Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging.

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types.

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co-transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10-4 pmol of siRNA, and 1 × 10-3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo.

HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis.

Kidney development depends crucially on proper ureteric bud branching giving rise to the entire collecting duct system. The transcription factor HNF1B is required for the early steps of ureteric bud branching, yet the molecular and cellular events regulated by HNF1B are poorly understood. We report that specific removal of Hnf1b from the ureteric bud leads to defective cell-cell contacts and apicobasal polarity during the early branching events. High-resolution ex vivo imaging combined with a membranous fluorescent reporter strategy show decreased mutant cell rearrangements during mitosis-associated cell dispersal and severe epithelial disorganization. Molecular analysis reveals downregulation of Gdnf-Ret pathway components and suggests that HNF1B acts both upstream and downstream of Ret signaling by directly regulating Gfra1 and Etv5 Subsequently, Hnf1b deletion leads to massively mispatterned ureteric tree network, defective collecting duct differentiation and disrupted tissue architecture, which leads to cystogenesis. Consistently, mRNA-seq analysis shows that the most impacted genes encode intrinsic cell-membrane components with transporter activity. Our study uncovers a fundamental and recurring role of HNF1B in epithelial organization during early ureteric bud branching and in further patterning and differentiation of the collecting duct system in mouse.

Renal carcinoma/kidney progenitor cell chimera organoid as a novel tumorigenesis gene discovery model.

Three-dimensional (3D) organoids provide a new way to model various diseases, including cancer. We made use of recently developed kidney-organ-primordia tissue-engineering technologies to create novel renal organoids for cancer gene discovery. We then tested whether our novel assays can be used to examine kidney cancer development. First, we identified the transcriptomic profiles of quiescent embryonic mouse metanephric mesenchyme (MM) and of MM in which the nephrogenesis program had been induced ex vivo The transcriptome profiles were then compared to the profiles of tumor biopsies from renal cell carcinoma (RCC) patients, and control samples from the same kidneys. Certain signature genes were identified that correlated in the developmentally induced MM and RCC, including components of the caveolar-mediated endocytosis signaling pathway. An efficient siRNA-mediated knockdown (KD) of Bnip3, Gsn, Lgals3, Pax8, Cav1, Egfr or Itgb2 gene expression was achieved in mouse RCC (Renca) cells. The live-cell imaging analysis revealed inhibition of cell migration and cell viability in the gene-KD Renca cells in comparison to Renca controls. Upon siRNA treatment, the transwell invasion capacity of Renca cells was also inhibited. Finally, we mixed E11.5 MM with yellow fluorescent protein (YFP)-expressing Renca cells to establish chimera organoids. Strikingly, we found that the Bnip3-, Cav1- and Gsn-KD Renca-YFP+ cells as a chimera with the MM in 3D organoid rescued, in part, the RCC-mediated inhibition of the nephrogenesis program during epithelial tubules formation. Altogether, our research indicates that comparing renal ontogenesis control genes to the genes involved in kidney cancer may provide new growth-associated gene screens and that 3D RCC-MM chimera organoids can serve as a novel model with which to investigate the behavioral roles of cancer cells within the context of emergent complex tissue structures.

CD146(+) cells are essential for kidney vasculature development.

The kidney vasculature is critical for renal function, but its developmental assembly mechanisms remain poorly understood and models for studying its assembly dynamics are limited. Here, we tested whether the embryonic kidney contains endothelial cells (ECs) that are heterogeneous with respect to VEGFR2/Flk1/KDR, CD31/PECAM, and CD146/MCAM markers. Tie1Cre;R26R(YFP)-based fate mapping with a time-lapse in embryonic kidney organ culture successfully depicted the dynamics of kidney vasculature development and the correlation of the process with the CD31(+) EC network. Depletion of Tie1(+) or CD31(+) ECs from embryonic kidneys, with either Tie1Cre-induced diphtheria toxin susceptibility or cell surface marker-based sorting in a novel dissociation and reaggregation technology, illustrated substantial EC network regeneration. Depletion of the CD146(+) cells abolished this EC regeneration. Fate mapping of green fluorescent protein (GFP)-marked CD146(+)/CD31(-) cells indicated that they became CD31(+) cells, which took part in EC structures with CD31(+) wild-type ECs. EC network development depends on VEGF signaling, and VEGF and erythropoietin are expressed in the embryonic kidney even in the absence of any external hypoxic stimulus. Thus, the ex vivo embryonic kidney culture models adopted here provided novel ways for targeting renal EC development and demonstrated that CD146(+) cells are critical for kidney vasculature development.

Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract.

The Müllerian duct (MD) is the anlage of the oviduct, uterus and upper part of the vagina, the main parts of the female reproductive tract. Several wingless-type mouse mammary tumor virus (MMTV) integration site family member (Wnt) genes, including Wnt4, Wnt5a and Wnt7a, are involved in the development of MD and its derivatives, with Wnt4 particularly critical, since the MD fails to develop in its absence. We use, here, Wnt4(EGFPCre)-based fate mapping to demonstrate that the MD tip cells and the subsequent MD cells are derived from Wnt4+ lineage cells. Moreover, Wnt4 is required for the initiation of MD-forming cell migration. Application of anti-Wnt4 function-blocking antibodies after the initiation of MD elongation indicated that Wnt4 is necessary for the elongation as well, and consistent with this, cell culture wound-healing assays with NIH3T3 cells overexpressing Wnt4 promoted cell migration by comparison with controls. In contrast to the Wnt4 null embryos, some Wnt4(monomeric cherry/monomeric cherry) (Wnt4(mCh/mCh)) hypomorphic mice survived to adulthood and formed MD in ∼45% of cases. Nevertheless, the MD of the Wnt4(mCh/mCh) females had altered cell polarization and basement membrane deposition relative to the controls. Examination of the reproductive tract of the Wnt4(mCh/mCh) females indicated a poorly coiled oviduct, absence of the endometrial glands and an undifferentiated myometrium, and these mice were prone to develop a hydro-uterus. In conclusion, the results suggest that the Wnt4 gene encodes signals that are important for various aspects of female reproductive tract development.

Conditional expression of Lodavin, an avidin-tagged LDL receptor, for biotin-mediated applications in vivo.

Lodavin represents an engineered fusion protein that consists of a cytoplasmic and a transmembrane domain of the human low-density lipoprotein receptor coupled to an extracellular avidin monomer. Biotinylated compounds have been successfully targeted to Lodavin-expressing cells that have been transduced by a Lodavin-containing virus, and the targeting is based on the high affinity between biotin and avidin. We engineered a Rosa26 (R26R) knock-in Lodavin mouse to develop biotin-based applications such as targeted drug delivery, cell purification, and tissue imaging in vivo. A cDNA encoding Lodavin was inserted downstream of a floxed ßgeo resistance gene in the R26R locus in embryonic stem cells, and a germ line-derived R26RLodavin mouse line was generated. Efficient removal of the floxed ßgeo cassette and conditional activation of Lodavin expression was achieved as a result of crossing the R26RLodavin mice with HoxB7-Cre, Wnt4-Cre, or Tie1-Cre mice. In summary, the R26RLodavin mouse line may provide a useful tool for testing and developing applications with the aid of avidin and biotin interaction.

Novel perspectives for investigating congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the commonest cause of chronic kidney disease in children. Structural anomalies within the CAKUT spectrum include renal agenesis, kidney hypo-/dysplasia, multicystic kidney dysplasia, duplex collecting system, posterior urethral valves and ureter abnormalities. While most CAKUT cases are sporadic, familial clustering of CAKUT is common, emphasizing a strong genetic contribution to CAKUT origin. Animal experiments demonstrate that alterations in genes crucial for kidney development can cause experimental CAKUT, while expression studies implicate mislocalization and/or aberrant levels of the encoded proteins in human CAKUT. Further insight into the pathogenesis of CAKUT will improve strategies for early diagnosis, follow-up and treatment. Here, we outline a collaborative approach to identify and characterize novel factors underlying human CAKUT. This European consortium will share the largest collection of CAKUT patients available worldwide and undertake multidisciplinary research into molecular and genetic pathogenesis, with extension into translational studies to improve long-term patient outcomes.