Novel culture and organoid technologies to study mammalian kidney development

Abstract Kidney diseases affect an increasing number of people worldwide, and there is a growing demand to develop new treatments and increase the number of transplantable organs. New treatments can be designed when new knowledge is gained by studying the details of kidney development. The ex vivo culture techniques have been used for over a century to study the development of kidneys, but they are not optimal for long-term imaging and following the nephrogenesis process over time. Kidney organoids, which are cellular aggregates resembling the in vivo kidney, together with intact embryonic kidneys, present a platform for these studies. However, there are limitations when working with primary embryonic kidney cells. Primary embryonic metanephric mesenchymal cells are usually low in number and lose the ability to undergo nephrogenesis rapidly. New ways to culture, biobank, and transfect cells can offer ways for functional testing of the effects of different genes on the nephrogenesis. This study presents new tools for studying nephrogenesis. Time-lapse imaging of organ development may be enhanced by using a Fixed Z-direction (FiZD) culture system where the kidney explant is grown in a restricted 70μm space. The technique enables the segmentation of the individual cells in a two-dimensional image and a dynamic analysis of the time-lapse data. This study also presents a technique of dissociation and reaggregation of the uninduced kidney metanephric mesenchyme (MM). With this novel method of culturing the dissociated MM cells in a growth factor medium for 24 hours, the cells can keep their competence for nephrogenesis. This technique allows the genetic manipulation of the MM cells before the induction to form nephrons, allowing functional testing of genes in the metanephric mesenchyme. This study further presents different techniques for gene editing of MM cells and introduces biobanking of primary kidney cells. It is shown here that the MM and ureteric bud (UB) cells have the capability to remember their fates and build nephron-like structures or continue branching after the cryopreservation in the liquid nitrogen. The methods introduced here provide new ways to create kidney organoids, manipulate their genome, and biobank the primary embryonic kidney cells. The developed FiZD culture system enhances the imaging of kidney development compared to the previously used culture methods. Using this method, the morphogenesis of the developing kidney can be followed more precisely, even in a single cell level. This culture method may also be used to culturing other organs, such as ovary, and may help provide insights into the development of other tissues as well.Tiivistelmä Munuaissairauksiin sairastuvien määrä on lisääntynyt maailmanlaajuisesti, ja se on aikaansaanut tarpeen uusien hoitokeinojen sekä siirtoelimien kehitykseen. Näiden kehittämiseksi tarvitsemme uutta tietoa munuaisen kehityksestä ja toiminnasta. Munuaisen kehitystä on tutkittu ex vivo -viljelyn avulla jo yli vuosisadan ajan, mutta nykyiset elinviljelytekniikat eivät ole kuitenkaan optimaalisia pitkäkestoiseen time-lapse-kuvaukseen. Tässä työssä käytetään munuaisen kehityksen tutkimiseen hiiren alkion munuaisia sekä munuaisorganoideja, jotka ovat munuaissoluista koostuvia ja aitoa munuaista mallintavia soluaggregaatteja. Primaaristen munuaissolujen käyttöön sisältyy rajoitteita, ja tämä luo tarpeen uusien organoiditekniikoiden kehitykseen ja optimointiin. Primaarisia munuaissoluja on yleensä käytettävissä pieniä määriä, ja ne eivät myöskään sovellu pitkäkestoiseen kasvatukseen, koska ne menettävät nopeasti kykynsä muodostaa nefroneita. Uusien tekniikoiden avulla voidaan parantaa näiden solujen kasvatusta, säilytystä ja transfektointia ja edistää eri geenien vaikutuksia tutkivat funktionaaliset testaukset. Tässä tutkimuksessa esitetään uusia työkaluja nefrogeneesin tutkimiseen. Elinten kehitystä seuraavan time-lapse-kuvauksen laatua voidaan parantaa käyttämällä tässä työssä esitettyä FiZD-kasvatusmenetelmää, jossa munuaiseksplantti kasvaa rajoitetussa 70μm:n tilassa. Kuvat ovat korkealaatuisia, ja se mahdollistaa 2D-kuvan yksittäisten solujen segmentoinnin ja solujen liikkeiden dynaamisen analyysin. Lisäksi tässä tutkimuksessa esitetään ei-indusoidun munuaismesenkyymin käsittelyyn kehitetty dissosiaatio- ja reaggregaatiomenetelmä. Munuaisen kehityksen alkuvaiheessa on mahdollistaerottaa nefroneja muodostava metanefrinen mesenkyymi (MM) sekä munuaisen kokoajaputkiston muodostava ureterin silmu. Metanefrinen mesenkyymi voidaan hajottaa yksisolususpensioksi, säilyttää 24 tuntia kasvutekijämediumissa ja tämän jälkeen reaggregoida ja indusoida muodostamaan nefroneita. Tämä tekniikka mahdollistaa MM-solujen geneettisen muokkauksen, ennen kuin munuaisen kehitys alkaa. Tämä tekniikka mahdollistaa myös dissosioitujen MM solujen geneettiset muokkaukset. Geenien yliekspression tai hiljentämisen avulla voidaan tehdä funktionaalisia kokeita näiden muutosten vaikutuksesta nefrogeneesiin. Lisäksi tässä työssä esitetään munuaisprogenitorisolujen säilömistä syväjäädytyksellä. Munuaisprogenitorisolut voidaan säilöä nestetyppeen, minkä jälkeen ne ovat edelleen kykeneviä muodostamaan nefronirakenteita tai haarautumaan. Tässä väitöskirjatyössä esitettyjen menetelmien avulla on tulevaisuudessa mahdollista saada lisätietoa munuaisten kehitysprosessista. Kehitetty FiZD-kasvatusmenetelmä parantaa munuaisen kehityksen kuvantamista ja mahdollistaa yksittäisten solujen seuraamisen. Tämä kasvatusmenetelmä sopii myös muiden elinten, kuten munarauhasten, ja kudosten kasvatukseen, ja sen avulla voidaan saada tietoa myös niiden kehityksestä

Mittaussarjoista saatavan tiedon kehittäminen – SCOAP-lämpötilaprofiilin pakkaus ja purku

Tiivistelmä START-projektissa kehitettiin useita erilaisia kuumanauhan lämpötilamittauksien käsittelyja analysointimenetelmiä. Menetelmäkehityksen rinnalla luotiin Strip Temperature Toolbox (STT), joka sisältää käyttöliittymän kuumanauhojen käsittelyyn ja analysointiin. Nämä asiat on raportoitu muissa raporttisarjan julkaisuissa. START-projektissa tutkittiin lisäksi kaksiulotteisen datan pakkautuvuutta, koska huolimatta suurista tallennuskapasiteeteista prosessiautomaatiossa, toisinaan on tarpeen pystyä pakkaamaan dataa, jotta sitä voidaan säilyttää tietokannoissa pidempään. Datan pakkauksen tarve kasvaa koko ajan, mm. data, kuvat ja videokuvat vievät suuren osan tietokoneiden muistitilasta. Pakkaustekniikat perustuvat toistuvan tai turhan informaation poistoon tiedostosta. Häviöllisellä datan pakkauksella saadaan tiivistettyä tiedosto pienempään osaan kuin häviöttömällä, mutta tällöin alkuperäisestä signaalista häviää joitakin piirteitä. Häviöllinen pakkaus sopii yleensä hyvin kuvien ja videokuvan pakkaukseen. Usein häviävä informaatio voi olla esim. kohinaa. Toisaalta tekstin pakkauksessa pienetkin virheet voivat aiheuttaa väärinkäsityksiä ja silloin on hyvä käyttää häviöttömiä pakkaustekniikoita. Tässä raportissa on vertailtu kolmea erilaista wavelet-tyyppiä sekä diskreettiä kosinimuunnosta kaksiulotteisen datan pakkaukseen. Nämä ovat kuvan pakkauksessa yleisesti käytettyjä muunnostekniikoita. Sovellusympäristönä on käytetty Rautaruukki Oyj:n Raahen terästehtaan nauhavalssaamon kaksiulotteisen SCOAP-lämpötilapyrometrin tuottamaa lämpötiladataa. Toteutustyökaluna käytettiin Matlab®-ohjelmistoa toolboxeineen

Modelling of a fed-batch fermentation process

Abstract This report describes the building of a simulator for prediction of the dissolved oxygen concentration, the oxygen transfer rate and the concentration of carbon dioxide in a fermentation process. The steady state models were made using the linguistic equations method. The dynamic models were made using Simulink® toolbox in the Matlab®. At the beginning, some basics about fermentation and microbiological reactions are stated. In the third chapter the modelling methods are presented. The modelling experiments are presented in chapter four and after that the results are stated. Chapter six includes discussion about the results and the conclusions. The simulation results were good

Internationalisation of microenterprises:systematic literature review

Abstract Microenterprises are the most common type of firm within the European Union, accounting for 93.2% of all enterprises. This study’s aim is to obtain an overview of the literature on microenterprises’ internationalisation; this is done by conducting a systematic literature review (SLR). The research question of this study is as follows: what is studied in an academic context about the internationalisation of microenterprises? The findings highlight that there is a lack of academic research focused on the internationalisation of microenterprises as a special group of enterprises. The study contributes to the field of international entrepreneurship and literature on microenterprises

Exploring and validating observations of non-local species in eDNA samples

Abstract The development of DNA-based methods in recent decades has opened the door to numerous new lines of research in the biological sciences. While the speed and accuracy of DNA methodologies are clearly beneficial, the sensitivity of these methods has the adverse effect of increased susceptibility to false positives resulting from contamination in field or lab. Here, we present findings from a metabarcoding study on the diet of and food availability for five insectivorous birds, in which multiple lepidopteran species not known to occur locally were discovered. After describing the pattern of occurrences of these non-local species in the samples, we discuss various potential origins of these sequences. First, we assessed that the taxonomic assignments appeared reliable, and local occurrences of many of the species could be plausibly ruled out. Then, we looked into the possibilities of natural environmental contamination, judging it to be unlikely, albeit impossible to fully falsify. Finally, while dissimilar combinations of non-local species’ occurrences across the samples did not initially suggest lab contamination, we found overlap with taxa and sequences handled in the same lab, which was undoubtedly not coincidental. Even so, not all exact sequences were accounted for in these locally conducted studies, nor was it clear if these and other sequences could remain detectable years later. Although the full explanation for the observations of non-local species remains inconclusive, these findings highlight the importance of critical examination of metabarcoding results, and showcase how species-level taxonomic assignments utilizing comprehensive reference libraries may be a tool in detecting potential contamination events, and false positives in general

The functional ability of older adults with visual impairments:a 2-year follow-up study

Abstract This study describes the self-estimated functional ability of older adults with visual impairments (VI) living at home prior to and after 24 months of individual low vision rehabilitation (LVR) according to the International Classification of Functioning, Disability and Health (ICF) framework. The LVR was carried out according to regular standard of care in Finland. The study provides knowledge that is relevant for improving both LVR as well as other services for older adults living with VI. Thirty-nine older adults with VI initially participated in the study with 28 remaining for the follow-up at 24 months of LVR. Data were collected by an orally administered questionnaire including items from the Oldwellactive Wellness Profile instrument. Data were analyzed using the marginal homogeneity test, and the outcomes were divided into four categories according to the ICF framework. Comparisons between the baseline and 2-year follow-up revealed statistically significant decreases in daily functions, including going outdoors (p = .011), washing oneself (p = .016), taking care for personal hygiene (p = .046), dressing (p = .034), preparing meals (p = .041), and doing heavy housework (p = .046), following 2 years of received LVR. A statistically significant increase in the need for help was also observed during the study period (p = .025). The independence of older adults with VI decreased, and the need for external services or help increased during 24 months after the onset of receiving LVR. Visual problems were shown to widely affect functional ability. Activities and participation dimension together with loneliness are most affected and need attention in individual LVR

Exosomes as secondary inductive signals involved in kidney organogenesis

Abstract 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 ex vivo 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

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

Abstract 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

Long-term follow up of families with pathogenic NFKB1 variants reveals incomplete penetrance and frequent inflammatory sequelae

Abstract Nuclear factor κ light-chain enhancer of activated B cells (NF-κB) family of evolutionarily conserved transcription factors are involved in key cellular signaling pathways. Previously, hypogammaglobulinemia and common variable immunodeficiency (CVID)-like phenotypes have been associated with NFKB1 variants and loss-of-function NFKB1 variants have been reported as the most common monogenic cause for CVID among Europeans. Here, we describe a Finnish cohort of NFKB1 carriers consisting of 31 living subjects in six different families carrying five distinct heterozygous variants. In contrast to previous reports, the clinical penetrance was not complete even with advancing age and the prevalence of CVID/hypogammaglobulinemia was significantly lower, whereas (auto)inflammatory manifestations were more common (42% of the total cohort). At current stage of knowledge, routine genetic screening of asymptomatic individuals is not recommended, but counseling of potential adult carriers seems necessary