Browsing by Subject "differentiation"

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  • Grönlund, Matti (Helsingfors universitet, 2016)
    The purpose of this study was to examine how students' in faculty of behavioural sciences understand and determine concept of giftedness, gifted student and teaching methods of gifted students. Research questions were (1) How do you understand and determine the concept of giftedness? (2) How do you recognize a gifted student? (3) How should the teaching of gifted students be differentiated and with what kind of methods? The first section of theory defined giftedness and what is giftedness and also teachers being determiners of giftedness. The second section of theory defined differentiation teaching in gifted students' point of view. The third section defined boundaries for teaching in elementary school. This study was a qualitative research completed with quantitative figures. The research material was gathered from students in faculty of behavioural sciences with a questionnaire. Materials were analyzed with theory-based content analyses. Similarities and differences was found between the research material and the research theory. As was assumed the was no inclusive concept of giftedness to be found based on this study. Also, the recognition of gifted student was found to be difficult and more or less the gifted seemed to be a student who was performing academically well. Problem solving, differentiation in lessons and extra assignments seemed to be the best methods of teaching to gifted students. The least effective was acceleration and quantitative augmentation of assignments.
  • Gonzalez, Marta Lopez; Oosterhoff, Dinja; Lindenberg, Jelle J.; Milenova, Ioanna; Lougheed, Sinead M.; Martianez, Tania; Dekker, Henk; Quixabeira, Dafne Carolina Alves; Hangalapura, Basav; Joore, Jos; Piersma, Sander R.; Cervera-Carrascon, Victor; Santos, Joao Manuel; Scheper, Rik J.; Verheul, Henk M. W.; Jimenez, Connie R.; Van De Ven, Rieneke; Hemminki, Akseli; Van Beusechem, Victor W.; De Gruijl, Tanja D. (2019)
    In patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivo vaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3 beta (GSK3 beta) as a pivotal kinase in both DC development and suppression. GSK3 beta inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3 beta induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivo melanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3 beta activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.
  • Vuorio, Emma (Helsingfors universitet, 2017)
    This study aims to find out if differentiation of the features of the 4P of the product milk can create value to customers in a particular customer segment. Creating value is in the core in every company´s actions. Customers buy products and services that create value to them and are willing to pay from the value they get. The more the product creates value, the more the customers are willing to pay. This study focused on the customer segment of young women with academic background who live in the capital region. Milk is a bulk product consumed widely in Finland. The product also has significant role in the nutritional history of the country and has a big role in Finnish nutritional recommendations. However the consumption of milk has decreased over the years. The theoretical background of this study is based on the following theories: marketing strategy and the 4P, customer value and differentiation. These theories formed the theoretical framework which gave the focus to the empirical experiment. The approach of the study was qualitative because qualitative research aims to understand the phenomenon it studies and the approach is more suitable for analysing verbal data. Qualitative research wants to get a deeper understanding and it gives room for individual´s thoughts and experience. The study was executed and a group interview using theme interview methods. For the study chosen features of the 4P of the product milk were differentiated and group interview focused on them. The differentiated features were the following. For product organic milk and milk with added protein, for price milk with a lower price, for place online shopping and for promotion advertising milk on social media. The findings of this study were that in some cases differentiation of the features if the 4P of the product milk can create value for customers but in most cases it does not. The focus group felt that the most valuable differentiated feature of the 4P was organically produced milk and other factors that supported the well-being of the production animals and the environment. If a company selling or producing milk tries to create value to its customers through differentiation of the features of the 4P, it needs to consider carefully which features to focus on.
  • Dekoninck, Sophie; Hannezo, Edouard; Sifrim, Alejandro; Miroshnikova, Yekaterina A.; Aragona, Mariaceleste; Malfait, Milan; Gargouri, Souhir; de Neunheuser, Charlotte; Dubois, Christine; Voet, Thierry; Wickström, Sara A.; Simons, Benjamin D.; Blanpain, Cédric (2020)
    Summary During embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.
  • Valtonen, Katja (Helsingin yliopisto, 2021)
    Lukivaikeus on verrattain yleinen oppimisvaikeus, johon jokainen opettaja luultavasti törmää uransa aikana. Koska lukivaikeuden taustalla on kielitietoisuuteen ja erityisesti äännetietoisuuteen liittyviä ongelmia, se aiheuttaa erityisiä hankaluuksia lukemisen ja kirjoittamisen lisäksi myös vieraiden kielten oppimisessa. Näin ollen olisi tärkeää, että vieraiden kielten opettajat ymmärtäisivät lukivaikeuden taustoja sekä eriyttämiskeinoja, jotka hyödyttävät erityisesti lukivaikeudesta kärsiviä vieraiden kielten oppijoita. Siksi tämän tutkimuksen tarkoituksena oli kartoittaa suomalaisten englanninopettajien kokemuksia lukivaikeuteen liittyen. Tutkimuksen tavoitteena oli selvittää, (1) mitä englanninopettajat tietävät lukivaikeudesta ja mistä he ovat tietonsa saaneet, (2) millaisia asenteita heillä on lukivaikeudesta kärsiviä oppilaita ja opiskelijoita kohtaan, (3) millaisina he näkevät lukivaikeuden vaikutukset englannin oppimiseen ja (4) miten he ovat ottaneet lukivaikeuden huomioon opetuksessaan. Kysymyksiin etsittiin vastausta kyselytutkimuksen keinoin. Kyselyä jaettiin sosiaalisessa mediassa ja sen täyttäminen tapahtui internetissä. Kaikki osallistujat (n = 72) olivat muodollisesti päteviä opettajia ja opettivat englantia suomenkielisissä alakouluissa, yläkouluissa, lukioissa ja/tai ammattikouluissa. Kyselyllä kerätty aineisto sisältää sekä määrällisiä että laadullisia tietoja. Määrällinen aineisto analysoitiin deskriptiivisin tilastollisin menetelmin ja laadullinen aineisto sisällönanalyysin keinoin. Tutkimukseen osallistuneilla opettajilla oli verrattain hyvä tietämys lukivaikeudesta, vaikkakaan he eivät kokeneet omaavansa tarvittavaa tietotaitoa lukivaikeudesta kärsivien oppijoiden tukemiseen. Lukivaikeutta ei joko ollut käsitelty ollenkaan tai ei riittävästi osallistujien opettajaopinnoissa. Sen sijaan osallistujat olivat saaneet tietonsa lukivaikeudesta muista lähteistä, kuten erityisopettajalta tai kirjallisuudesta oman aktiivisuutensa turvin. Osallistujien asenne lukivaikeutta ja siitä kärsiviä oppijoita kohtaan oli laajasti ottaen positiivinen. Opettajan näkökulmasta lukivaikeus vaikeuttaa englannin oppimista sanaston, kieliopin, kirjoittamisen, lukemisen, kuuntelun, ääntämisen ja äänne-erittelyn osalta. Oppijoilla saattaa myös olla erilaisia negatiivisia tunteita itseään, kieliä tai oppimista kohtaan. Lisäksi lukivaikeudesta kärsivät tarvitsevat enemmän aikaa tehtävien tekemiseen kuin luokkatoverinsa. Lähes kaikki osallistujat ovat ottaneet lukivaikeuden huomioon opetuksessaan esimerkiksi eriyttämällä arviointia, materiaaleja ja opetustaan. Osallistujille tutuimpia eriyttämisen keinoja olivat arvioinnin eriyttämiseen liittyvät keinot sekä sellaiset keinot, joita yleisesti käytetään kaikkien oppimisvaikeuksien huomioinnissa ja kaikkien oppiaineiden opetuksessa. Osallistujille vähemmän tuttuja olivat opetuksen eriyttämiseen liittyvät keinot, joita suositellaan nimenomaan lukivaikeudesta kärsiville vieraiden kielten oppijoille. Tutkielmassa pohditaan, miten hyvin opettajakoulutus valmistaa opettajia käytännön työhön, ja kritisoidaan sitä lähtökohtaa, että lukivaikeudesta kärsiviä kielten oppijoita tuetaan kouluissa ensisijaisesti arvioinnin keinoin oppimisen edesauttamisen sijaan.
  • Gómez Sánchez, Celia (Helsingin yliopisto, 2022)
    Kv7.1 is a potassium ion channel comprised of the KCNQ1 protein, which can coassemble with distinct β-subunits modulating the channel functions in different tissues. In 2017, Raivio’s group (from the University of Helsinki) found two missense mutations in the KCNQ1 gene, p.(Arg116Leu) and p.(Pro369Leu), responsible for causing pituitary hormone deficiency and maternally inherited gingival fibromatosis. The facial features and bone structure pointed to a cranial neural crest (CNC)-derived phenotype caused by an alteration in the potassium channel balance, given that these cells form the bone and cartilage of the cranial zone. To understand the implication of the CNC in the KCNQ1 syndrome, I attempted to replicate the CNC differentiation protocol of Suga and Furue (2019) with the aim of obtaining cranial neural crest cells (CNCCs). This would enable future generation of a KCNQ1-related disease model. The differentiation process was carried out thrice, and two BMP4 concentrations (10 and 100 ng/ml) were assayed. The differentiated cells exhibited a CNC-like morphology as well as upregulation of the marker genes (TFAP2A, SOX10, DLX1, MSX1, and DLX2) associated to this cell lineage. However, the gene expression was low according to the qRT-PCR Ct values, which were in most cases higher than 30. Additionally, no differences were found between the two BMP4 treatments. Furthermore, the cells did not express KCNQ1, and thus the impact of the two KCNQ1 mutations was not investigated under this protocol. In conclusion, the protocol had a low efficiency in the generation of CNCCs that was not improved by increasing the BMP4 concentration. Further optimization of the protocol, such as the BMP4 concentration or the cell density of the culture, will be needed to improve its efficiency and obtain an adequate disease model.
  • Porola, Pauliina (Helsingfors universitet, 2012)
    Hepatotoxicity is an undesired feature of many drugs and is one of the main reasons for attrition during the drug development process. Although an in vitro model can never totally correspond to or replace a whole organism, a reliable in vitro model for liver toxicity screening would help to detect liver toxicity earlier in the development process. Effective and early in vitro screening would reduce the need of animal subjects and clinical trials and thus would be both ethically more acceptable and more cost-effective. Currently mostly used models for liver metabolism and toxicity studies are primary hepatocytes, hepatic cell lines and animal models. However, these models have many drawbacks and are not considered reliable. Human embryonic stem cells (hESCs) are pluripotent cells that can be differentiated into many specialized cell types including hepatocytes. They are also self-renewable and thus represent an unlimited and promising source of hepatocytes to be used as a tool in in vitro liver toxicity testing of drug candidates. The aim of this study was to produce hepatocytes from hESCs via multiple steps following the in vivo pathway of developing hepatocytes: first hESCs were differentiated into definitive endoderm cells, after which they were differentiated into hepatic progenitor cells. Finally, hepatocyte-like cells (HLCs) were induced from the progenitor cells. Our specific interest was the use of hepatic cell derived acellular matrix as a differentiation basis for hepatic progenitors and hepatocytes. We also studied the effect of Matrigel overlay on the hepatic differentiation. Differentiation method without the Matrigel overlay was promising. HLCs showed correct hepatocyte-like morphology and expressed hepatocyte markers such as albumin, α-antitrypsin, CYP3A4 and HNF4α both on mRNA and protein level shown by qPCR and flow cytometry and immunofluorescence staining, respectively. Accordingly, the expression of stem cells marker SSEA-3 showed a tendency to decrease as the differentiation proceeded. HLCs also functionally resembled hepatocytes shown by albumin production. However, we could not detect other hepatocyte functions such as urea production or CYP activity. With Matrigel overlay, the hepatocyte-like morphology of the cells was lost, no albumin production was shown and the expression of several hepatocyte markers was lower than in the experiment done without the Matrigel overlay. Thus, Matrigel overlay was shown to be unbeneficial for hepatocyte differentiation. In conclusion, we showed that differentiation of hESCs on the acellular matrix with specific growth factors and without the Matrigel overlay seems promising as a method to produce HLCs. This preliminary study serves as a basis for future studies, in which the differentiation method should still be further studied and developed to yield functional HLCs of uniform quality.
  • Kuisma, Saara (Helsingfors universitet, 2012)
    Pharmaceutical companies are currently facing increasing developmental costs, and at the same time, less new compounds are being brought to the market. In vitro -metabolism studies and toxicity assessment of new drug candidates are crucial, as early as possible, to prevent their withdrawal in later development phases. Used study systems are, however, limited and new improved technologies are being investigated. Notable, drug induced liver toxicity and alterations in the liver function are frequent reasons for the drug removals from the development. Human embryonic stem cell (hESC) is one of the most powerful cell types known. hESCs have not only the possibility to divide indefinitely but these cells have also the ability to differentiate to all mature cell types of the human body, such as hepatocytes. This makes them potentially very valuable for pharmaceutical development, in order to create a functional in vitro -model, mimicking the liver tissue. In the literature part, the three dimensional (3D) -hepatic differentiation of mouse and human ESCs in vitro, are discussed. Traditional 2D-culture systems do not adequately mimic the microenvironment of three dimensionally organized native tissue. In 2D-cultures cells grow as a monolayer, when the cell morphology is flattened leading to poor cell-cell and cell-matrix contacts and preventing from the tissue formation. In 3D-culture systems, cells are able to form tissue-like cell integrations, spheroids, and thus, remain their functionality and viability significantly longer. Hydrogels are commonly used biomaterials in 3D-cell cultivation and well known in various areas of tissue engineering for their nano scale porosity and ability to surround cells in 3D-polymer network. In addition, they are capable to absorb large volumes of water and functionalized, in various ways, to improve the required biological or mechanical properties. In the experimental part, the main purpose was to differentiate human hepatic progenitor cells to mature hepatocyte-like cells in three dimensional (3D) -biomaterials. Overall, four different hydrogels (cellulose nanofiber (CNF) hydrogel, HydroMatrixTM, ExtracelTM and PuraMatrixTM) were used as 3D-cell culture scaffolds. Several hepatic cell functions (albumin and urea production and cytochrome P450 (CYP) 3A4 activity) were measured in 2D- and 3D-cultures and compared with the human hepatic carcinoma cells, HepG2, which are often used in drug development. Differentiated hepatocyte-like cells did not show CYP3A4 activity and they produced less albumin and urea compared with HepG2 cells. However, working with hESCs is very demanding and the research in this area is only in the beginning. Therefore, the poor cell functionality results did not come up as a surprise.
  • Hellinen, Laura; Hongisto, Heidi; Ramsay, Eva; Kaarniranta, Kai; Vellonen, Kati-Sisko; Skottman, Heli; Ruponen, Marika (2020)
    The retinal pigment epithelial (RPE) cell monolayer forms the outer blood-retinal barrier and has a crucial role in ocular pharmacokinetics. Although several RPE cell models are available, there have been no systematic comparisons of their barrier properties with respect to drug permeability. We compared the barrier properties of several RPE secondary cell lines (ARPE19, ARPE19mel, and LEPI) and both primary (hfRPE) and stem-cell derived RPE (hESC-RPE) cells by investigating the permeability of nine drugs (aztreonam, ciprofloxacin, dexamethasone, fluconazole, ganciclovir, ketorolac, methotrexate, voriconazole, and quinidine) across cell monolayers. ARPE19, ARPE19mel, and hfRPE cells displayed a narrow P-app value range, with relatively high permeation rates (5.2-26 x 10(-6) cm/s. In contrast, hESC-RPE and LEPI cells efficiently restricted the drug flux, and displayed even lower P-app values than those reported for bovine RPE-choroid, with the range of 0.4-32 cm(-6)/s (hESC-RPE cells) and 0.4-29 x 10(-6) cm/s, (LEPI cells). Therefore, ARPE19, ARPE19mel, and hfRPE cells failed to form a tight barrier, whereas hESC-RPE and LEPI cells restricted the drug flux to a similar extent as bovine RPE-choroid. Therefore, LEPI and hESC-RPE cells are valuable tools in ocular drug discovery.
  • Li, Hao; Hohenstein, Peter; Kuure, Satu (2021)
    The adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could replenish functional homeostasis similarly to, e.g., skin or the hematopoietic system. Unlike a mature kidney, the embryonic kidney hosts at least three types of lineage-specific stem cells that give rise to (a) a ureter and collecting duct system, (b) nephrons, and (c) mesangial cells together with connective tissue of the stroma. Extensive interest has been raised towards these embryonic progenitor cells, which are normally lost before birth in humans but remain part of the undifferentiated nephrogenic rests in the pediatric renal cancer Wilms tumor. Here, we discuss the current understanding of kidney-specific embryonic progenitor regulation in the innate environment of the developing kidney and the types of disruptions in their balanced regulation that lead to the formation of Wilms tumor.
  • Peteri, Ulla-Kaisa; Pitkonen, Juho; Utami, Kagistia Hana; Paavola, Jere; Roybon, Laurent; Pouladi, Mahmoud A.; Castren, Maija L. (2021)
    Astrocytes form functionally and morphologically distinct populations of cells with brainregion-specific properties. Human pluripotent stem cells (hPSCs) offer possibilities to generate astroglia for studies investigating mechanisms governing the emergence of astrocytic diversity. We established a method to generate human astrocytes from hPSCs with forebrain patterning and final specification with ciliary neurotrophic factor (CNTF). Transcriptome profiling and gene enrichment analysis monitored the sequential expression of genes determining astrocyte differentiation and confirmed activation of forebrain differentiation pathways at Day 30 (D30) and D60 of differentiation in vitro. More than 90% of astrocytes aged D95 in vitro co-expressed the astrocytic markers glial fibrillary acidic protein (GFAP) and S100 beta. Intracellular calcium responses to ATP indicated differentiation of the functional astrocyte population with constitutive monocyte chemoattractant protein-1 (MCP-1/CCL2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) expression. The method was reproducible across several hPSC lines, and the data demonstrated the usefulness of forebrain astrocyte modeling in research investigating forebrain pathology.
  • Peltoniemi, Pasi (Helsingfors universitet, 2012)
    Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have two unique properties: the self-renewal capacity and the broad developmental potential. They both have their advantages and disadvantages, but the current perception is that hESCs and hiPSCs complement rather than replace each other. New scientific problems and ethical challenges will arise because stem cell research is developing rapidly. The potential of hiPSC and hESC technologies in drug discovery is tremendous. The human pluripotent stem cell (hPSC)-derived cells have a potential to replace a part of the current preclinical toxicity and efficacy screening tests and to prevent misrouted drug development and use for lead optimization at phases before clinical trials. The hPSC-based disease models can also narrow the gap between traditional animal models and clinical trials. One major challenge is the differentiation process of hPSCs into cells of the relevant tissue. The recent study of our laboratory shows that the liver cell-deried acellular matrix (ACM) promotes the hepatic commitment of hESCs. To create chemically defined, xeno-free and feeder-free culture matrices for the differentiation of the hESCs into hepatocyte-like cells (HLCs), the ECM components of the ACM were characterized. The results suggest that the ACM contains fibronectin, laminins. After the characterization, the object was to identify which of the ECM proteins are essential and effective in the differentiation. A three-step differentiation protocol with differenent ECM protein solutions was used to produce HLCs. The hESCs were first induced into definitive endoderm (DE) cells. The DE cells were committed to the bipotential hepatic progenitors positive for HNF4α and AFP. Finally the progenitors were differentiated into HLCs. The mRNA expression of albumin, CK8, CK18, AAT, and BCRP was increased in HLCs. All the derived HLCs were albumin positive. The hESCderived HLCs showed hepatic morphology, cytoplasmic vacuole characteristics, and functional albumin secretion. The chemically defined matrices showed a supportive role in the differentiation of the hESCs into HLCs. This study establishes an efficient, chemically defined, xeno-free system to produce HLCs as a cell source for pharmaceutical and developmental studies.
  • Chen, Yan; Stegaev, Vasily; Kouri, Vesa-Petteri; Sillat, Tarvo; Chazot, Paul L.; Stark, Holger; Wen, Jian Guo; Konttinen, Yrjo T. (2015)
    To date, conventional and/or novel histamine receptors (HRs) have not been investigated in mouse skeletal myogenesis. Therefore, the present study aimed to investigate the HR-subtypes in skeletal myogenesis. The myogenesis of C2C12 skeletal myoblasts was evaluated using desmin, myogenin and myosin heavy chain (Myh) as early, intermediate and late differentiation markers, respectively. Reverse transcription-quantitative polymerase chain reaction and immunostaining were performed and the messenger RNA (mRNA) expression levels of the HR-subtypes and markers were determined. H1R mRNA was found to be highly expressed in myoblasts at day 0; however, the expression levels were reduced as differentiation progressed. By contrast, H2R mRNA expression remained constant, while H3R mRNA expression increased by 28-, 103- and 198-fold at days 2, 4 and 6 compared with the baseline level (day 0), respectively. In addition, Myh expression increased by 7,718-, 94,487- and 286,288-fold on days 2, 4 and 6 compared with the baseline expression level (day 0). Weak positive staining of the cells for H3R protein was observed on day 2, whereas highly positive staining was observed on days 4 and 6. HR expression during myogenesis was, in part, regulated by the stage of differentiation. These results along with previous findings indicated possible involvement of H1R in the regulation of progenitor cell mitogenesis and of H2R in the relaxation of acetylcholine-stimulated contraction of muscle cells, following the activation of professional histamine-producing cells, including mast cells. By contrast, H3R may participate in the regulation of specialized myocyte functions, potentially by maintaining the relaxed state under the influence of constitutive H3R activity and low histamine concentrations, locally produced/released by non-professional histamine-producing cells.
  • Tikker, Laura; Casarotto, Plinio; Singh, Parul; Biojone, Caroline; Piepponen, T. Petteri; Estartus, Nuri; Seelbach, Anna; Sridharan, Ravindran; Laukkanen, Liina; Castren, Eero; Partanen, Juha (2020)
    Serotonergic neurons in the dorsal raphe (DR) nucleus are associated with several psychiatric disorders including depression and anxiety disorders, which often have a neurodevelopmental component. During embryonic development, GATA transcription factors GATA2 and GATA3 operate as serotonergic neuron fate selectors and regulate the differentiation of serotonergic neuron subtypes of DR. Here, we analyzed the requirement of GATA cofactor ZFPM1 in the development of serotonergic neurons using Zfpm1 conditional mouse mutants. Our results demonstrated that, unlike the GATA factors, ZFPM1 is not essential for the early differentiation of serotonergic precursors in the embryonic rhombomere 1. In contrast, in perinatal and adult male and female Zfpm1 mutants, a lateral subpopulation of DR neurons (ventrolateral part of the DR) was lost, whereas the number of serotonergic neurons in a medial subpopulation (dorsal region of the medial DR) had increased. Additionally, adult male and female Zfpm1 mutants had reduced serotonin concentration in rostral brain areas and displayed increased anxiety-like behavior. Interestingly, female Zfpm1 mutant mice showed elevated contextual fear memory that was abolished with chronic fluoxetine treatment. Altogether, these results demonstrate the importance of ZFPM1 for the development of DR serotonergic neuron subtypes involved in mood regulation. It also suggests that the neuronal fate selector function of GATAs is modulated by their cofactors to refine the differentiation of neuronal subtypes.
  • Kurtzeborn, Kristen; Kwon, Hyuk Nam; Kuure, Satu (2019)
    Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects derived from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that the MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer, Wilms' tumor.
  • Hellinen, Laura; Pirskanen, Lea; Tengvall-Unadike, Unni; Urtti, Arto; Reinisalo, Mika (2019)
    Retinal pigment epithelium (RPE) acts as an outer blood-retinal barrier that limits the access of circulating xenobiotics to the eye. In addition, the RPE limits posterior elimination of intravitreally injected drugs to circulation. Thus, permeation in the RPE has a significant effect on ocular pharmacokinetics. The RPE is also a potentially important drug target in age-related macular degeneration. Therefore, the cell models of the RPE are important tools in ocular drug development, but poor availability and problems in reproducibility limit the use of primary RPE cell cultures. Furthermore, the best and widely used human cell line ARPE19 requires specialized culture conditions and a long time for cellular differentiation. In this paper, we describe a cell population arisen from the ARPE19 culture, with fast differentiation and improved barrier properties. This cell line, LEPI, forms clear microvilli and rapidly displays RPE-like cobblestone morphology after subculture in simple culture conditions. The LEPI cells show RPE-specific functions and expression of RPE65, ezrin, and BEST1 proteins. On filter, the LEPI cells develop tighter barrier than the ex vivo bovine RPE-choroid: permeability coefficients of beta-blockers (atenolol, nadolol, timolol, pindolol, metoprolol, betaxolol) ranged from 0.4 x 10(-6 )cm/sec to 2.3 x 10(-6) cm/sec depending on the drug lipophilicity. This rapidly differentiating cell line will be an asset in ocular studies since it is easily maintained, it grows and differentiates quickly and does not require specialized culture conditions for differentiation. Thus, this cell line is suitable for both small scale assays and high throughput screening in drug discovery and development.
  • Lintala, Annika (Helsingin yliopisto, 2020)
    Bipotential gonads are precursor structures for testes and ovaries. Steroidogenic factor-1 (SF1) is one of the most important transcription factors in an embryo needed for the development and maintenance of bipotential gonads. If SF1 is not expressed, bipotential gonads fail to develop, and genitalia and kidneys are not formed. Later, SF1 expression persists high in testes, where it supports Sertoli and Leydig cell formation and development. If SF1 is not expressed enough in males, the bipotential gonads differentiate into ovaries. The factors activating and regulating SF1 are not currently fully known. By getting more knowledge of how SF1 is controlled, regulatory mechanisms behind normal fetal development of gonads and disorders of sex development (DSD) can be understood better. The aims of this thesis were to study whether growth factors, that naturally regulate differentiation of developing gonads, promote differentiation of human induced pluripotent stem cells (hiPSC) into Sertoli-like cells (SLCs) and whether SF1 expression is induced by the addition of these growth factors. For conducting the study, we used hiPSCs, which have an SF1 activation domain cassette previously introduced to the cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) method. SF1 could be activated by adding doxycycline (DOX) and trimethoprim (TMP). These hiPSCs were differentiated into intermediate mesoderm (IM) on the first four days according to the protocol published earlier by the group. After this, the differentiation to SLCs was guided by adding growth factors to the culture medium. Basic fibroblast growth factor (bFGF), fibroblast growth factor 9 (FGF9) and prostaglandin-2 (PGD2) were tested separately and in a combined cocktail also including follicle stimulating (FSH) and glial cell-derived neurotrophic factor (GDNF). In a control condition, cells were differentiated without additional growth factors. In all tested conditions, cells first differentiated into IM were further differentiated either in the presence or absence of DOX and TMP for 8 days. The differentiation medias were changed to the cells every day and lysis samples for quantitative PCR (qRT-PCR) were taken every other day. The relative gene expression levels of bipotential gonad, testis and steroidogenic gene markers from each condition were monitored with qRT-PCR and compared to the levels of the undifferentiated hiPSCs. Immunocytochemistry was performed to see the changes in protein production. Against our hypothesis and the previous studies by others, none of the tested growth factors induced the cells to differentiate into SLCs. However, SF1 expression was triggered by chemical induction with DOX and TMP. Also, the expression levels of bipotential gonadal and testicular gene markers increased in control conditions with/without chemical induction. PGD2 conditions were the only ones to resemble the gene expression and morphology of control conditions while the others differed. These results indicated that the addition of bFGF, FGF9, FSH and GDNF did not improve the differentiation of iPSCs into SLCs and in fact, bFGF and FGF9 hindered their differentiation into SLCs. As a future perspective the optimal concentrations for each growth factor and the duration of growth factor supplementation ought to be tested to refine the protocol.
  • Jekunen, Jaakko (Helsingin yliopisto, 2020)
    In my Master’s thesis, I offer a novel interpretation of Gilles Deleuze’s (1925-1995) conception of transcendent thinking. As a first approximation, transcendent thinking is an unconscious disruption of quotidian thinking (i.e. empirical thinking). Deleuze’s conception is an important attempt at explaining the emergence of thought from material reality. Additionally, it offers insights into the conditions of creating something new in thinking. In Deleuze’s account, these two are closely connected. My interpretation is mainly based on Deleuze’s Difference and Repetition (1968), but I also draw from Deleuze’s other works and philosophers he discusses. Deleuze’s reading of Immanuel Kant (1724–1804) is important for my interpretation. I proceed by close readings of Deleuze and compare my interpretations to others from secondary literature. My thesis is divided into five chapters and I begin by introducing my reading of the relevant features of Deleuze’s overall project in Difference and Repetition. In chapter one, I introduce Deleuze’s novel philosophy of difference. According to Deleuze, all continuity we experience is constituted by the interplay of internal difference and hidden repetition. In chapter two, I introduce the relevant features of Deleuze’s ontological scheme in Difference and Repetition. According to it, actual objects are constituted through the process of different/ciation; two figures of internal difference, the differential relations of virtual Ideas and intensive differences, produce the actual objects we perceive in our experience. Situating Deleuze’s transcendent thinking into his overall project is necessary to interpret it correctly and to grasp its significance. Next, I interpret what Deleuze means by thinking. In chapter three, I read Immanuel Kant’s (1724–1804) determining judgment (e.g. “This is a dog”) as providing a case of Deleuze’s empirical thinking. This kind of thinking is what human subjects experience in the quotidian. However, transcendent thinking goes beyond empirical thinking. In chapter four, I show how transcendent thinking is comprised of a series of encounters where the different faculties (i.e. cognitive capabilities) of the thinker are elevated to their transcendent exercise. This series starts as sensibility encounters sensible intensity and it continues as subsequent faculties are traversed by a virtual Idea. In these encounters, the faculties confront their internal differences, which reveal their limits and what is most singular to them. However, intermediary encounters do not correspond to any conscious empirical experiences, nor does the whole of transcendent thinking either. In the final chapter of my Master’s thesis, I begin by arguing that my interpretation ameliorates on previous readings. First, it reveals that transcendent thinking is a case of different/ciation unravelling through the faculties of a psychic system. Second, my reading distinguishes between empirical thinking and transcendent thinking—both being kinds of thinking, for Deleuze. Third, it clarifies that learning is an instance of transcendent thinking (not vaguely thinking in general). Next, I discuss how transcendent thinking reveals the possibility of creation in thinking. Empirical thinking is incapable of change because in it, the faculties function according to the model of recognition: the thinker only recognizes what is already known using pregiven concepts. Transcendent thinking, as a case of different/ciation progressing through the faculties, changes the faculties and, in doing so, transforms the composition of the psychic system. This process is carried out on the level of being and results in something new emerging in thinking. However, transcendent thinking is involuntary and unconscious, leaving the conception of creative agency in Difference and Repetition restricted.
  • Karhunen, Emilia (Helsingfors universitet, 2018)
    Functional in vitro cultured human hepatocytes are needed in different applications in biomedical research. Treatment for liver diseases is usually liver transplantation, but due to the lack of healthy donors, cell therapy using hepatocytes is considered as a better option. Drug industry will also need representative liver models to test metabolic profiles of drug molecules. Primary human hepatocytes are studied in cell therapy and disease modelling, but they have also drawbacks. In vitro they do not proliferate efficiently, and they are short-lived. In vitro differentiated human pluripotent stem cells (hPSCs) to hepatic fate are an alternative for the primary human hepatocytes. Especially human induced pluripotent stem cells (hiPSCs) are widely studied because they are easily available, and they even make personalized therapy possible without problems with ethical issues related to the human embryonic stem cells (hESCs). Differentiation to hepatic fate includes several steps before mature functional hepatocyte-like cells are formed. Hepatocytes are derived from the definitive endoderm (DE) which is one of the germ layers formed in the gastrulation process. Efficient induction of hPSCs into DE lineage would be a good starting point for generating mature hepatocyte-like cells in further hepatic differentiation. Different protocols to differentiate hPSCs in vitro into DE have been published. In vitro cell culture systems should well represent the environment of the target tissue because signals from the environment guide the differentiation. Three-dimensional (3D) cell culture systems are widely studied, because they better mimic the in vivo microenvironment of cells than two-dimensional (2D) cell culture. The aim of the thesis was to study the efficacy of the 3D differentiation of hiPSCs into DE. Before starting the 3D differentiation, differentiation protocol was optimized and the effect of ROCK inhibitor Y-27632 was investigated. Differentiation medium was supplemented with Y-27632 during the whole 6 days differentiation, because survival of the cells and formation of the spheroids were improved, and gene expression studies of pluripotency markers and several DE markers did not show evident effect of Y-27632 on the gene expression of hiPSCs. The main objective in the studies was also to investigate possible differences between different 3D culture conditions on hiPSCs differentiation into DE. Also, the effect of the spheroid size on differentiation was examined. Two different hydrogels were used as a matrix material in the experiments: basement membrane extract (BME) and nanofibrillar cellulose (NFC) hydrogels. Suspension culture was used as a biomaterial-free 3D culture system. Experiments were performed with three spheroid sizes: 200 cells/spheroid, 500 cells/spheroid and 1000 cells/spheroid. Efficacy of differentiation to DE lineage was estimated by studying protein and mRNA expression of some of the DE markers (HNF3B, SOX17, CXCR4, CER1), pluripotency marker OCT4, mesendoderm marker Brachyury and hepatoblast marker HNF4A in the cells. Spheroids differentiated in suspension and NFC were analysed by flow cytometry to get the number of DE positive live cells and dead cells using CXCR4 and 7-AAD double staining. Besides flow cytometry, protein expression of some of the key markers were studied by immunofluorescent staining and further confocal imaging. Viability of the spheroids in BME hydrogel culture were investigated using live/dead staining followed by confocal imaging. BME hydrogel culture was left out from the further experiments due to the morphology of the spheroids and results from viability and protein expression studies. Spheroids in suspension started DE differentiation faster compared to NFC culture. Suspension and NFC cultures yielded high number of double positive cells in flow cytometry and bright fluorescence of other DE markers was seen in the confocal images. NFC hydrogel proved to be a promising 3D culture system by supporting the differentiation of hiPSCs. Flow cytometry results and gene expression studies propose that four days long 3D differentiation would be efficient to produce sufficient number of DE cells. Smaller spheroids showed higher number of DE positive cells than bigger spheroids on day 2 but gene expression studies showed difference in DE marker expression between size conditions rather in later days in differentiation and it was the opposite. Experiments showed signs of more efficient differentiation of the smaller sized spheroids in the beginning of differentiation. But further studies are needed to verify the obtained results and both draw conclusions about the possible differences between different 3D culture systems and explore the best size of the spheroid for hepatic differentiation. However, results obtained from the studies are useful for designing further experiments.