Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations.

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.

Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex.

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.

In situ Hybridization of miRNAs in Human Embryonic Kidney and Human Pluripotent Stem Cell-derived Kidney Organoids.

MicroRNAs are small RNAs that negatively regulate gene expression and play an important role in fine-tuning molecular pathways during development. There is increasing interest in studying their function in the kidney, but the majority of studies to date use kidney cell lines and assess the total amounts of miRNAs of interest either by qPCR or by high-throughput methods such as next generation sequencing. However, this provides little information as to the distribution of the miRNAs in the developing kidney, which is crucial in deciphering their role, especially as there are multiple kidney cell types, each with its own specific transcriptome. Thus, we present a protocol for obtaining spatial information for miRNA expression during kidney development by in situ hybridization (ISH) of anti-miRNA, digoxigenin-labelled (DIG), Locked Nucleic Acid (LNA®) probes on (i) native human embryonic tissue and (ii) human pluripotent stem cell (hPSC)-derived 3D kidney organoids that model kidney development. We found that the method reveals the precise localization of miRNA in specific anatomical structures and/or cell types and confirms their absence from others, thus informing as to their specific role during development.

The miR-199a/214 Cluster Controls Nephrogenesis and Vascularization in a Human Embryonic Stem Cell Model.

MicroRNAs (miRNAs) are gene expression regulators and they have been implicated in acquired kidney diseases and in renal development, mostly through animal studies. We hypothesized that the miR-199a/214 cluster regulates human kidney development. We detected its expression in human embryonic kidneys by in situ hybridization. To mechanistically study the cluster, we used 2D and 3D human embryonic stem cell (hESC) models of kidney development. After confirming expression in each model, we inhibited the miRNAs using lentivirally transduced miRNA sponges. This reduced the WT1+ metanephric mesenchyme domain in 2D cultures. Sponges did not prevent the formation of 3D kidney-like organoids. These organoids, however, contained dysmorphic glomeruli, downregulated WT1, aberrant proximal tubules, and increased interstitial capillaries. Thus, the miR-199a/214 cluster fine-tunes differentiation of both metanephric mesenchymal-derived nephrons and kidney endothelia. While clinical implications require further study, it is noted that patients with heterozygous deletions encompassing this miRNA locus can have malformed kidneys.

Aberrant Differentiation of Human Pluripotent Stem Cell-Derived Kidney Precursor Cells inside Mouse Vascularized Bioreactors.

BACKGROUND: Numerous studies have documented the in vitro differentiation of human pluripotent stem cells (hPSCs) into kidney cells. Fewer studies have followed the fates of such kidney precursor cells (KPCs) inside animals, a more life-like setting. Here, we tested the hypothesis that implanting hPSC-derived KPCs into an in vivo milieu surgically engineered to be highly vascular would enhance their maturation into kidney tissues. METHODS: 3D printed chambers containing KPCs were implanted into the thighs of adult immunodeficient mice. In some chambers, an arterial and venous flow-through (AVFT) was surgically fashioned. After 3 weeks and 3 months, implants were studied by histology, using qualitative and quantitative methods. RESULTS: After 3 weeks, chambers containing AVFTs were richer in small vessels than contralateral chambers without AVFTs. Glomeruli with capillary loops and diverse types of tubules were detected in all chambers. At 3 months, chambers contained only rudimentary tubules and glomeruli that appeared avascular. In chambers with AVFTs, prominent areas of muscle-like cells were also detected near tubules and the abnormal tissues immunostained for transforming growth factor ß1. These features have similarities to renal dysplasia, a typical histological signature of human congenital kidney malformations. CONCLUSIONS: This study urges a note of caution regarding the in vivo fates of hPSC-derived kidney precursors, with pathological differentiation appearing to follow a period of increased vascularity.

Formation of Mature Nephrons by Implantation of Human Pluripotent Stem Cell-Derived Progenitors into Mice.

In the future, stem cell-based technologies may be harnessed to replace conventional dialysis and transplantation in patients with diabetic nephropathy. Recently, there has been considerable effort to improve methods for the differentiation of human pluripotent stem cells (hPSCs) into kidney cells in culture. Here, we present a protocol for obtaining more advanced kidney structures than have currently been possible in vitro, including vascularized glomeruli and tubular elements. HPSCs are first differentiated in 2D culture to a kidney progenitor stage. These cells are then dissociated and injected subcutaneously into immunocompromised mice. Twelve weeks later, the cells have developed into mature kidney structures and are excised for further characterization. This method constitutes a significant improvement on protocols that involve either exclusively a 2D culture or placing the cells in 3D organoid culture at the air-liquid interface in vitro.

Generation of Functioning Nephrons by Implanting Human Pluripotent Stem Cell-Derived Kidney Progenitors.

Human pluripotent stem cells (hPSCs) hold great promise for understanding kidney development and disease. We reproducibly differentiated three genetically distinct wild-type hPSC lines to kidney precursors that underwent rudimentary morphogenesis in vitro. They expressed nephron and collecting duct lineage marker genes, several of which are mutated in human kidney disease. Lentiviral-transduced hPSCs expressing reporter genes differentiated similarly to controls in vitro. Kidney progenitors were subcutaneously implanted into immunodeficient mice. By 12 weeks, they formed organ-like masses detectable by bioluminescence imaging. Implants included perfused glomeruli containing human capillaries, podocytes with regions of mature basement membrane, and mesangial cells. After intravenous injection of fluorescent low-molecular-weight dextran, signal was detected in tubules, demonstrating uptake from glomerular filtrate. Thus, we have developed methods to trace hPSC-derived kidney precursors that formed functioning nephrons in vivo. These advances beyond in vitro culture are critical steps toward using hPSCs to model and treat kidney diseases.

Comparability: manufacturing, characterization and controls, report of a UK Regenerative Medicine Platform Pluripotent Stem Cell Platform Workshop, Trinity Hall, Cambridge, 14-15 September 2015.

This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this 'may be difficult for cell-based medicinal products'. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates.

MiR-132 Is Upregulated by Ischemic Preconditioning of Cultured Hippocampal Neurons and Protects them from Subsequent OGD Toxicity.

We explored the response of a panel of selected microRNAs (miRNAs) in neuroprotection produced by ischemic preconditioning. Hippocampal neuronal cultures were exposed to a 30-min oxygen-glucose deprivation (OGD). In our hands, this duration of OGD does not result in neuronal loss in vitro but significantly reduces neuronal death from a subsequent 'lethal' OGD insult. RT-qPCR was used to determine the expression of 16 miRNAs of interest at 1 and 24-h post-OGD. One miRNA (miR-98) was significantly decreased at 1-h post-OGD. Ten miRNAs (miR-9, miR-21, miR-29b, miR-30e, miR-101a, miR-101b, miR-124a, miR-132, miR-153, miR-204) were increased significantly at 24-h post-OGD. No miRNAs were decreased at 24-h. The increases observed in the 24-h group suggested that these miRNAs might play a role in preconditioning-induced neuroprotection. We selected the widely studied miR-132, a brain enriched, CREB regulated miRNA, to explore its role in simulated ischemic insults. We found that hippocampal neurons transduced with lentiviral vectors expressing miR-132 were protected from OGD and NMDA treatment, but not hydrogen peroxide. These findings add to the growing literature that targeting neuroprotective pathways controlled by miRNAs may represent a therapeutic strategy for the treatment of ischemic brain injury.

Regulation of axon growth by the JIP1-AKT axis.

The polarisation of developing neurons to form axons and dendrites is required for the establishment of neuronal connections leading to proper brain function. The protein kinase AKT and the MAP kinase scaffold protein JNK-interacting protein-1 (JIP1) are important regulators of axon formation. Here we report that JIP1 and AKT colocalise in axonal growth cones of cortical neurons and collaborate to promote axon growth. The loss of AKT protein from the growth cone results in the degradation of JIP1 by the proteasome, and the loss of JIP1 promotes a similar fate for AKT. Reduced protein levels of both JIP1 and AKT in the growth cone can be induced by glutamate and this coincides with reduced axon growth, which can be rescued by a stabilized mutant of JIP1 that rescues AKT protein levels. Taken together, our data reveal a collaborative relationship between JIP1 and AKT that is required for axon growth and can be regulated by changes in neuronal activity.

Epigenetic regulation of miR-21 in colorectal cancer: ITGB4 as a novel miR-21 target and a three-gene network (miR-21-ITGΒ4-PDCD4) as predictor of metastatic tumor potential.

Previous studies have uncovered several transcription factors that determine biological alterations in tumor cells to execute the invasion-metastasis cascade, including the epithelial-mesenchymal transition (EMT). We sought to investigate the role of miR-21 in colorectal cancer regulation. For this purpose, miR-21 expression was quantified in a panel of colorectal cancer cell lines and clinical specimens. High expression was found in cell lines with EMT properties and in the vast majority of human tumor specimens. We demonstrate in a cell-specific manner the occupancy of MIR-21 gene promoter by AP-1 and ETS1 transcription factors and, for the first time, the pattern of histone posttranslational modifications necessary for miR-21 overexpression. We also show that Integrin-ß4 (ITGß4), exclusively expressed in polarized epithelial cells, is a novel miR-21 target gene and plays a role in the regulation of EMT, since it is remarkably de-repressed after transient miR-21 silencing and downregulated after miR-21 overexpression. miR-21-dependent change of ITGß4 expression significantly affects cell migration properties of colon cancer cells. Finally, in a subgroup of tumor specimens, ROC curve analysis performed on quantitative PCR data sets for miR-21, ITGß4, and PDCD4 shows that the combination of high miR-21 with low ITGß4 and PDCD4 expression is able to predict presence of metastasis. In conclusion, miR-21 is a key player in oncogenic EMT, its overexpression is controlled by the cooperation of genetic and epigenetic alterations, and its levels, along with ITGß4 and PDCD4 expression, could be exploited as a prognostic tool for CRC metastasis.

MiR-3120 is a mirror microRNA that targets heat shock cognate protein 70 and auxilin messenger RNAs and regulates clathrin vesicle uncoating.

We show that a single gene locus gives rise to two fully processed and functional miRNAs, i.e. that due to imperfect base pairing, two distinct microRNAs (miRNAs) can be produced from the fully complementary DNA strands. The antisense strand encodes miR-214, which is transcribed by its own promoter, whereas a novel miRNA, miR-3120, is co-expressed with its host gene mRNA. We also found that miR-3120 regulates important aspects of cellular function that are similar to that of its host gene, dynamin-3. miR-3120 was found to be located in neuronal cell bodies and to target Hsc70 and auxilin, and its lentivirus-mediated expression inhibited the uncoating of clathrin-coated vesicles. Finally, mirror miRNAs are likely to represent a new group of miRNAs with complex roles in coordinating gene expression.

Twist-1 regulates the miR-199a/214 cluster during development.

MicroRNAs are known to regulate developmental processes but their mechanism of regulation remains largely uncharacterized. We show the transcription factor Twist-1 drives the expression of a 7.9-kb noncoding RNA transcript (from the Dynamin-3 gene intron) that encodes a miR-199a and miR-214 cluster. We also show that knocking down Twist-1 with shRNAs decreased miR-199a/214 levels and that Twist-1 bound an E-Box promoter motif to developmentally regulate the expression of these miRNAs. The expression of HIF-1 (known to mediate Twist-1 transcription), miR-199a and miR-214 was maximal at E12.5 and the miRNAs were expressed specifically in mouse cerebellum, midbrain, nasal process and fore- and hindlimb buds. This study shows the expression of the miR199a/214 cluster is controlled by Twist-1 via an E-Box promoter element and supports a role for these miRNAs as novel intermediates in the pathways controlling the development of specific neural cell populations.

Twist induces reversal of myotube formation.

Mammals possess reduced ability to regenerate lost tissue, compared with other vertebrates, which can regenerate through differentiation of precursor cells or de-differentiation. Mammalian multinucleated myotube formation is a differentiation process, which arises from the fusion of mononucleated myoblasts and is thought to be an irreversible process toward muscle formation. By overexpressing the Twist gene in terminally differentiated myotubes, we managed to induce reversal of cell differentiation. More specifically, following expression of the Twist gene, myotubes underwent morphological changes that caused them to cleave. This was accompanied by a reduction in the expression of certain myogenic markers. Interestingly, Twist overexpression also caused a reduction in the muscle transcription factor MyoD. Further experiments showed an increase in the cell cycle entry molecule, cyclin D1 and initiation of DNA synthesis, due to Twist overexpression. The exploitation of Twist-mediated reversal of differentiation and the study of its specific mechanism would be important in order to study mammalian cellular de-differentiation and determine its potential in muscle regeneration.

Assessing adenoviral hammerhead ribozyme and small hairpin RNA cassettes in neurons: inhibition of endogenous caspase-3 activity and protection from apoptotic cell death.

Antisense technology, including ribozyme and small interfering RNA, is being developed to mediate the down-regulation of specific intracellular genes. It was observed in this study that both antiluciferase ribozymes and short hairpin RNAs (shRNAs) could significantly reduce the activity of exogenously expressed luciferase in primary hippocampal neurons in a viral titer-dependent manner. shRNAs were more effective gene-silencing agents than ribozymes, although they exhibited some nonspecific gene-silencing effects at high viral titers. We also attempted to increase ribozyme efficacy by using a woodchuck hepatitis posttranscriptional regulatory element (WPRE) in the ribozyme expression cassette. The results showed that adenoviral vectors encoding specific ribozymes could silence the cellular expression of luciferase and endogenous procaspase-3 significantly. Furthermore, the antiprocaspase-3 ribozyme was shown to inhibit staurosporine-mediated cell death. The addition of a WPRE did not, however, increase or decrease ribozyme activity. As far as we are aware, this is the first example of adenovirally mediated delivery of hammerhead ribozymes being used to manipulate gene expression in primary neurons. The results therefore suggest that hammerhead ribozymes may be useful tools for studying neuronal gene function and have potential as therapeutic agents to treat CNS diseases.