Sukurauhasten kehityksen ja toiminnan mallintaminen monikykyisten humaanikantasolujen avulla

Human gonads (testes and ovaries) develop from mesoderm as paired organs and are initially identical, or bipotent, in males and females. Upon sex determination, the gonads initiate differential transcription, molecular signaling, and hormonal patterns and begin to display their sex-specific characteristics. Occasionally, these complex processes become disrupted, causing impaired gonadal development at embryonic and early fetal stages and altered gonadal functions, which may lead to infertility or even abnormal sex development. The molecular mechanisms delineating early gonadal development and sex determination are not thoroughly understood and have been challenging to investigate in humans. Human pluripotent stem cells (hPSCs), comprising human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are a versatile tool for studying regulatory mechanisms of human organ development and related disease processes. hPSCs can form all three germ layers of an early embryo and have an endless capacity to self-renew, providing an infinite source of cells equivalent to the undifferentiated cells of an embryo. Follicle-stimulating hormone (FSH) and its receptor (FSHR) are necessary for normal development and function of the reproductive system. Any disruptions in FSH-FSHR signaling may cause severe fertility issues. For example, inactivating FSHR mutation resulting in an alanine to valine (A189V) amino acid substitution leads in women to amenorrhea and infertility due to blocked follicle development and in men to markedly reduced spermatogenic capacity but partially preserved fertility. The disease mechanisms for the observed phenotypes remain obscure. In this study, the first aim was to reveal regulatory pathways and signaling mechanisms involved in early gonadal development by developing an hPSC-based in vitro model. The second aim was to evaluate the regulatory role of nuclear receptor subfamily 5 group A member 1 (NR5A1), a gene encoding steroidogenic factor 1 (SF1), in early gonadal cell transcriptomics. The third aim was to examine the function of human wild-type (WT) FSHR and FSHR with an inactivating A189V mutation. To model gonadal development, we differentiated hPSCs into bipotential gonadal-like cells via primitive streak- and intermediate mesoderm-like stages by stimulating activin, bone morphogenetic protein, and Wingless-related integration site-dependent signaling, demonstrating the importance of the proper balance between these signaling pathways in early gonadal differentiation. hiPSCs of male origin, differentiated to the bipotential gonadal-like stage, were further directed towards more mature gonadal-like anti-Müllerian hormone-producing and steroidogenic cells by activating endogenous NR5A1 with CRISPR-Cas9 technology. Cells differentiated in the absence of NR5A1 activation did not exhibit these characteristics, as assayed by gene expression and immunodetection-based methods and mass spectrometry. Moreover, by performing whole sample RNA sequencing analysis for the cells in the presence and absence of NR5A1 activation, we identified many novel targets of NR5A1 and altered gene expression patterns of many gonad-related genes and genes not previously associated with gonads in response to NR5A1. We also established a protocol for directed differentiation of hPSCs into cells endogenously expressing FSHR. By differentiating hESCs expressing WT FSHR and patient-specific hiPSCs expressing mutated A189V FSHR and by performing immunosorbent-based functional assays in the differentiated cells, we demonstrated distinct functional response to FSH between the mutated and WT receptors. In addition, we identified several novel FSHR-protein interactions by using affinity proximity mass spectrometry in HEK293 cells stably expressing WT or A189V FSHRs in stimulated and unstimulated conditions. This research project provided novel insights into signaling and gene regulatory mechanisms during early gonadal development. The established differentiation protocols can serve as promising tools also in future studies. For example, the gonadal models can be used to examine gonadal dysgenesis or causes of infertility with patient-derived hiPSCs. This project also yielded a considerable amount of publicly available transcriptomics and proteomics data, which can be utilized in evaluating SF1 and FSHR biology.Miehen ja naisen sukurauhaset (kivekset, munasarjat) ovat alun perin samankaltaiset. Sukupuolenmääräytymisestä lähtien sukurauhasten geeniekspressio, signalointi- ja hormonitoiminta alkavat ilmentää kunkin sukupuolen piirteitä. Toisinaan nämä toiminnot häiriintyvät johtaen sukurauhasten varhaiskehityksen häiriöihin ja normaalista poikkeavaan toimintaan, mikä puolestaan saattaa johtaa hedelmättömyyteen tai häiriöihin sukupuolenmääräytymisessä. Sukurauhasten varhaiskehitykseen ja sukupuolenmääräytymiseen liittyviä mekanismeja tunnetaan edelleen huonosti. Ihmisen monikykyiset kantasolut (hPS-solut), joihin luetaan sikiöaikaiset kantasolut (hES-solut) ja indusoidut monikykyiset kantasolut (hiPS-solut), ovat hyödyllisiä selvitettäessä ihmisen elinten kehitykseen liittyvien säätelytekijöiden vaikutuksia ja tautimekanismien mallinnuksessa. Follikkeleita stimuloiva hormoni (FSH) ja sen reseptori (FSHR) ovat välttämättömiä lisääntymiselinten normaalin kehityksen ja toiminnan kannalta. Eräs FSH-reseptorin inaktivoiva mutaatio (A189V), joka johtaa alaniinin korvautumiseen valiini-aminohapolla, johtaa naisilla follikkeleiden kehityksen pysähtymiseen ja sen seurauksena kuukautisten puuttumiseen ja hedelmättömyyteen. Vastaava mutaatio miehillä heikentää siittiötuotantoa. Näihin poikkeaviin ilmiasuihin johtavat tautimekanismit eivät ole vielä kovin hyvin selvillä. Tämän tutkimuksen ensimmäisenä tavoitteena oli selvittää varhaisten sukurauhasten kehityksen säätely-ja signalointimekanismeja ja kehittää sitä varten kantasoluihin perustuva soluviljelymalli. Toisena tavoitteena oli tutkia tumareseptori 5 A 1 (NR5A1)-geenin säätelyvaikutusta kehittyvien sukurauhasten geenien ilmentymisessä. Kolmantena tavoitteena oli tutkia villityypin ja A189V mutantti-FSHR:n toimintaa. Tutkimuksessa mallinsimme sukurauhasten kehitystä ohjaamalla hPS-solujen kehitystä sukurauhasen kaltaisiksi soluiksi stimuloimalla solunsisäisiä signalointireittejä. Lisäksi erilaistimme anti-müllerian hormonia ja sukupuolihormoneja tuottavia sukurauhasen kaltaisia soluja aktivoimalla NR5A1-geeniä CRISPR-Cas9-teknologian avulla. Solujen RNA sekvensointi-analyysi osoitti useita uusia NR5A1:n säätelykohteita ja geenejä, joiden yhteyttä NR5A1:n ja sukurauhasten välillä ei ole aiemmin osoitettu. Kehitimme myös menetelmän hPS-solujen erilaistamiseksi FSHR-geeniä ilmentäviksi soluiksi. Erilaistimme villityypin ja mutantti FSH-reseptoria ilmentäviä soluja ja osoitimme ELISA vasta-ainemäärityksin niiden erilaisen vasteen FSH:lle. Lisäksi identifioimme affiniteettimassaspektrometrialla useita uusia vuorovaikutussuhteita FSH-reseptorin ja muiden proteiinien välillä FSH:lla stimuloiduissa ja kontrolli HEK293 soluissa, jotka ilmensivät joko villityypin tai mutantti FSH-reseptoria. Tämän työn ansiosta saatiin tietoa sukurauhasten varhaiskehityksen säätelystä geeni-ja signalointitasoilla. Kehitettyjä menetelmiä voidaan hyödyntää selvitettäessä sukurauhasten kehityshäiriöiden ja hedelmättömyyden syitä. Projektista saatiin runsaasti transkriptomiikka-ja proteomiikka-aineistoa, jota voidaan hyödyntää jatkotutkimuksissa

An Analytic Model for Nano Confined Fluids Phase-Transition (Applications for Confined Fluids in Nanotube and Nanoslit)

In this report, an analytic model to predict phase transitions of confined fluids in nano systems is presented and it is used to predict the behavior of the confined fluid in nanotubes and nanoslits. In our approach besides including a third degree of freedom due to wall effect to define the state of the system, the tensorial character for pressure is considered. Using the perturbation theory of statistical mechanics it is shown that the van der Waals equation of state is equally valid for small as well as large systems. The model proposed is shown to predict the liquid-vapor phase transition as well as the critical point in any size confined fluid systems. It is also shown that the critical temperature increases with the size of the nano system and finally it reaches the macroscopic critical temperature value as the diameter of the nanotube (or width of the nanoslit) approaches infinity. The proposed model can also demonstrate the existence of the local density and phase fragmentations during phase transitions in a confined nano system.Comment: 28 pages, 10 figure

Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells

Peer reviewe

Valtion tutkimuslaitosten ja -rahoituksen kokonaisuudistuksen (TULA-uudistus) arviointi

Arvioinnin kohteena on ollut tutkimuslaitosten ja tutkimusrahoituksen kokonaisuudistus (TULA-uudistus), josta valtioneuvosto teki periaatepäätöksen vuonna 2013. Uudistuksen toimenpiteet koskivat tutkimuslaitosten yhdistämistä ja yhtiöittämistä, tutkimuslaitosten ja korkeakoulujen välisen yhteistyön syventämistä, toimintojen kehittämistä sekä uusia päätöksentekoa tukevia tutkimuksen rahoitusmuotoja kuten strategisen tutkimuksen rahoitus (STN) ja valtioneuvoston selvitys- ja tutkimustoiminta (VN TEAS). Arvioinnissa selvitettiin, miten TULA-uudistus on toteutunut, ja miten se on edesauttanut tutkimuksen toimimista yhteiskunnan kehittämisen ja päätöksenteon strategisena resurssina. Arviointi perustui dokumenttianalyysin, kyselyjen, haastattelujen ja muun asiantuntijatyön tuottaman laadullisen ja määrällisen aineiston analyysiin. Henkilöstökyselyn aineisto on saatavilla avoimena datana: https://www.ulap-land.fi/opendata. Arvioinnin tuloksena syntyi tietoa uudistuksen tuloksellisuudesta ja tehokkuudesta, selkeydestä, yhtenäisyydestä, relevanssista sekä vaikutuksista yhteiskunnan tulevaisuuden osaamis- ja tutkimusvalmiuksiin. Arvioinnin perusteella tutkimuskentässä ei ole tarvetta uusille mittaville rakenteellisille muutoksille, vaan suositukset annettiin nykyisen järjestelmän edelleen kehittämiseksi

Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans.</p

Localization of I-BAR domain proteins in invasive cancer cells

An important step in cancer metastasis is the ability of the cancer cells to degrade the surrounding extracellular matrix (ECM) and migrate through it. To accomplish this, invasive cancer cells generate protrusive structures called invadopodia, which are dynamic cell protrusions that secrete matrix metalloproteinases (MMPs) responsible for the degradation of the ECM. Invadopodia formation employs the actin polymerization machinery and several cell adhesion, signal transduction and matrix degradation proteins. In addition, mechanisms involving membrane deformation may play a major role in their formation. Recently, an evolutionary conserved group of proteins involved in the formation of cell protrusions was characterized. These proteins were found to contain an inverse Bin-Amphiphysin-Rvs167 (I-BAR) domain. The I-BAR domain is capable of bending the plasma membrane outwards and via accessory domains, the I-BAR proteins link this membrane bending activity to actin cytoskeleton remodeling to generate cell protrusions. In the present study, the aim was to examine the localization of I-BAR domain proteins in invasive cancer cell lines. First, I studied the expression of the members of the I-BAR gene family (ABBA, IRTKS, IRSp53) in MDA-MB-231 breast cancer cells and SNB-19 glioma cells by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The results indicated that I-BAR domain proteins were expressed at the messenger RNA (mRNA) and protein level in both cell lines. Then, to investigate the sub-cellular localization of these proteins to invasive sites, an ECM degradation assay was set up to distinguish invadopodias from other actin-rich sub-cellular structures. Using this assay, the localization of both endogenous and green fluorescent protein (GFP)-tagged I-BAR domain proteins to invadopodia was studied by light microscopy. Endogenous I-BAR domain proteins were examined both in two dimensional (2D) and three dimensional (3D) environments, but the antibodies used in this study appeared not to be suitable for their detection. However, the intensity profiles of invadopodia showed that in cells expressing I-BAR domain proteins fused to a GFP, these proteins accumulated either on the edge or at the centre of an invadopodium. The results suggest that the I-BAR domain proteins are likely to be recruited to distinct sites of invadopodia where they can induce or stabilize plasma membrane curvature and link it to actin cytoskeleton remodeling during the formation of invasive cell protrusions