Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

Human Transplant Kidneys on Normothermic Machine Perfusion Display Endocrine Activity.

Normothermic machine perfusion (NMP) is an alternative to hypothermic machine perfusion (HMP) for donor kidney preservation before transplantation. Contrary to HMP, NMP allows for functional assessment of donor kidneys because normothermic conditions allow for metabolic activity. The kidneys are key producers of hormones. Yet, it remains unknown whether donor kidneys during NMP display endocrine functions. Methods: Fifteen donor kidneys were subjected to HMP followed by 2 h of NMP before transplantation. NMP perfusate was collected at 3 time points (0, 1, 2 h) for the measurements of prorenin/renin, erythropoietin (EPO), and vitamin D, and urine samples were collected at 1 h and 2 h for urodilatin measurement. Fifteen HMP perfusate samples were collected for the same measurements. Results: Kidneys on NMP secreted significantly more prorenin, renin, EPO, and active vitamin D than during HMP. EPO and vitamin D secretion remained stable during 2 h of NMP, whereas the prorenin release rate increased and renin release rate decreased after 1 h. Donation after brain death kidneys secreted more vitamin D and less EPO during NMP than donation after circulatory death kidneys. Twelve donor kidneys produced urine during NMP and released detectable levels of urodilatin. Kidneys exhibited a large variation in hormone release rates. No significant differences were found in hormone release capacity between delayed graft function (DGF) and non-DGF kidneys, and no significant correlations were found between hormone release rates and the duration of DGF or 1-mo posttransplant serum creatinine levels. Conclusions: Human transplant kidneys display endocrine activity during NMP. To explore whether correlations exist between hormone release rates and posttransplant kidney function, large numbers of kidneys are required.

Kidney Organoids Are Capable of Forming Tumors, but Not Teratomas.

Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry-/-RAG2-/- mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids.

Identification of Predictive Markers for the Generation of Well-Differentiated Human Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Human-induced pluripotent stem cell (iPSC)-derived kidney organoids have the potential to advance studies to kidney development and disease. However, reproducible generation of kidney organoids is a challenge. A large variability in the percentage of nephron structures and the expression of kidney-specific genes was observed among organoids, showing no association with iPSC lines. To associate the quality of kidney organoid differentiation with predictive markers, a ranking system was developed based on the ratio of nephron structure determined by histological examination. Well-differentiated organoids were defined as organoids with >30% nephron structure and vice versa. Subsequently, correlations were made with expression profiles of iPSC markers, early kidney development markers, and fibrosis markers. Higher expression of sex-determining region Y-box 2 (SOX2) during differentiation was associated with poorly differentiated kidney organoid. Furthermore, early secretion of basic fibroblast growth factor (FGF2) predicted poorly differentiated kidney organoid. Of interest, whereas cadherin-1 (CDH1) expression in kidney organoids indicates distal tubules formation, onefold higher CDH1 expression in iPSC predicted poor differentiation. High expression of the stromal progenitor marker Forkhead Box D1 (FOXD1) and significantly increased TGFß levels were found in well-differentiated kidney organoids. These early expression profiles could predict the outcome of kidney organoid formation. This study helps to improve the robustness of kidney organoid protocols.

p57 Suppresses the Pluripotency and Proliferation of Mouse Embryonic Stem Cells by Positively Regulating p53 Activation.

Embryonic stem cells (ESCs) are pluripotent stem cells that have indefinite self-renewal capacities under appropriate culture conditions in vitro. The pluripotency maintenance and proliferation of these cells are delicately governed by the concert effect of a complex transcriptional regulatory network. Herein, we discovered that p57Kip2 (p57), a cyclin-dependent kinase inhibitor canonically inhibiting cell proliferation, played a role in suppressing the pluripotency state of mouse ESCs (mESCs). p57 knockdown significantly stimulated the expressions of core pluripotency factors NANOG, OCT4, and SOX2, while p57 overexpression inhibited the expressions of these factors in mESCs. In addition, consistent with its function in somatic cells, p57 suppressed mESC proliferation. Further analysis showed that p57 could interact with and contribute to the activation of p53 in mESCs. In conclusion, the present study showed that p57 could antagonize the pluripotency state and the proliferation process of mESCs. This finding uncovers a novel function of p57 and provides new evidence for elucidating the complex regulatory of network of mESC fate.

Human kidney organoids produce functional renin.

Renin production by the kidney is of vital importance for salt, volume, and blood pressure homeostasis. The lack of human models hampers investigation into the regulation of renin and its relevance for kidney physiology. To develop such a model, we used human induced pluripotent stem cell-derived kidney organoids to study the role of renin and the renin-angiotensin system in the kidney. Extensive characterization of the kidney organoids revealed kidney-specific cell populations consisting of podocytes, proximal and distal tubular cells, stromal cells and endothelial cells. We examined the presence of various components of the renin-angiotensin system such as angiotensin II receptors, angiotensinogen, and angiotensin-converting enzymes 1 and 2. We identified by single-cell sequencing, immunohistochemistry, and functional assays that cyclic AMP stimulation induces a subset of pericytes to increase the synthesis and secretion of enzymatically active renin. Renin production by the organoids was responsive to regulation by parathyroid hormone. Subcutaneously implanted kidney organoids in immunodeficient IL2Ry-/-Rag2-/- mice were successfully vascularized, maintained tubular and glomerular structures, and retained capacity to produce renin two months after implantation. Thus, our results demonstrate that kidney organoids express renin and provide insights into the endocrine potential of human kidney organoids, which is important for regenerative medicine in the context of the endocrine system.

Self-assembly of carbon nanotube/graphitic-like flake/BiOBr nanocomposite with 1D/2D/3D heterojunctions for enhanced photocatalytic activity.

A self-assembled nanocomposite of lamellar BiOBr covalently bonded with conductive network of dispersive one-dimensional carbon nanotubes (1D CNT) and two-dimensional reduced graphitic-like flakes (2D GF) had been in situ constructed using one-pot facile solvothermal technique. Through self-assembly, BiOBr/CNT/GF (BiOBr/CG) displayed three-dimensional architectures in which a strong interfacial contact interaction and covalent banding between BiOBr nanostructures and CNT/GF network appeared. Furthermore, visible-light-driven catalytic activity of BiOBr/CG for RhB dye degradation was superior to that of pure BiOBr or BiOBr/C. Interestingly, the photodegradation activity of the BiOBr/CG nanocomposite could be improved further by subsequent facile annealing treatment, in which the annealed BiOBr/CG-DS had degraded almost 97.9% of RhB dye within only 100 min of visible-light irradiation. Moreover, analysis of the photodegradation mechanism revealed that the repression of electron-hole recombination in the nanocomposites, with sufficient covalent interfacial contact with CNT/GF as effective electron collecting and transferring system, were responsible for the outstanding photocatalytic performance. This effect, in turn, led to the continuous generation of O2- and OH reactive oxygen species for the degradation of RhB dye, which was verified by active species trapping and ESR spectra.

Reconstitution of male germline cell specification from mouse embryonic stem cells using defined factors in vitro.

In vitro induction of functional haploid cells from embryonic stem cells (ESCs) has been reported by several groups. However, these reports either involve complex induction process with undefined induction factors or show low-induction efficiency. Here, we report complete meiosis in vitro from ESCs with defined induction factors. ESCs were first induced into primordial germ cell-like cells, which were further induced into male germline cells, including spermatogonial stem cell-like cells (SSCLCs) and spermatid-like cells. Importantly, the obtained SSCLCs were functional as infertile male mice sired healthy offspring via SSCLC transplantation. Further, we found that eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) was essential for spermatogenesis. Eif2s3y-overexpressing ESCs showed enhanced spermatogenesis in vitro, as demonstrated by higher expression levels of SSC-specific markers during SSCLC induction process, improved reproductive ability recovery of infertile male mice, and increased efficiency of haploid cell induction. Our work provides a convenient and efficient approach to obtain functional male germline cells.

Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells.

Diabetes mellitus affects a large number of men of reproductive age and it usually leads to serious reproductive disorders. However, the underlying mechanisms and specific therapies still remain largely unknown. We observed Leydig cell loss in the testes of diabetic mice. Continuous high glycemic status of testes stimulated expression of Caspase12, Grp78, and Chop, the three ERS response factors; this might induce cell cycle arrest and apoptosis of Leydig cells in response to ERS. In these diabetic mouse models, melatonin alleviated apoptosis of testicular stromal cell induced by ERS, and promoted SSCs self-renewal by recovering Leydig cells secretion of CSF1 after 8 weeks of treatment. To explore the relationship between CSF-1 and ERS in Leydig cells, we treated Leydig tumor cell line with an activator Tuniamycin and an inhibitor 4-Phenylbutyrate of ERS. Our data showed that the CSF-1 expression in mouse Leydig cell lines decreased six-fold while reversely increasing five-fold in the 4-Phenylbutyrate-treated group. Thus, melatonin likely alleviates the loss of Leydig cells in diabetic testes and provides a healthier niche for SSCs to self-renew and continually provide healthy sperm for male fertility.

Resveratrol Enhances Self-Renewal of Mouse Embryonic Stem Cells.

Resveratrol (RSV) has been shown to affect the differentiation of several types of stem cells, while the detailed mechanism is elusive. Here, we aim to investigate the function of RSV in self-renewal of mouse embryonic stem cells (ESCs) and the related mechanisms. In contrast with its reported roles, we found unexpectedly that differentiated ESCs or iPSCs treated by RSV would not show further differentiation, but regained a naïve pluripotency state with higher expressions of core transcriptional factors and with the ability to differentiate into all three germ layers when transplanted in vivo. In accordance with these findings, RSV also enhanced cell cycle progression of ESCs via regulating cell cycle-related proteins. Finally, enhanced activation of JAK/STAT3 signaling pathway and suppressed activation of mTOR were found essential in enhancing the self-renewal of ESCs by RSV. Our finding discovered a novel function of RSV in enhancing the self-renewal of ESCs, and suggested that the timing of treatment and concentration of RSV determined the final effect of it. Our work may contribute to understanding of RSV in the self-renewal maintenance of pluripotent stem cells, and may also provide help to the generation and maintenance of iPSCs in vitro. J. Cell. Biochem. 118: 1928-1935, 2017. © 2017 Wiley Periodicals, Inc.