Mitochondria Transplantation Mitigates Damage in an In Vitro Model of Renal Tubular Injury and in an Ex Vivo Model of DCD Renal Transplantation.

OBJECTIVES: To test whether mitochondrial transplantation (MITO) mitigates damage in 2 models of acute kidney injury (AKI). BACKGROUND: MITO is a process where exogenous isolated mitochondria are taken up by cells. As virtually any morbid clinical condition is characterized by mitochondrial distress, MITO may find a role as a treatment modality in numerous clinical scenarios including AKI. METHODS: For the in vitro experiments, human proximal tubular cells were damaged and then treated with mitochondria or placebo. For the ex vivo experiments, we developed a non-survival ex vivo porcine model mimicking the donation after cardiac death renal transplantation scenario. One kidney was treated with mitochondria, although the mate organ received placebo, before being perfused at room temperature for 24 hours. Perfusate samples were collected at different time points and analyzed with Raman spectroscopy. Biopsies taken at baseline and 24 hours were analyzed with standard pathology, immunohistochemistry, and RNA sequencing analysis. RESULTS: In vitro, cells treated with MITO showed higher proliferative capacity and adenosine 5'-triphosphate production, preservation of physiological polarization of the organelles and lower toxicity and reactive oxygen species production. Ex vivo, kidneys treated with MITO shed fewer molecular species, indicating stability. In these kidneys, pathology showed less damage whereas RNAseq analysis showed modulation of genes and pathways most consistent with mitochondrial biogenesis and energy metabolism and downregulation of genes involved in neutrophil recruitment, including IL1A, CXCL8, and PIK3R1. CONCLUSIONS: MITO mitigates AKI both in vitro and ex vivo.

Identification and Characterization of the Wilms Tumor Cancer Stem Cell.

A nephrogenic progenitor cell (NP) with cancer stem cell characteristics driving Wilms tumor (WT) using spatial transcriptomics, bulk and single cell RNA sequencing, and complementary in vitro and transplantation experiments is identified and characterized. NP from WT samples with NP from the developing human kidney is compared. Cells expressing SIX2 and CITED1 fulfill cancer stem cell criteria by reliably recapitulating WT in transplantation studies. It is shown that self-renewal versus differentiation in SIX2+CITED1+ cells is regulated by the interplay between integrins ITGß1 and ITGß4. The spatial transcriptomic analysis defines gene expression maps of SIX2+CITED1+ cells in WT samples and identifies the interactive gene networks involved in WT development. These studies define SIX2+CITED1+ cells as the nephrogenic-like cancer stem cells of WT and points to the renal developmental transcriptome changes as a possible driver in regulating WT formation and progression.

The spatially resolved transcriptional profile of acute T cell-mediated rejection in a kidney allograft.

Analysis of the transcriptional profile of graft biopsies represents a promising strategy to study T cell-mediated-rejection (TCMR), also known as acute cellular rejection. However, bulk RNA sequencing of graft biopsies may not capture the focal nature of acute rejection. Herein, we used the whole exome GeoMX Digital Space Profiling platform to study five tubular and three glomerular regions of interest in the kidney graft biopsy from a patient with a chronic-active TCMR episode and in analogous areas from two different normal kidney control biopsies. All kidney sections were from paraffin blocks. Overall, inflammatory genes were significantly upregulated in the tubular areas of the TCMR biopsy and showed an enrichment for gene-ontology terms associated with T-cell activation, differentiation, and proliferation. Enrichment analysis of the 100 genes with the highest coefficient of variation across the TCMR tubular regions of interest revealed that these highly variable genes are involved in kidney development and injury and interestingly do not associate with the 2019 Banff classification pathology scores within the individual regions of interest. Spatial transcriptomics allowed us to unravel a previously unappreciated variability across different areas of the TCMR biopsy related to the graft response to the alloimmune attack, rather than to the immune cells. Thus, our approach has the potential to decipher clinically relevant, new pathogenic mechanisms, and therapeutic targets in acute cellular rejection and other kidney diseases with a focal nature.

Glomerular endothelial cell heterogeneity in Alport syndrome.

Glomerular endothelial cells (GEC) are a crucial component of the glomerular physiology and their damage contributes to the progression of chronic kidney diseases. How GEC affect the pathology of Alport syndrome (AS) however, is unclear. We characterized GEC from wild type (WT) and col4α5 knockout AS mice, a hereditary disorder characterized by progressive renal failure. We used endothelial-specific Tek-tdTomato reporter mice to isolate GEC by FACS and performed transcriptome analysis on them from WT and AS mice, followed by in vitro functional assays and confocal and intravital imaging studies. Biopsies from patients with chronic kidney disease, including AS were compared with our findings in mice. We identified two subpopulations of GEC (dimtdT and brighttdT) based on the fluorescence intensity of the TektdT signal. In AS mice, the brighttdT cell number increased and presented differential expression of endothelial markers compared to WT. RNA-seq analysis revealed differences in the immune and metabolic signaling pathways. In AS mice, dimtdT and brighttdT cells had different expression profiles of matrix-associated genes (Svep1, Itgß6), metabolic activity (Apom, Pgc1α) and immune modulation (Apelin, Icam1) compared to WT mice. We confirmed a new pro-inflammatory role of Apelin in AS mice and in cultured human GEC. Gene modulations were identified comparable to the biopsies from patients with AS and focal segmental glomerulosclerosis, possibly indicating that the same mechanisms apply to humans. We report the presence of two GEC subpopulations that differ between AS and healthy mice or humans. This finding paves the way to a better understanding of the pathogenic role of GEC in AS progression and could lead to novel therapeutic targets.

Amniotic fluid cells: current progress and emerging challenges in renal regeneration.

Amniotic fluid (AF) contains a heterogeneous population of cells that have been identified to possess pluripotent and progenitor-like characteristics. These cells have been applied in various regenerative medicine applications ranging from in vitro cell differentiation to tissue engineering to cellular therapies for different organs including the heart, the liver, the lung, and the kidneys. In this review, we examine the different methodologies used for the derivation of amniotic fluid stem cells and renal progenitors, and their application in renal repair and regeneration. Moreover, we discuss the recent achievements and newly emerging challenges in our understanding of their biology, their immunoregulatory characteristics, and their paracrine-mediated therapeutic potential for the treatment of acute and chronic kidney diseases.

Thyroid Hormone Receptor Alpha is Essential to Maintain the Satellite Cell Niche During Skeletal Muscle Injury and Sarcopenia of Aging.

BACKGROUND: Myopathic changes are commonly described in hypothyroid and hyperthyroid patients, including muscular atrophy and weakness. Satellite cells (SCs) play a major role in skeletal muscle maintenance and regeneration after injury. A mouse model of resistance to thyroid hormone-TRα1PV demonstrated impaired skeletal muscle regeneration after injury with significant reduction of SCs, suggesting that exhaustion of the SC pool contributes to the impaired regeneration. To test this hypothesis, SC activation and proliferation were analyzed in vivo in response to skeletal muscle injury and during aging. METHODS: SCs of TRα1PV male mice were analyzed four days after cardiotoxin-induced muscle injury, and they were compared to wild-type (WT) male animals. TRα-knockdown C2C12 myoblasts were injected into injured skeletal muscle, and four days after transplantation, the in vivo behavior was compared to control C2C12 myoblasts. Skeletal muscle regeneration was compared in younger and older TRα1PV and WT animals. RESULTS: The total number of SCs in skeletal muscle of TRα1PV mice was significantly lower than control, both before and shortly after muscle injury, with significant impairment of SC activation, consistent with SC pool exhaustion. TRα-knockdown myoblasts showed impaired in vivo proliferation and migration. TRα1PV mice had skeletal muscle loss and significant impairment in skeletal muscle regeneration with aging. This translated to a significant reduction of the SC pool with aging compared to WT mice. CONCLUSION: TRα plays an important role in the maintenance of the SC pool. Impaired skeletal muscle regeneration in TRα1PV mice is associated with insufficient SC activation and proliferation, as well as the progressive loss of the SC pool with aging. Regulation of the SC pool and SC proliferation provides a therapeutic target to enhance skeletal muscle regeneration and possibly slow age-associated sarcopenia.

Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury.

Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and ß, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and ß, increased myoblast differentiation whereas GC1, a selective TRß agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/ß-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRß PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration.

A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan.

Prolonged fasting (PF) promotes stress resistance, but its effects on longevity are poorly understood. We show that alternating PF and nutrient-rich medium extended yeast lifespan independently of established pro-longevity genes. In mice, 4 days of a diet that mimics fasting (FMD), developed to minimize the burden of PF, decreased the size of multiple organs/systems, an effect followed upon re-feeding by an elevated number of progenitor and stem cells and regeneration. Bi-monthly FMD cycles started at middle age extended longevity, lowered visceral fat, reduced cancer incidence and skin lesions, rejuvenated the immune system, and retarded bone mineral density loss. In old mice, FMD cycles promoted hippocampal neurogenesis, lowered IGF-1 levels and PKA activity, elevated NeuroD1, and improved cognitive performance. In a pilot clinical trial, three FMD cycles decreased risk factors/biomarkers for aging, diabetes, cardiovascular disease, and cancer without major adverse effects, providing support for the use of FMDs to promote healthspan.

Amniotic fluid stem cells prevent ß-cell injury.

BACKGROUND AIMS: The contribution of amniotic fluid stem cells (AFSC) to tissue protection and regeneration in models of acute and chronic kidney injuries and lung failure has been shown in recent years. In the present study, we used a chemically induced mouse model of type 1 diabetes to determine whether AFSC could play a role in modulating ß-cell injury and restoring ß-cell function. METHODS: Streptozotocin-induced diabetic mice were given intracardial injection of AFSC; morphological and physiological parameters and gene expression profile for the insulin pathway were evaluated after cell transplantation. RESULTS: AFSC injection resulted in protection from ß-cell damage and increased ß-cell regeneration in a subset of mice as indicated by glucose and insulin levels, increased islet mass and preservation of islet structure. Moreover, ß-cell preservation/regeneration correlated with activation of the insulin receptor/Pi3K/Akt signaling pathway and vascular endothelial growth factor-A expression involved in maintaining ß-cell mass and function. CONCLUSIONS: Our results suggest a therapeutic role for AFSC in preserving and promoting endogenous ß-cell functionality and proliferation. The protective role of AFSC is evident when stem cell transplantation is performed before severe hyperglycemia occurs, which suggests the importance of early intervention. The present study demonstrates the possible benefits of the application of a non-genetically engineered stem cell population derived from amniotic fluid for the treatment of type 1 diabetes mellitus and gives new insight on the mechanism by which the beneficial effect is achieved.

A novel source of cultured podocytes.

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.

Amniotic fluid stem cells inhibit the progression of bleomycin-induced pulmonary fibrosis via CCL2 modulation in bronchoalveolar lavage.

The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.

Injection of amniotic fluid stem cells delays progression of renal fibrosis.

Injection of amniotic fluid stem cells ameliorates the acute phase of acute tubular necrosis in animals by promoting proliferation of injured tubular cells and decreasing apoptosis, but whether these stem cells could be of benefit in CKD is unknown. Here, we used a mouse model of Alport syndrome, Col4a5(-/-) mice, to determine whether amniotic fluid stem cells could modify the course of progressive renal fibrosis. Intracardiac administration of amniotic fluid stem cells before the onset of proteinuria delayed interstitial fibrosis and progression of glomerular sclerosis, prolonged animal survival, and ameliorated the decline in kidney function. Treated animals exhibited decreased recruitment and activation of M1-type macrophages and a higher proportion of M2-type macrophages, which promote tissue remodeling. Amniotic fluid stem cells did not differentiate into podocyte-like cells and did not stimulate production of the collagen IVa5 needed for normal formation and function of the glomerular basement membrane. Instead, the mechanism of renal protection was probably the paracrine/endocrine modulation of both profibrotic cytokine expression and recruitment of macrophages to the interstitial space. Furthermore, injected mice retained a normal number of podocytes and had better integrity of the glomerular basement membrane compared with untreated Col4a5(-/-) mice. Inhibition of the renin-angiotensin system by amniotic fluid stem cells may contribute to these beneficial effects. In conclusion, treatment with amniotic fluid stem cells may be beneficial in kidney diseases characterized by progressive renal fibrosis.

Stem cells as a therapeutic approach to chronic kidney diseases.

Much attention recently has been focused on stem cell technology as a possible alternative modality of treatment of a variety of diseases. Chronic kidney disease is a serious health problem and most chronic kidney diseases share in common the presence of interstitial and glomerular fibrosis, regardless of the underlying cause. To date there are no specific therapies aimed at treating fibrosis in the kidney. In a novel effort to address the underlying pathology in kidney disease, researchers are demonstrating that stem cell therapy can attenuate fibrosis in chronic kidney disease in animal models. This review will focus on the recent developments in stem cell research and their possible implications to treat chronic kidney disease.

Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis.

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kit(pos) stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.

Human amniotic fluid as a potential new source of organ specific precursor cells for future regenerative medicine applications.

PURPOSE: Human amniotic fluid contains multiple cell types, including pluripotent and committed progenitor cells, and fully differentiated cells. We characterized various cell populations in amniotic fluid. MATERIALS AND METHODS: Optimum culture techniques for multiple cell line passages with minimal morphological change were established. Cell line analysis and characterization were done with reverse transcriptase and real-time polymerase chain reaction. Immunoseparation was done to distinguish native progenitor cell lines and their various subpopulations. RESULTS: Endodermal and mesodermal marker expression was greatest in samples of early gestational age while ectodermal markers showed a constant rate across all samples. Pluripotent and mesenchymal cells were always present but hematopoietic cell markers were expressed only in older samples. Specific markers for lung, kidney, liver and heart progenitor cells were increasingly expressed after 18 weeks of gestation. We specifically focused on a CD24+OB-cadherin+ population that could identify uninduced metanephric mesenchyma-like cells, which in vivo are nephron precursors. The CD24+OB-cadherin+ cell line was isolated and subjected to further immunoseparation to select 5 distinct amniotic fluid kidney progenitor cell subpopulations based on E-cadherin, podocalyxin, nephrin, TRKA and PDGFRA expression, respectively. CONCLUSIONS: These subpopulations may represent different precursor cell lineages committed to specific renal cell fates. Committed progenitor cells in amniotic fluid may provide an important and novel resource of useful cells for regenerative medicine purposes.

Human amniotic fluid stem cells can integrate and differentiate into epithelial lung lineages.

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein-injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10-kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung. Disclosure of potential conflicts of interest is found at the end of this article.

Stem cell and regenerative science applications in the development of bioengineering of renal tissue.

A rising number of patients with acute and chronic renal failure worldwide have created urgency for clinicians and investigators to search out alternative therapies other than chronic renal dialysis and/or organ transplantation. This review focuses on the recent achievements in this area, and discusses the various approaches in the development of bioengineering of renal tissue including recent discoveries in the field of regenerative medicine research and stem cells. A variety of stem cells, ranging from embryonic, bone marrow, endogenous, and amniotic fluid, have been investigated and may prove useful as novel alternatives for organ regeneration both in vitro and in vivo. Tissue engineering, developmental biology, and therapeutic cloning techniques have significantly contributed to our understanding of some of the molecular mechanisms involved in renal regeneration and have demonstrated that renal tissue can be generated de novo with similar physiologic functions as native tissue. Ultimately all of these emerging technologies may provide viable therapeutic options for regenerative medicine applications focused on the bioengineering of renal tissue for the future.

Characterization of human amniotic fluid stem cells and their pluripotential capability.

Over the past decade, there has been ever-increasing emphasis placed on stem cells and their potential role in regenerative medicine for reconstruction of bio-artificial tissues and organs. Scientists have looked at various sources for pluripotential cells ranging from embryonic stem cells to adult stem cells. Amniocentesis is a well-established technique for the collection of cells derived from the human embryo. In this chapter, we are going to describe how to isolate, maintain in culture, and characterize the pluripotential capabilities of stem cells derived from amniocentesis in an in vitro and in vivo system. Cell samples are obtained from human pregnancies, and the progenitor cells are isolated from male fetuses with a normal karyotype in order to confirm the absence of maternal admixed cells. Progenitor cells express embryonic-specific cell markers, they show a high self-renewal capacity with 350 population doublings, and normal ploidy is confirmed by cell-cycle analyses. They maintain their undifferentiated state, pluripotential ability, clonogenicity, and telomere length over the population doublings. The progenitor cells are inducible to different cell lineages (osteogenic, adipogenic, skeletal muscle, endothelial, neuronal, and hepatic cells) under specific growth conditions. The ability to induce cell-type-specific differentiation is confirmed by phenotypic changes, immunocytochemistry, gene expression, and functional analyses. In addition, we will describe an application of these cells in an ex vivo and in vivo system for potential in organ (renal) regeneration. The progenitor cells described in this chapter have a high potential for expansion, and may be a good source for research and therapeutic applications where large numbers of cells are needed. Progenitor cells isolated during gestation may be beneficial for fetuses diagnosed with malformations and could be cryopreserved for future self-use.