optimization-renal-organoid-organotypic-culture-for-vascularization

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 ...

University of Oulu

University of oulu

The Wnt4 gene encodes a secreted signaling molecule controlling the development of several organs, such as the kidney, adrenal gland, ovary, mammary gland and pituitary gland. It is thought to act in the embryonic kidney as an auto-inducer of nephrogenesis controlling mesenchyme-to-epithelium transition, and Wnt4-deficient mice die soon after birth, probably of kidney failure. Given the requirement for Wnt4 signaling in the control of organogenesis, the targeting of Cre recombinase under the control of the Wnt4 promoter would provide a valuable tool for fate mapping and functional genomics. We report here on the generation and characterization of a Wnt4(EGFPCre) knock-in allele where the EGFPCre fusion cDNA and Neo selection cassette were targeted into the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates of the E14.5 embryonic kidney that normally express Wnt4 mRNA. Characterization of the pattern of recombination of the floxed Rosa26(LacZ) reporter with the Wnt4(EGFPCre) allele revealed that in addition to the embryonic kidney, reporter-derived staining was observed in the embryonic gonad, spinal cord, lung and adrenal gland, i.e. the sites of Wnt4 gene expression. Time-lapse fate mapping of the Wnt4(EGFPCre)-activated yellow fluorescent protein (YFP) from the Rosa26 locus in organ culture revealed that the cells that had expressed the Wnt4 gene contributed to the nephrons, some of the cells around the stalk of the developing ureter and also certain presumptive medullary stromal cells. Moreover, the time-lapse movies suggested that the first few pretubular cell aggregates may not mature into nephrons but instead appear to disintegrate. In association with this, Rosa26(YFP)-positive stromal cells emerge around these disintegrating structures. Such cells may be transient, since their derivatives are neither detected later in the more mature kidney nor is there an overlap of the Wnt4(EGFPCre); Rosa26(LacZ)-marked cells with those of the endothelial cells, the smooth muscle cells or the macrophages. The Wnt4(EGFPCre) allele provides a useful new tool for conditional mutagenesis and provides the first time-lapse-based map of the fate of nephron precursor cells.

Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development.

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.

A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development.

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 βgeo 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 βgeo 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.

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

The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.

Functional Genetic Targeting of Embryonic Kidney Progenitor Cells Ex Vivo.

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. 

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

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.

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

Tissue, organ and organoid cultures provide suitable models for developmental studies, but our understanding of how the organs are assembled at the single-cell level still remains unclear. We describe here a novel fixed -direction (FiZD) culture setup that permits high-resolution confocal imaging of organoids and embryonic tissues. In a FiZD culture a permeable membrane compresses the tissues onto a glass coverslip and the spacers adjust the thickness, enabling the tissue to grow for up to 12 days. Thus, the kidney rudiment and the organoids can adjust to the limited -directional space and yet advance the process of kidney morphogenesis, enabling long-term time-lapse and high-resolution confocal imaging. As the data quality achieved was sufficient for computer-assisted cell segmentation and analysis, the method can be used for studying morphogenesis  at the level of the single constituent cells of a complex mammalian organogenesis model system.

Novel fixed -direction (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging.

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  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 , , , , ,  or  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 ,  and -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.

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

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  from the ureteric bud leads to defective cell-cell contacts and apicobasal polarity during the early branching events. High-resolution  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  and  Subsequently,  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.

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

Time-lapse imaging is a technique of frequent imaging and following a course of a process. Because the development of the embryonic kidney can proceed ex vivo after dissection, it is possible to study the morphogenesis by culturing the kidney in the onstage incubator of a microscope and follow the developmental process by imaging. Confocal microscopes and other three-dimensional imaging systems offer the possibility for tracking the development process in four dimensions-3D and the time.

Time-Lapse Technologies and 4D Imaging of Kidney Development.

In this work, several optical-spectroscopic methods have been used to visualize and investigate the penetration of diamond nanoparticles (NPs) of various sizes (3-150 nm), surface structures and fluorescence properties into the animal skin in vitro. Murine skin samples have been treated with nanodiamond (ND) water suspensions and studied using optical coherence tomography (OCT), confocal and two-photon fluorescence microscopy and fluorescence lifetime imaging (FLIM). An analysis of the optical properties of the used nanodiamonds (NDs) enables the selection of optimal optical methods or their combination for the study of nanodiamond-skin interaction. Among studied NDs, particles of 100 nm in nominal size were shown to be appropriate for multimodal imaging using all three methods. All the applied NDs were able to cross the skin barrier and penetrate the different layers of the epidermis to finally arrive in the hair follicle niches. The results suggest that NDs have the potential for multifunctional applications utilizing multimodal imaging.

Optical Studies of Nanodiamond-Tissue Interaction: Skin Penetration and Localization.

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 pmol of siRNA, and 1 × 10 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.

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

In the framework of novel medical paradigm the red blood cells (RBCs) have a great potential to be used as drug delivery carriers. This approach requires an ultimate understanding of the peculiarities of mutual interaction of RBC influenced by nano-materials composed the drugs. Optical tweezers (OT) is widely used to explore mechanisms of cells' interaction with the ability to trap non-invasively, manipulate and displace living cells with a notably high accuracy. In the current study, the mutual interaction of RBC with polymeric nano-capsules (NCs) is investigated utilizing a two-channel OT system. The obtained results suggest that, in the presence of NCs, the RBC aggregation in plasma satisfies the 'cross-bridges' model. Complementarily, the allocation of NCs on the RBC membrane was observed by scanning electron microscopy (SEM), while for assessment of NCs-induced morphological changes the tests with the human mesenchymal stem cells (hMSC) was performed. The combined application of OT and advanced microscopy approaches brings new insights into the conception of direct observation of cells interaction influenced by NCs for the estimation of possible cytotoxic effects.

Impact of Nanocapsules on Red Blood Cells Interplay Jointly Assessed by Optical Tweezers and Microscopy.

Generation of kidney organoids from pluripotent stem cells (PSCs) is regarded as a potentially powerful way to study kidney development, disease, and regeneration. Direct differentiation of PSCs towards renal lineages is well studied; however, most of the studies relate to generation of nephron progenitor population from PSCs. Until now, differentiation of PSCs into ureteric bud (UB) progenitor cells has had limited success. Here, we describe a simple, efficient, and reproducible protocol to direct differentiation of mouse embryonic stem cells (mESCs) into UB progenitor cells. The mESC-derived UB cells were able to induce nephrogenesis when co-cultured with primary metanephric mesenchyme (pMM). In generated kidney organoids, the embryonic pMM developed nephron structures, and the mESC-derived UB cells formed numerous collecting ducts connected with the nephron tubules. Altogether, our study established an uncomplicated and reproducible platform to generate ureteric bud progenitors from mouse embryonic stem cells.

Article	Year
Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development. Differentiation; research in biological diversity\| PubMed ID: 19740593	2010
A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development. PloS one\| PubMed ID: 22114682	2011
Conditional expression of Lodavin, an avidin-tagged LDL receptor, for biotin-mediated applications in vivo. Genesis (New York, N.Y. : 2000)\| PubMed ID: 22467513	2012
Functional Genetic Targeting of Embryonic Kidney Progenitor Cells Ex Vivo. Journal of the American Society of Nephrology : JASN\| PubMed ID: 25201883	2014
Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract. Human molecular genetics\| PubMed ID: 26721931	2016
CD146(+) cells are essential for kidney vasculature development. Kidney international\| PubMed ID: 27165833	2016
Novel fixed -direction (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging. Development (Cambridge, England)\| PubMed ID: 28219945	2017
Renal carcinoma/kidney progenitor cell chimera organoid as a novel tumorigenesis gene discovery model. Disease models & mechanisms\| PubMed ID: 29084770	2017
HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis. Development (Cambridge, England)\| PubMed ID: 29158444	2017
Time-Lapse Technologies and 4D Imaging of Kidney Development. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30742271	2019
Optical Studies of Nanodiamond-Tissue Interaction: Skin Penetration and Localization. Materials (Basel, Switzerland)\| PubMed ID: 31731700	2019
Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 31840408	2020
Impact of Nanocapsules on Red Blood Cells Interplay Jointly Assessed by Optical Tweezers and Microscopy. Micromachines\| PubMed ID: 31878030	2019
Mouse Embryonic Stem Cell-Derived Ureteric Bud Progenitors Induce Nephrogenesis. Cells\| PubMed ID: 32023845	2020

Ilya Skovorodkin

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