肾脏损伤中应用的研究进展

终末期肾病加剧了世界医疗资源的负担。现在迫切需要有效的策略,即通过肾脏再生来预防进一步的肾损伤以及恢复肾功能。除防止肾脏损伤外,再生受损的肾组织对于延缓慢性肾脏病发展到终末期肾衰竭也是非常重要的。肾脏再生的最新进展包括了胚胎干细胞向肾脏细胞定向诱导分化;肾小管损伤后的增殖加强;内皮祖细胞和肾祖细胞在肾损伤修复中的作用;采用间充质干细胞治疗肾病和肾脏组织工程等。然而就目前的研究,在上述过程中依然存在一些争议,如成体上皮干细胞究竟是不是存在;如何探寻并研究最好的肾损伤治疗策略以及如何以最佳的方式使用间充质干细胞来预防肾损伤等。本文总结了干细胞和再生方法在肾损伤中应用研究的最新进展。     

Progenitor/stem cells in renal regeneration and mass lesions

Adult kidneys have limited regenerative capacity following a prominent acute kidney injury. As understanding regenerative mechanisms is the key to discovering therapeutic strategies for preventing and treating renal diseases, in recent years, researchers have hotly debated whether progenitor/stem cells offer a support system for renal repair. The Romagnani-led group identified CD133 stem cells in the parietal epithelium and renal tubules, and their data indicate that these progenitor/stem cells support renal repair in the glomeruli and renal tubules. The Humphreys and Bonventre group used the lineage-tracing technique. They observed no contribution of progenitor cells to tubular repair, but they later reported that the differentiated tubular cells responsible for tubular repair actually gained some characteristics of progenitor cells. This review article will focus on major updates regarding the controversial progenitor/stem cells in renal regeneration and highlight some new progenitor cell issues in renal mass lesions.     

Progenitor cells in the kidney: biology and therapeutic perspectives

The stem cell may be viewed as an engineer who can read the blue print and become the building. The role of this fascinating cell in physiology and pathophysiology has recently attracted a great deal of interest. The archetype of stem cells is the zygote: one cell capable of endless proliferation and differentiation into all tissue types in the human body. Historically, the differentiation of embryonic stem cells is seen as an irreversible process with restricting possibilities for differentiation leading finally to a terminally differentiated cell type. Stem cells have also been described in the adult. They were first defined in tissues with a high cell turnover like skin and gut. Today, stem cells have also been shown in tissues with no or low regenerative potential and turnover, like the kidney. Traditionally, adult stem cells were thought to be restricted in their differentiative and regenerative potential to the tissues in which they reside. However, the stem cell concept is changing rapidly as evidence is mounting that adult stem cells not only reside locally in specific niches, but may also be recruited from the circulation to actively participate in the regeneration of various tissues. Furthermore, reverse differentiation has been demonstrated. This means that highly specialized cell types are able to dedifferentiate and engage in stem cell like activities. Moreover, transdifferentiation of mature cells into different cell types has been reported. This paper will review our current knowledge on renal stem cells and progenitor cells. Specifically, it will discuss the role of progenitor cells and transdifferentiation in renal repair and maintenance. Finally, the potential clinical implications of these findings will be discussed.     

Stem cells and kidney injury

PURPOSE OF REVIEW: The most commonly used therapies in nephrology target the reduction of acute injury, reduction of the rate of progression, or renal replacement therapy. The purpose of this review is to examine new evidence that renal progenitors can be used for therapeutic purposes. Stem cells possess two characteristics, self-renewal and the capacity for multilineage differentiation. They are typically classified as derived from embryos or from the adult. RECENT FINDINGS: New studies on embryonic stem cells show that they can be use to enrich for specific renal progenitors, which integrate into mature structures. Studies on adult stem cells show that almost all kidney cell types can be renewed by adult stem cells originating in bone marrow. Moreover, some animal studies demonstrate that a phenotype such as the aging and diabetic phenotype can be transferred from progenitors residing in the bone marrow, suggesting that the bone marrow contains renal progenitors that may be useful for therapeutic purposes. SUMMARY: Stem cell therapy opens the door to regenerative nephrology. Embryonic stem cells are a useful tool to determine the pathways to convert a pluripotent stem cell into renal progenitors. Adult stem cells in the bone marrow or in a specific kidney niche may provide a source of stem cells with a therapeutic potential.     

The contribution of adult stem cells to renal repair

The kidney undergoes continuous, slow cellular turnover for tissue maintenance and rapid cell replacement after injury. The cellular origin of newly differentiated tubular epithelium remains controversial. In some non-renal organs, adult stem cells are recognized as the cell of origin for tissue replacement, such as the hematopoietic system, intestine and skin. These findings have prompted intense investigation for evidence of renal stem cells because of the great need for new therapeutic approaches to treat acute kidney injury and chronic kidney disease. Early excitement at reports that bone marrow-derived cells transdifferentiate into renal epithelial cells has been tempered by findings that show such events to be rare or potentially explained by cell fusion. More recent studies have focused on the possibility that renal progenitors exist within the kidney. In this review we compare data supporting the existence of adult renal stem cells with the body of evidence indicating that the kidney regenerates by self-duplication of differentiated cells. The identification of adult renal epithelial progenitor cells will ultimately determine the future direction of renal regenerative medicine.     

Lessons learned about adult kidney stem cells from the malpighian tubules of Drosophila

All multicellular organisms have a specialized organ to concentrate and excrete wastes from the body. The kidneys in vertebrates and the malpighian tubules in Drosophila accomplish these functions. Mammals and Drosophila share some similar features during renal tubular development. Vertebrate kidneys are derived through the mutual induction of the ureteric bud and metanephric mesoderm, whereas the malpighian tubules of Drosophila develop from the hindgut primordium and visceral mesoderm. The vertebrate kidney also has the capacity to recover and regenerate following episodes of acute injury. Previous studies suggest that stem cells and progenitor cells may be involved in the repair and regeneration of injured renal tissue. However, studies differ as to the source of the regenerating renal cells. Recently, multipotent stem cells in Drosophila malpighian tubules were identified, and it was demonstrated that several differentiated cells in the malpighian tubules arise from these stem cells. In this article, the current understanding of kidney development and stem cell fate in mammal and Drosophila is compared. Furthermore, the potential application of the adult renal stem cells in kidney repair and the treatment of kidney cancers are discussed.     

Experimental renal progenitor cells: Repairing and recreating kidneys?

Strategies to facilitate repair or generate new nephrons are exciting prospects for acute and chronic human renal disease. Repair of kidney injury involves not just local mechanisms but also mobilisation of progenitor/stem cells from intrarenal niches, including papillary, tubular and glomerular locations. Diverse markers characterise these unique cells, often including CD24 and CD133. Extrarenal stem cells may also contribute to repair, with proposed roles in secreting growth factors, transfer of microvesicles and exosomes and immune modulation. Creating new nephrons from stem cells is beginning to look feasible in mice in which kidneys can be dissociated into single cells and will then generate mature renal structures when recombined. The next step is to identify the correct human markers for progenitor cells from the fetus or mature kidney with similar potential to form new kidneys. Intriguingly, development can continue in vivo: whole foetal kidneys and recombined organs engraft, develop a blood supply and grow when xenotransplanted, and there are new advances in decellularised scaffolds to promote differentiation. This is an exciting time for human kidney repair and regeneration. Many of the approaches and techniques are in their infancy and based on animal rather than human work, but there is a rapid pace of discovery, and we predict that therapies based on advances in this field will come into clinical practice in the next decade.     

Innovation in the Treatment of Uremia: Proceedings from the Cleveland Clinic Workshop: Cellular Therapy of Kidney Diseases

The understanding of cellular sources of kidney regeneration has rapidly evolved in the last decade. It is now believed that regeneration occurs predominantly from cells that reside within the injured kidney, with minimal contribution from extra‐renal cells. We now know that improved kidney regeneration seen following exogenous administration of stem cells occur predominantly by noncellular paracrine mechanisms. Of all extra‐renal stem cells, mesenchymal stem cells (MSC) are the most promising stem cell type for treating kidney diseases. There is an ongoing clinical trial evaluating safety and efficacy of MSC in treating acute kidney injury (AKI). Results of this trial are expected to bring use of MSC closer to the clinical realm. An improved understanding of the small molecules that facilitate kidney regeneration and are secreted by MSC will likely result in the development of new therapies for treating AKI. Identification of adult stem cell markers will result in improved understanding of pathophysiology of kidney diseases and could lead to the development of new cellular therapies. Directed differentiation of stem cells into desired cell types such as erythropoietin producing cells will allow selective replacement of lost kidney function. Cell‐based therapies for patients with chronic kidney disease are presently in proof‐of‐principle stage and are expected to evolve in the coming years with improved understanding of stem cell biology. Technological advancement in cellular therapy is expected to provide improved therapeutic options for patients with kidney diseases in the near future.     

Adult stem cells in renal injury and repair

There has been considerable focus on the ability of bone marrow-derived cells to differentiate into non-haematopoietic cells of various tissue lineages, including cells of the kidney. This growing evidence has led to a reconsideration of the source of cells contributing to renal repair following injury. The kidney has an inherent ability for recovery and regeneration following acute damage. It is thought that dedifferentiation of glomerular and tubular cells to a more embryonic/mesenchymal phenotype represent key processes for recovery in response to damage. However, there has been much contention as to the source of regenerating renal cells. The present review focuses on new aspects of the plasticity of intrinsic renal cells and their role in renal remodelling and scarring. Growing support also suggests that bone marrow-derived cells have the ability to contribute to structural and functional repair following acute renal failure. Evidence for bone marrow cell engraftment in the repairing kidney leading to incorporation into a variety of tissue types is discussed. Because cell death and fibrosis is a common end-point in a variety of acute and chronic renal nephropathies, the paradigm of stem cell plasticity may have important implications in the cellular and pathological mechanisms of renal injury and repair. A better understanding of the processes controlling extra-renal cell engraftment and intrinsic renal cell differentiation may provide important clues for the development of new cell-based therapies in the field of renal reparative medicine.     

Renal differentiation from adult spermatogonial stem cells

Abstract

There is considerable interest in the use of multi-potent stem cells in kidney tissue regeneration. We studied if spermatogonial stem cells have the ability to undergo kidney differentiation. Spermatogonial stem cell differentiation was induced using in vitro and ex vivo co-culture techniques. Conditioned media from human kidney fibroblasts induced the expression of epithelial and endothelial lineages in spermatogonial stem cells, consistent with nephrogenesis. Furthermore, we showed that these cells up-regulated renal tubular-specific markers alkaline phosphatase, mineralocorticoid receptor, renal epithelial sodium channel and sodium-glucose transporter-2 (p?<?0.05). GFP-labeled spermatogonial stem cells were engrafted into metanephric kidney organ cultures harvested from E12.5 mouse embryos. After 5 days of organ culture, focal anti-GFP staining was detectable in all inoculated kidneys demonstrating integration of spermatogonial stem cells into the developing kidney (p?<?0.01). Histological assessment showed early nephron-like architecture. In summary, we show that spermatogonial stem cells have the potential to generate renal tissue and lay the foundations for further investigations into a novel therapeutic approach for renal insufficiency.     

Normal distribution and medullary-to-cortical shift of Nestin-expressing cells in acute renal ischemia

Nestin, a marker of multi-lineage stem and progenitor cells, is a member of intermediate filament family, which is expressed in neuroepithelial stem cells, several embryonic cell types, including mesonephric mesenchyme, endothelial cells of developing blood vessels, and in the adult kidney. We used Nestin-green fluorescent protein (GFP) transgenic mice to characterize its expression in normal and post-ischemic kidneys. Nestin-GFP-expressing cells were detected in large clusters within the papilla, along the vasa rectae, and, less prominently, in the glomeruli and juxta-glomerular arterioles. In mice subjected to 30 min bilateral renal ischemia, glomerular, endothelial, and perivascular cells showed increased Nestin expression. In the post-ischemic period, there was an increase in fluorescence intensity with no significant changes in the total number of Nestin-GFP-expressing cells. Time-lapse fluorescence microscopy performed before and after ischemia ruled out the possibility of engraftment by the circulating Nestin-expressing cells, at least within the first 3 h post-ischemia. Incubation of non-perfused kidney sections resulted in a medullary-to-cortical migration of Nestin-GFP-positive cells with the rate of expansion of their front averaging 40 microm/30 min during the first 3 h and was detectable already after 30 min of incubation. Explant matrigel cultures of the kidney and aorta exhibited sprouting angiogenesis with cells co-expressing Nestin and endothelial marker, Tie-2. In conclusion, several lines of circumstantial evidence identify a sub-population of Nestin-expressing cells with the mural cells, which are recruited in the post-ischemic period to migrate from the medulla toward the renal cortex. These migrating Nestin-positive cells may be involved in the process of post-ischemic tissue regeneration.     

Insights into the role of bone marrow-derived stem cells in renal repair

Acute kidney injury (AKI) is a frequent clinical problem with a high mortality rate, generally caused by ischemic insults. Nevertheless, the kidney has a remarkably high capacity to regenerate after ischemic injury. Tubular cells can restore renal function by proliferation and dedifferentiation into a mesenchymal cell type, but also stem cells residing in bone marrow may contribute. We compiled a protocol from several published methods to study the contribution of bone marrow-derived cells to renal regeneration. Bone marrow was isolated from donor FVB mice and labeled with enhanced green fluorescent protein (eGFP) through adenovirus transduction. After cell sorting, eGFP-labeled cells were transplanted in sublethally irradiated recipient FVB mice. Four weeks after transplantation, we provoked AKI in mice by inducing unilateral ischemic-reperfusion injury for 30 min. Seven days after the injury, eGFP-positive bone marrow-derived cells were clearly detectable in ischemic kidney tissue, and they contribute to the regeneration of approximately 10% of proximal tubular mass. In this review the advantages and shortcomings of our procedure are critically discussed and compared with other methods described.     

Stem cell-based cell therapy for spinal cord injury

Traumatic injuries to the spinal cord lead to severe and permanent neurological deficits. Although no effective therapeutic option is currently available, recent animal studies have shown that cellular transplantation strategies hold promise to enhance functional recovery after spinal cord injury (SCI). This review is to analyze the experiments where transplantation of stem/progenitor cells produced successful functional outcome in animal models of SCI. There is no consensus yet on what kind of stem/progenitor cells is an ideal source for cellular grafts. Three kinds of stem/progenitor cells have been utilized in cell therapy in animal models of SCI: embryonic stem cells, bone marrow mesenchymal stem cells, and neural stem cells. Neural stem cells or fate-restricted neuronal or glial progenitor cells were preferably used because they have clear capacity to become neurons or glial cells after transplantation into the injured spinal cord. At least a part of functional deficits after SCI is attributable to chronic progressive demyelination. Therefore, several studies transplanted glial-restricted progenitors or oligodendrocyte precursors to target the demyelination process. Directed differentiation of stem/progenitor cells to oligodendrocyte lineage prior to transplantation or modulation of microenvironment in the injured spinal cord to promote oligodendroglial differentiation seems to be an effective strategy to increase the extent of remyelination. Transplanted stem/progenitor cells can also contribute to promoting axonal regeneration by functioning as cellular scaffolds for growing axons. Combinatorial approaches using polymer scaffolds to fill the lesion cavity or introducing regeneration-promoting genes will greatly increase the efficacy of cellular transplantation strategies for SCI.     

Bioartificial kidney for full renal replacement therapy

The rapid understanding of the cellular and molecular basis of organ function and disease processes will be translated in the next millennium into new therapeutic approaches to a wide range of clinical disorders, including acute and chronic renal failure. Central to these new therapies are the developing fields of gene therapy, cell therapy, and tissue engineering. These new technologies are based on the ability to expand stem or progenitor cells in tissue culture to perform differentiated tasks and to introduce these cells into the patient either in extracorporeal circuits or as implantable constructs. Cell therapy devices are currently being developed to replace the filtrative, metabolic, and endocrinologic functions of the kidney lost in both acute and chronic renal failure. This article summarizes the current state of device development for a renal tubule assist device, a bioartificial hemofilter, and a regulatable erythropoietin cell therapy device. These individual devices have the promise to be combined to produce a wearable or implantable bioartificial kidney for full renal replacement therapy. These new approaches may result in therapeutic modalities that significantly diminish the morbidity and mortality in patients with acute renal failure or end-stage renal disease.     

Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia

Embryonic stem (ES) cells have been induced to differentiate in vitro into a broad spectrum of specialized cell types, including hematopoietic, pancreatic, and neuronal cell types. Such ES-derived cells can provide a valuable source of progenitor cell types. Whereas undifferentiated ES cells can become integrated into a developing kidney and contribute to tubular epithelia, the ability to generate renal precursor cells in vitro has not been reported. This study used a combination of nephrogenic growth factors to differentiate ES cells into renal epithelial cells that are capable of integrating into a developing kidney with very high efficiency. Using a combination of retinoic acid, Activin-A, and Bmp7, cultured ES cells can be induced to express markers specific for the intermediate mesoderm, from which the kidneys arise. Embryoid bodies that are cultured in the presence of nephrogenic factors can respond to inductive signals and form epithelial structures in vitro. When injected into developing kidney rudiments, treated ES cells contribute to tubular epithelia with near 100% efficiency. These methods may facilitate the large-scale culture of renal epithelial precursor cells for a variety of applications.