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.

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.

Kinase-independent inhibition of cyclophosphamide-induced pathways protects the ovarian reserve and prolongs fertility.

Premature ovarian failure and infertility are adverse effects of cancer therapies. The mechanism underlying chemotherapy-mediated depletion of the ovarian reserve remains unclear. Here, we aim to identify the signaling pathways involved in the loss of the ovarian reserve to prevent the damaging effects of chemotherapy. We evaluated the effects of cyclophosphamide, one of the most damaging chemotherapeutic drugs, against follicle reserve. In vivo studies showed that the cyclophosphamide-induced loss of ovarian reserve occurred through a sequential mechanism. Cyclophosphamide exposure induced the activation of both DNAPK-γH2AX-checkpoint kinase 2 (CHK2)-p53/TAp63α isoform and protein kinase B (AKT)-forkhead box O3 (FOXO3a) signaling axes in the nucleus of oocytes. Concomitant administration of an allosteric ABL inhibitor and cyclophosphamide modulated both pathways while protecting the ovarian reserve from chemotherapy assaults. As a consequence, the fertility of the treated mice was prolonged. On the contrary, the administration of an allosteric ABL activator enhanced the lethal effects of cyclophosphamide while shortening mouse fertility. Therefore, kinase-independent inhibition may serve as an effective ovarian-protective strategy in women under chemotherapy.

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.

ß-Cell regeneration mediated by human bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into ß-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous ß-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF) or pancreatic-duodenal homeobox 1 (PDX1) to reverse diabetes and whether these cells were differentiated into ß-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo ß-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous ß-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining ß-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of ß-cell recovery after injury mediated by hBMSC therapy.