Publications by authors named "Seppo J Vainio"

44 Publications

Identification of extracellular nanoparticle subsets by nuclear magnetic resonance.

Chem Sci 2021 Apr 29;12(24):8311-8319. Epub 2021 Apr 29.

NMR Research Unit, University of Oulu Finland

Exosomes are a subset of secreted lipid envelope-encapsulated extracellular vesicles (EVs) of 50-150 nm diameter that can transfer cargo from donor to acceptor cells. In the current purification protocols of exosomes, many smaller and larger nanoparticles such as lipoproteins, exomers and microvesicles are typically co-isolated as well. Particle size distribution is one important characteristics of EV samples, as it reflects the cellular origin of EVs and the purity of the isolation. However, most of the physicochemical analytical methods today cannot illustrate the smallest exosomes and other small particles like the exomers. Here, we demonstrate that diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) method enables the determination of a very broad distribution of extracellular nanoparticles, ranging from 1 to 500 nm. The range covers sizes of all particles included in EV samples after isolation. The method is non-invasive, as it does not require any labelling or other chemical modification. We investigated EVs secreted from milk as well as embryonic kidney and renal carcinoma cells. Western blot analysis and immuno-electron microscopy confirmed expression of exosomal markers such as ALIX, TSG101, CD81, CD9, and CD63 in the EV samples. In addition to the larger particles observed by nanoparticle tracking analysis (NTA) in the range of 70-500 nm, the DOSY distributions include a significant number of smaller particles in the range of 10-70 nm, which are visible also in transmission electron microscopy images but invisible in NTA. Furthermore, we demonstrate that hyperpolarized chemical exchange saturation transfer (Hyper-CEST) with Xe NMR indicates also the existence of smaller and larger nanoparticles in the EV samples, providing also additional support for DOSY results. The method implies also that the Xe exchange is significantly faster in the EV pool than in the lipoprotein/exomer pool.
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http://dx.doi.org/10.1039/d1sc01402aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221169PMC
April 2021

Characterization of nucleic acids from extracellular vesicle-enriched human sweat.

BMC Genomics 2021 Jun 9;22(1):425. Epub 2021 Jun 9.

Faculty of Biochemistry and Molecular Medicine, Disease Networks Research Unit, Laboratory of Developmental Biology, Kvantum Institute, Infotech Oulu, University of Oulu, 90014 University of Oulu, Oulu, Finland.

Background: The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles.

Results: We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7-45%), with 50-60% of those reads mapping to unannotated region of the genome and 30-55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA.

Conclusions: Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.
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http://dx.doi.org/10.1186/s12864-021-07733-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188706PMC
June 2021

Secreted Extracellular Vesicle Molecular Cargo as a Novel Liquid Biopsy Diagnostics of Central Nervous System Diseases.

Int J Mol Sci 2021 Mar 23;22(6). Epub 2021 Mar 23.

Cancer Biology & Epigenetics Group, Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal.

Secreted extracellular vesicles (EVs) are heterogeneous cell-derived membranous granules which carry a large diversity of molecules and participate in intercellular communication by transferring these molecules to target cells by endocytosis. In the last decade, EVs' role in several pathological conditions, from etiology to disease progression or therapy evasion, has been consolidated, including in central nervous system (CNS)-related disorders. For this review, we performed a systematic search of original works published, reporting the presence of molecular components expressed in the CNS via EVs, which have been purified from plasma, serum or cerebrospinal fluid. Our aim is to provide a list of molecular EV components that have been identified from both nonpathological conditions and the most common CNS-related disorders. We discuss the methods used to isolate and enrich EVs from specific CNS-cells and the relevance of its components in each disease context.
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http://dx.doi.org/10.3390/ijms22063267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004928PMC
March 2021

Extracellular Vesicles as Innovative Tool for Diagnosis, Regeneration and Protection against Neurological Damage.

Int J Mol Sci 2020 Sep 18;21(18). Epub 2020 Sep 18.

Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, 44121 Ferrara, Italy.

Extracellular vesicles (EVs) have recently attracted a great deal of interest as they may represent a new biosignaling paradigm. According to the mode of biogenesis, size and composition, two broad categories of EVs have been described, exosomes and microvesicles. EVs have been shown to carry cargoes of signaling proteins, RNA species, DNA and lipids. Once released, their content is selectively taken up by near or distant target cells, influencing their behavior. Exosomes are involved in cell-cell communication in a wide range of embryonic developmental processes and in fetal-maternal communication. In the present review, an outline of the role of EVs in neural development, regeneration and diseases is presented. EVs can act as regulators of normal homeostasis, but they can also promote either neuroinflammation/degeneration or tissue repair in pathological conditions, depending on their content. Since EV molecular cargo constitutes a representation of the origin cell status, EVs can be exploited in the diagnosis of several diseases. Due to their capability to cross the blood-brain barrier (BBB), EVs not only have been suggested for the diagnosis of central nervous system disorders by means of minimally invasive procedures, i.e., "liquid biopsies", but they are also considered attractive tools for targeted drug delivery across the BBB. From the therapeutic perspective, mesenchymal stem cells (MSCs) represent one of the most promising sources of EVs. In particular, the neuroprotective properties of MSCs derived from the dental pulp are here discussed.
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http://dx.doi.org/10.3390/ijms21186859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555813PMC
September 2020

MicroRNAs in Extracellular Vesicles in Sweat Change in Response to Endurance Exercise.

Front Physiol 2020 15;11:676. Epub 2020 Jul 15.

Faculty of Sport and Health Sciences, University of Jyväskylä, Jyväskylä, Finland.

Background: To date, microRNAs (miRs) carried in extracellular vesicles (EVs) in response to exercise have been studied in blood but not in non-invasively collectable body fluids. In the present study, we examined whether six exercise-responsive miRs, miRs-21, -26, -126, -146, -221, and -222, respond to acute endurance exercise stimuli of different intensities in sweat.

Methods: We investigated the response of miRs isolated from sweat and serum EVs to three endurance exercise protocols: (1) maximal aerobic capacity (VO ), (2) anaerobic threshold (AnaT), and (3) aerobic threshold (AerT) tests. Sauna bathing was used as a control test to induce sweating through increased body temperature in the absence of exercise. All protocols were performed by the same subjects ( = 8, three males and five females). The occurrence of different miR carriers in sweat and serum was investigated via EV markers (CD9, CD63, and TSG101), an miR-carrier protein (AGO2), and an HDL-particle marker (APOA1) with Western blot. Correlations between miRs in sweat and serum (post-sample) were examined.

Results: Of the studied miR carrier markers, sweat EV fractions expressed CD63 and, very weakly, APOA1, while the serum EV fraction expressed all the studied markers. In sweat EVs, miR-21 level increased after AerT and miR-26 after all the endurance exercise tests compared with the Sauna ( < 0.050). miR-146 after AnaT correlated to sweat and serum EV samples ( = 0.881, = 0.004).

Conclusion: Our preliminary study is the first to show that, in addition to serum, sweat EVs carry miRs. Interestingly, we observed that miRs-21 and -26 in sweat EVs respond to endurance exercise of different intensities. Our data further confirmed that miR responses to endurance exercise in sweat and serum were triggered by exercise and not by increased body temperature. Our results highlight that sweat possesses a unique miR carrier content that should be taken into account when planning analyses from sweat as a substitute for serum.
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http://dx.doi.org/10.3389/fphys.2020.00676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373804PMC
July 2020

Erbb4 regulates the oocyte microenvironment during folliculogenesis.

Hum Mol Genet 2020 10;29(17):2813-2830

Organogenesis Laboratory, Department of Medical Biochemistry and Molecular Biology, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders leading to infertility in women affecting reproductive, endocrine and metabolic systems. Recent genomewide association studies on PCOS cohorts revealed a single nucleotide polymorphism (SNP) in the ERBB4 receptor tyrosine kinase 4 gene, but its role in ovary development or during folliculogenesis remains poorly understood. Since no genetic animal models mimicking all PCOS reproductive features are available, we conditionally deleted Erbb4 in murine granulosa cells (GCs) under the control of Amh promoter. While we have demonstrated that Erbb4 deletion displayed aberrant ovarian function by affecting the reproductive function (asynchronous oestrous cycle leading to few ovulations and subfertility) and metabolic function (obesity), their ovaries also present severe structural and functional abnormalities (impaired oocyte development). Hormone analysis revealed an up-regulation of serum luteinizing hormone, hyperandrogenism, increased production of ovarian and circulating anti-Müllerian hormone. Our data implicate that Erbb4 deletion in GCs leads to defective intercellular junctions between the GCs and oocytes, causing changes in the expression of genes regulating the local microenvironment of the follicles. In vitro culture assays reducing the level of Erbb4 via shRNAs confirm that Erbb4 is essential for regulating Amh level. In conclusion, our results indicate a functional role for Erbb4 in the ovary, especially during folliculogenesis and its reduced expression plays an important role in reproductive pathophysiology, such as PCOS development.
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http://dx.doi.org/10.1093/hmg/ddaa161DOI Listing
October 2020

Trisk 95 as a novel skin mirror for normal and diabetic systemic glucose level.

Sci Rep 2020 07 22;10(1):12246. Epub 2020 Jul 22.

Laboratory of Developmental Biology, Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, Aapistie 5A, 90220, Oulu, Finland.

Developing trustworthy, cost effective, minimally or non-invasive glucose sensing strategies is of great need for diabetic patients. In this study, we used an experimental type I diabetic mouse model to examine whether the skin would provide novel means for identifying biomarkers associated with blood glucose level. We first showed that skin glucose levels are rapidly influenced by blood glucose concentrations. We then conducted a proteomic screen of murine skin using an experimental in vivo model of type I diabetes and wild-type controls. Among the proteins that increased expression in response to high blood glucose, Trisk 95 expression was significantly induced independently of insulin signalling. A luciferase reporter assay demonstrated that the induction of Trisk 95 expression occurs at a transcriptional level and is associated with a marked elevation in the Fluo-4AM signal, suggesting a role for intracellular calcium changes in the signalling cascade. Strikingly, these changes lead concurrently to fragmentation of the mitochondria. Moreover, Trisk 95 knockout abolishes both the calcium flux and the mitochondrial phenotype changes indicating dependency of glucose flux in the skin on Trisk 95 function. The data demonstrate that the skin reacts robustly to systemic blood changes, and that Trisk 95 is a promising biomarker for a glucose monitoring assembly.
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http://dx.doi.org/10.1038/s41598-020-68972-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376074PMC
July 2020

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

J Vis Exp 2020 03 28(157). Epub 2020 Mar 28.

Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu; InfoTech Oulu, University of Oulu; Biobank Borealis of Northern Finland, Oulu University Hospital;

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.
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http://dx.doi.org/10.3791/60995DOI Listing
March 2020

Mouse Embryonic Stem Cell-Derived Ureteric Bud Progenitors Induce Nephrogenesis.

Cells 2020 01 31;9(2). Epub 2020 Jan 31.

Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, Laboratory of Developmental Biology, Infotech Oulu, University of Oulu, Aapistie 5A, 90220 Oulu, Finland.

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.
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http://dx.doi.org/10.3390/cells9020329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072223PMC
January 2020

Self-assembled nanofibrils from RGD-functionalized cellulose nanocrystals to improve the performance of PEI/DNA polyplexes.

J Colloid Interface Sci 2019 Oct 3;553:71-82. Epub 2019 Jun 3.

Fibre and Particle Engineering Research Unit, University of Oulu, P.O. Box 4300, FI-90014 Oulu, Finland. Electronic address:

Cellulose nanocrystals (CNCs) are promising bio-derived nanomaterials for the bottom-up fabrication of biomedical constructs. In this report, dicarboxylic acid-functionalized CNC (DCC) was functionalized with arginylglycylaspartic acid (RGD) tripeptide as a motif for improved cell adhesion and targeting. The product (DCC-RGD) self-assembled into a more elongated nanofibrillar structure through lateral and end-to-end association. When added into poly(ethylene imine) (PEI)/pDNA polyplex solution, nanocelluloses interacted electrostatically with positively charged polyplexes without affecting their integrity. The constructs were tested for their potentials as non-viral transfection reagents. Cell viability and transfection efficiency of fibroblast NIH3T3 cells were monitored as a function of CNC concentration where, in general, viability increased as the CNC concentration increased, and transfection efficiency could be optimized. Using wild-type MDCK and αV-knockout MDCK cells, the construct was able to provide targeted uptake of polyplexes. The findings have potential applications, for example, cell-selective in vitro or ex vivo transfection of autologous mesenchymal stem cells for cell therapy, or bottom-up design of future innovative biomaterials.
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http://dx.doi.org/10.1016/j.jcis.2019.06.001DOI Listing
October 2019

Embryonic Stem Cells Derived Kidney Organoids as Faithful Models to Target Programmed Nephrogenesis.

Sci Rep 2018 11 9;8(1):16618. Epub 2018 Nov 9.

Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 5A, 90220, Oulu, Finland.

The kidney is a complex organ that is comprised of thousands of nephrons developing through reciprocal inductive interactions between metanephric mesenchyme (MM) and ureteric bud (UB). The MM undergoes mesenchymal to epithelial transition (MET) in response to the signaling from the UB. The secreted protein Wnt4, one of the Wnt family members, is critical for nephrogenesis as mouse Wnt4 mutants fail to form pretubular aggregates (PTA) and therefore lack functional nephrons. Here, we generated mouse embryonic stem cell (mESC) line lacking Wnt4 by applying the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems 9 (Cas9). We describe here, differentiation of the wild type and Wnt4 knockout mESCs into kidney progenitors, and such cells induced to undergo nephrogenesis by the mouse E11.5 UB mediated induction. The wild type three-dimensional (3D) self-organized organoids depict appropriately segmented nephron structures, while the Wnt4-deficient organoids fail to undergo the MET, as is the case in the phenotype of the Wnt4 knockout mouse model in vivo. In summary, we have established a platform that combine CRISPR/Cas9 and kidney organoid technologies to model kidney development in vitro and confirmed that mutant organoids are able to present similar actions as in the in vivo studies.
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http://dx.doi.org/10.1038/s41598-018-34995-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226521PMC
November 2018

Polyion complex hydrogels from chemically modified cellulose nanofibrils: Structure-function relationship and potential for controlled and pH-responsive release of doxorubicin.

Acta Biomater 2018 07 7;75:346-357. Epub 2018 Jun 7.

Fibre and Particle Engineering Research Unit, University of Oulu, P.O. Box 4300, FI-90014 Oulu, Finland. Electronic address:

Herein, we report the fabrication of a polyion complex hydrogel from two oppositely charged derivatives of cellulose nanofibrils (CNF). CNF was produced from dissolving pulp through subsequent periodate oxidation, chemical modification, and microfluidization. Three different durations for periodate oxidation (30 min, 120 min, and 180 min) resulted in three different aldehyde contents. Further, two types of chemical modifications were introduced to react with the resulting aldehydes: chlorite oxidation to yield anionic CNF with carboxylic acid groups (DCC) and imination with Girard's reagent T to yield cationic CNF containing quaternary ammonium groups (CDAC). Functional group contents were assessed using conductometric titration and elemental analysis, while nanofibril morphologies were assessed using atomic force microscopy (AFM). Longer durations of periodate oxidation did not yield different width profile but was found to decrease fibril length. The formation of self-standing hydrogel through mixing of DCC and CDAC dispersions was investigated. Oscillatory rheology was performed to assess the relative strengths of different gels. Self-standing hydrogels were obtained from mixture of DCC180 and CDAC180 dispersions in acetate buffer at pH 4 and 5 at a low concentration of 0.5% w/w that displayed approximately 10-fold increase in storage and loss moduli compared to those of the individual dispersions. Self-standing gels containing doxorubicin (an anticancer drug) displayed pH-responsive release profiles. At physiological pH 7.4, approximately 65% of doxorubicin was retained past a burst release regime, while complete release was observed within 5 days at pH 4. Biocompatibility of DCC180, CDAC180, and their mixture were investigated through quantification of the metabolic activity of NIH3T3 cells in vitro. No significant cytotoxicity was observed at concentrations up to 900 µg/mL. In short, the nanocellulose-based polyion complex hydrogels obtained in this study are promising nature-derived materials for biomedical applications.

Statement Of Significance: We demonstrate that polyion complex can be formed between two cellulose nanofibrils containing complementary charges. To the best of our knowledge, this is the first time that polyion complex formation between complementarily-modified cellulose nanofibrils has been reported, and the results may lead to new ideas on applications of the very promising nanocellulosic materials. The polyion complex helps form a self-standing network that is demonstrated to provide controlled and pH-responsive release of doxorubicin. Particularly, the report explores the connection between the physical properties of functionalizable nanocellulosic materials and their potential biomedical applications. Thus, the study encompasses several broad fields of materials science and engineering, chemistry, and biomedical science that we believe is in line with the readers' interests.
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http://dx.doi.org/10.1016/j.actbio.2018.06.013DOI Listing
July 2018

Exosomes as secondary inductive signals involved in kidney organogenesis.

J Extracell Vesicles 2018 23;7(1):1422675. Epub 2018 Jan 23.

Biocenter Oulu, Laboratory of Developmental Biology, InfoTech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland.

The subfraction of extracellular vesicles, called exosomes, transfers biological molecular information not only between cells but also between tissues and organs as nanolevel signals. Owing to their unique properties such that they contain several RNA species and proteins implicated in kidney development, exosomes are putative candidates to serve as developmental programming units in embryonic induction and tissue interactions. We used the mammalian metanephric kidney and its nephron-forming mesenchyme containing the nephron progenitor/stem cells as a model to investigate if secreted exosomes could serve as a novel type of inductive signal in a process defined as embryonic induction that controls organogenesis. As judged by several characteristic criteria, exosomes were enriched and purified from a cell line derived from embryonic kidney ureteric bud (UB) and from primary embryonic kidney UB cells, respectively. The cargo of the UB-derived exosomes was analysed by qPCR and proteomics. Several miRNA species that play a role in Wnt pathways and enrichment of proteins involved in pathways regulating the organization of the extracellular matrix as well as tissue homeostasis were identified. When labelled with fluorescent dyes, the uptake of the exosomes by metanephric mesenchyme (MM) cells and the transfer of their cargo to the cells can be observed. Closer inspection revealed that besides entering the cytoplasm, the exosomes were competent to also reach the nucleus. Furthermore, fluorescently labelled exosomal RNA enters into the cytoplasm of the MM cells. Exposure of the embryonic kidney-derived exosomes to the whole MM in an organ culture setting did not lead to an induction of nephrogenesis but had an impact on the overall organization of the tissue. We conclude that the exosomes provide a novel signalling system with an apparent role in secondary embryonic induction regulating organogenesis.
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http://dx.doi.org/10.1080/20013078.2017.1422675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795705PMC
January 2018

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

Development 2017 12 20;144(24):4704-4719. Epub 2017 Nov 20.

Sorbonne Universités, UPMC Université Paris 06, IBPS - UMR7622, F-75005 Paris, France

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.
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http://dx.doi.org/10.1242/dev.154336DOI Listing
December 2017

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

Dis Model Mech 2017 12 19;10(12):1503-1515. Epub 2017 Dec 19.

Biocenter Oulu, Laboratory of Developmental Biology, InfoTech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, Oulu University, FI-90014 Oulu, Finland

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.
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http://dx.doi.org/10.1242/dmm.028332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769601PMC
December 2017

Editorial: Organogenesis: From Development to Disease.

Front Cell Dev Biol 2017 20;5:85. Epub 2017 Sep 20.

Laboratory of Developmental Biology, Biocenter Oulu, InfoTech Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu UniversityOulu, Finland.

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http://dx.doi.org/10.3389/fcell.2017.00085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611542PMC
September 2017

Kidney development and perspectives for organ engineering.

Cell Tissue Res 2017 07 20;369(1):171-183. Epub 2017 Apr 20.

Laboratory of Developmental Biology, Biocenter Oulu, FIN-90014, Oulu, Finland.

Organ transplantation is currently the best strategy for treating end stage renal disease (ESRD) but the numbers of donor kidneys available are not sufficient to meet the needs of the ever-increasing ESRD population. Therefore, developments in the field of tissue engineering are necessary to provide alternative treatments. Decellularization and three-dimensional (3D) bioprinting strategies may serve as attractive novel options. Since successful tissue engineering requires an in -depth understanding of organ development and regulatory pathways, we discuss signaling in renal development and the composition of the renal extracellular matrix before presenting progress in the decellularization and 3D bioprinting fields.
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http://dx.doi.org/10.1007/s00441-017-2616-xDOI Listing
July 2017

BMP7 Induces Uterine Receptivity and Blastocyst Attachment.

Endocrinology 2017 04;158(4):979-992

Departments of Pathology and Immunology, Baylor College of Medicine, Houston, TX.

In women, the window of implantation is limited to a brief 2- to 3-day period characterized by optimal levels of circulating ovarian hormones and a receptive endometrium. Although the window of implantation is assumed to occur 8 to 10 days after ovulation in women, molecular markers of endometrial receptivity are necessary to determine optimal timing prior to embryo transfer. Previous studies showed that members of the bone morphogenetic protein (BMP) family are expressed in the uterus necessary for female fertility; however, the role of BMP7 during implantation and in late gestation is not known. To determine the contribution of BMP7 to female fertility, we generated Bmp7flox/flox-Pgr-cre+/- [BMP7 conditional knockout (cKO)] mice. We found that absence of BMP7 in the female reproductive tract resulted in subfertility due to uterine defects. At the time of implantation, BMP7 cKO females displayed a nonreceptive endometrium with elevated estrogen-dependent signaling. These implantation-related defects also affected decidualization and resulted in decreased expression of decidual cell markers such as Wnt4, Cox2, Ereg, and Bmp2. We also observed placental abnormalities in pregnant Bmp7 cKO mice, including excessive parietal trophoblast giant cells and absence of a mature placenta at 10.5 days post coitum. To establish possible redundant roles of BMP5 and BMP7 during pregnancy, we generated double BMP5 knockout/BMP7 cKO [BMP5/7 double knockout (DKO)] mice; however, we found that the combined deletion had no additive disruptive effect on fertility. Our studies indicate that BMP7 is an important factor during the process of implantation that contributes to healthy embryonic development.
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http://dx.doi.org/10.1210/en.2016-1629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460793PMC
April 2017

The CapZ interacting protein Rcsd1 is required for cardiogenesis downstream of Wnt11a in Xenopus laevis.

Dev Biol 2017 04 22;424(1):28-39. Epub 2017 Feb 22.

Institute of Biochemistry and Molecular Biology, Ulm University, Albert-Einstein-Allee 11, D-89081 Ulm, Germany. Electronic address:

Wnt proteins are critical for embryonic cardiogenesis and cardiomyogenesis by regulating different intracellular signalling pathways. Whereas canonical Wnt/β-catenin signalling is required for mesoderm induction and proliferation of cardiac progenitor cells, β-catenin independent, non-canonical Wnt signalling regulates cardiac specification and terminal differentiation. Although the diverse cardiac malformations associated with the loss of non-canonical Wnt11 in mice such as outflow tract (OFT) defects, reduced ventricular trabeculation, myofibrillar disorganization and reduced cardiac marker gene expression are well described, the underlying molecular mechanisms are still not completely understood. Here we aimed to further characterize Wnt11 mediated signal transduction during vertebrate cardiogenesis. Using Xenopus as a model system, we show by loss of function and corresponding rescue experiments that the non-canonical Wnt signalling mediator Rcsd1 is required downstream of Wnt11 for ventricular trabeculation, terminal differentiation of cardiomyocytes and cardiac morphogenesis. We here place Rcsd1 downstream of Wnt11 during cardiac development thereby providing a novel mechanism for how non-canonical Wnt signalling regulates vertebrate cardiogenesis.
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http://dx.doi.org/10.1016/j.ydbio.2017.02.014DOI Listing
April 2017

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

Development 2017 03 20;144(6):1113-1117. Epub 2017 Feb 20.

Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220 Oulu, Finland

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.
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http://dx.doi.org/10.1242/dev.142950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358112PMC
March 2017

Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis.

BMC Dev Biol 2016 08 31;16(1):30. Epub 2016 Aug 31.

Biocenter Oulu, Laboratory of Developmental Biology, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell Matrix Research, University of Oulu, Aapistie 5A, Oulu, 90014, Finland.

Background: Wnt11 is a member of the Wnt family of secreted signals controlling the early steps in ureteric bud (UB) branching. Due to the reported lethality of Wnt11 knockout embryos in utero, its role in later mammalian kidney organogenesis remains open. The presence of Wnt11 in the emerging tubular system suggests that it may have certain roles later in the development of the epithelial ductal system.

Results: The Wnt11 knockout allele was backcrossed with the C57Bl6 strain for several generations to address possible differences in penetrance of the kidney phenotypes. Strikingly, around one third of the null mice with this inbred background survived to the postnatal stages. Many of them also reached adulthood, but urine and plasma analyses pointed out to compromised kidney function. Consistent with these data the tubules of the C57Bl6 Wnt11 (-/-) mice appeared to be enlarged, and the optical projection tomography indicated changes in tubular convolution. Moreover, the C57Bl6 Wnt11 (-/-) mice developed secondary glomerular cysts not observed in the controls. The failure of Wnt11 signaling reduced the expression of several genes implicated in kidney development, such as Wnt9b, Six2, Foxd1 and Hox10. Also Dvl2, an important PCP pathway component, was downregulated by more than 90 % due to Wnt11 deficiency in both the E16.5 and NB kidneys. Since all these genes take part in the control of UB, nephron and stromal progenitor cell differentiation, their disrupted expression may contribute to the observed anomalies in the kidney tubular system caused by Wnt11 deficiency.

Conclusions: The Wnt11 signal has roles at the later stages of kidney development, namely in coordinating the development of the tubular system. The C57Bl6 Wnt11 (-/-) mouse generated here provides a model for studying the mechanisms behind tubular anomalies and glomerular cyst formation.
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http://dx.doi.org/10.1186/s12861-016-0131-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007805PMC
August 2016

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

Kidney Int 2016 08 18;90(2):311-324. Epub 2016 Apr 18.

Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland; Biocenter Oulu, Oulu, Finland; Center of Excellence in Cell-Extracellular Matrix Research, Oulu, Finland; InfoTech Oulu, Oulu, Finland. Electronic address:

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.
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http://dx.doi.org/10.1016/j.kint.2016.02.021DOI Listing
August 2016

Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning.

PLoS One 2016 21;11(1):e0147171. Epub 2016 Jan 21.

Laboratory of Developmental Biology, Oulu Centre for Cell-Matrix Research, Biocenter Oulu and Infotech Oulu, and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland.

The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to Wnt5a knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the Wnt5a knock out when compared to control. Expression of a panel of BM components such as laminin and of type IV collagen was also reduced due to the Wnt5a knock out. The P4ha1 gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in Wnt5a function from control. The compound Wnt5a+/-;P4ha1+/- embryos demonstrated Wnt5a-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling in vitro. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147171PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721645PMC
August 2016

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

Hum Mol Genet 2016 Mar 31;25(6):1059-73. Epub 2015 Dec 31.

Oulu Centre for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, Laboratory of Developmental Biology, InfoTech Oulu, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland and

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.
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http://dx.doi.org/10.1093/hmg/ddv621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764189PMC
March 2016

Exosomes as renal inductive signals in health and disease, and their application as diagnostic markers and therapeutic agents.

Front Cell Dev Biol 2015 20;3:65. Epub 2015 Oct 20.

Biocenter Oulu, Infotech Oulu, Developmental Biology Lab, Faculty of Biochemistry and Molecular Medicine, Center for Cell Matrix Research, University of Oulu Oulu, Finland.

Cells secrete around 30-1000 nm membrane-enclosed vesicles, of which members of the subgroup between 30 and 100 nm are termed exosomes (EXs). EXs are released into the extracellular space and are widely present in body fluids and incorporated mRNA, miRNA, proteins, and signaling molecules. Increasing amounts of evidence suggest that EXs play an important role not only in cell-to-cell communication but also in various physiological and disease processes. EXs secreted by kidney cells control nephron function and are involved in kidney diseases and cancers. This makes them potential targets for diagnostic and therapeutic applications such as non-invasive biomarkers and cell-free vaccines and for use as drug delivery vehicles. This review provides an overview on the known roles of EXs in kidney development and diseases, including renal cancer. Additionally, it covers recent findings on their significance as diagnostic markers and on therapeutic applications to renal diseases and cancers. The intention is to promote an awareness of how many questions still remain open but are certainly worth investigating.
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http://dx.doi.org/10.3389/fcell.2015.00065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4611857PMC
November 2015

ATGme: Open-source web application for rare codon identification and custom DNA sequence optimization.

BMC Bioinformatics 2015 Sep 21;16:303. Epub 2015 Sep 21.

Biocenter Oulu, Laboratory of Developmental Biology, InfoTech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 5A, FIN-90220, Oulu, Finland.

Background: Codon usage plays a crucial role when recombinant proteins are expressed in different organisms. This is especially the case if the codon usage frequency of the organism of origin and the target host organism differ significantly, for example when a human gene is expressed in E. coli. Therefore, to enable or enhance efficient gene expression it is of great importance to identify rare codons in any given DNA sequence and subsequently mutate these to codons which are more frequently used in the expression host.

Results: We describe an open-source web-based application, ATGme, which can in a first step identify rare and highly rare codons from most organisms, and secondly gives the user the possibility to optimize the sequence.

Conclusions: This application provides a simple user-friendly interface utilizing three optimization strategies: 1. one-click optimization, 2. bulk optimization (by codon-type), 3. individualized custom (codon-by-codon) optimization. ATGme is an open-source application which is freely available at: http://atgme.org.
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http://dx.doi.org/10.1186/s12859-015-0743-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4578782PMC
September 2015

Identification of the genes regulated by Wnt-4, a critical signal for commitment of the ovary.

Exp Cell Res 2015 Mar 30;332(2):163-78. Epub 2015 Jan 30.

Academy of Finland, Centre of Excellence of Cell-Extracellular Matrix Research, Biocenter Oulu & Infotech Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland. Electronic address:

The indifferent mammalian embryonic gonad generates an ovary or testis, but the factors involved are still poorly known. The Wnt-4 signal represents one critical female determinant, since its absence leads to partial female-to-male sex reversal in mouse, but its signalling is as well implicated in the testis development. We used the Wnt-4 deficient mouse as a model to identify candidate gonadogenesis genes, and found that the Notum, Phlda2, Runx-1 and Msx1 genes are typical of the wild-type ovary and the Osr2, Dach2, Pitx2 and Tacr3 genes of the testis. Strikingly, the expression of these latter genes becomes reversed in the Wnt-4 knock-out ovary, suggesting a role in ovarian development. We identified the transcription factor Runx-1 as a Wnt-4 signalling target gene, since it is expressed in the ovary and is reduced upon Wnt-4 knock-out. Consistent with this, introduction of the Wnt-4 signal into early ovary cells ex vivo induces Runx-1 expression, while conversely Wnt-4 expression is down-regulated in the absence of Runx-1. We conclude that the Runx-1 gene can be a Wnt-4 signalling target, and that Runx-1 and Wnt-4 are mutually interdependent in their expression. The changes in gene expression due to the absence of Wnt-4 in gonads reflect the sexually dimorphic role of this signal and its complex gene network in mammalian gonad development.
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http://dx.doi.org/10.1016/j.yexcr.2015.01.010DOI Listing
March 2015

Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk.

EMBO J 2015 Mar 20;34(5):641-52. Epub 2015 Jan 20.

Ecole Polytechnique Fédérale de Lausanne (EPFL) ISREC - Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland

Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf-κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR+ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal-myoepithelial crosstalk with Wnt4 acting independent of PR perinatally.
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http://dx.doi.org/10.15252/embj.201490434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365033PMC
March 2015

Functional genetic targeting of embryonic kidney progenitor cells ex vivo.

J Am Soc Nephrol 2015 May 8;26(5):1126-37. Epub 2014 Sep 8.

Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland;

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.
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http://dx.doi.org/10.1681/ASN.2013060584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413750PMC
May 2015

ErbB4, a receptor tyrosine kinase, coordinates organization of the seminiferous tubules in the developing testis.

Mol Endocrinol 2014 Sep 24;28(9):1534-46. Epub 2014 Jul 24.

Oulu Centre for Cell-Matrix Research (F.N., S.J.V.), Biocenter Oulu, Infotech Oulu, Laboratory of Developmental Biology, Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90220 Oulu, Finland; Department of Medical Biochemistry and Genetics (V.V., K.E.), and Medicity Research Laboratory, University of Turku, FI-20520 Turku, Finland; Electron Microscopy Unit (I.M.), FI-90220 Oulu, Finland; Laboratory Animal Center (P.S.), University of Helsinki, FIN-00014 Helsinki, Finland; Department of Physiology (M.P.), Turku University Hospital, FI-2001 4 Turku, Finland; and Department of Oncology (K.E.), Turku University Hospital, FI-20520 Turku, Finland.

Although close to every fifth couple nowadays has difficulty conceiving, the molecular mechanisms behind the decline in human reproduction remain poorly understood. We report here that the receptor tyrosine kinase Erbb4 is a candidate causal gene, because it is expressed in a sexually dimorphic manner and is abundant in the developing and adult testes in the mouse. Sertoli cell-specific Erbb4-knockout mice have a compromised 3-dimensional organization of the testicular seminiferous tubules that affects their fertility. More specifically, adhesion defects are observed in the absence of Erbb4, which are characterized by changes in the expression of laminin-1, N-cadherin, claudin-3, and certain cell-cell junction components between the Sertoli and germ cells. Interestingly, Erbb4 knockout also had an effect on the Leydig cells, which suggests a paracrine influence of Sertoli cells expressing ErbB4. Many of the defects observed in Erbb4-knockout mice are rescued in targeted ERBB4 gain-of-function mice, pointing to a coordination role for ErbB4 in the developing testis. Thus, the ErbB4 receptor tyrosine kinase promotes seminiferous tubule development by controlling Sertoli cell and germ cell adhesion.
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http://dx.doi.org/10.1210/me.2013-1244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414798PMC
September 2014
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