Bioinspired matrices for in vitro hepatic differentiation

Standard two-dimensional (2D) in vitro cell culture systems do not mimic the complexity found in the liver as three-dimensional (3D) cell-cell and cell-matrix interactions are missing. Although the concept of cell culturing was established over 100 years ago the currently used culture techniques are not yet ideal. In the field of pharmacy especially, the need of physiologically-relevant models to characterize biotransformation pathways during drug development is urgent. Hepatocytes, the main cell type of the liver, are essential components in these in vitro models. Liver cell lines and derivation of hepatocyte-like cells from stem cells are alternative sources to primary isolations for obtaining hepatocytes. In the liver, hepatocytes are in continuous interaction with other cells and surrounding extracellular matrix (ECM). Moreover, liver functions are strictly dependent on correct tissue architecture. One approach to improve the standard cell culture systems is to mimic the hepatocytes natural microenvironment and organization by culturing the cells within biomaterial matrices. Matrix-based culture systems for hepatocytes have been developed from natural, synthetic and hybrid biomaterials and the cells can be grown in 2D or 3D configuration. The aim of this thesis was to find new defined culture matrices for in vitro hepatic differentiation. First, we studied two biomaterials, nanofibrillar cellulose (NFC) hydrogel and hyaluronic acid-gelatin (HG) hydrogel, to construct functional liver 3D organoids. Both of the studied hydrogels supported 3D spheroid organization of human liver progenitor HepaRG cells and their functional polarization. The 3D culture systems promoted hepatic differentiation of progenitor cells faster than the standard 2D culture. However, the 3D hydrogels did not enhance hepatocyte-like properties if the HepaRG cells were pre-differentiated to hepatocyte-like cells in advance. Subsequently, we showed that NFC hydrogel culture can be combined with high-resolution imaging since the intact spheroids can be enzymatically released from the matrix. This was not possible with the HG hydrogel. We demonstrated that silica bioreplication preserved the 3D spheroid structure with its fine details and cellular antigens and allowed detailed morphological analysis of the spheroids cultured in NFC hydrogel. Next, we developed a xeno-free matrix for hepatic specification of human pluripotent stem cell-derived definite endoderm (DE) cells using a three-step approach. We first proved our hypothesis that a liver progenitor-like matrix, HepaRG-derived acellular matrix (ACM), supports hepatic lineage differentiation of DE cells. Then, we characterized the ECM proteins secreted by HepaRG cells, and finally we showed that the identified proteins, laminin-511 and laminin-521, can replicate the effect of HepaRG-ACM. The human pluripotent stem cell-derived hepatic cells expressed mature hepatocyte-like functions but the phenotype of the cells was eventually closer to fetal hepatocytes than mature cells. Thus, hepatic maturation should be further studied. In conclusion, this thesis describes new biomaterials for hepatic differentiation, a protocol to form 3D spheroids and to transfer intact spheroids to high-resolution imaging, and that the described three-step approach can guide the identification of new defined matrices.Perinteiset kaksiulotteiset (2D) in vitro -solumallit jäljittelevät heikosti oikeita kudoksia. 2D-kasvatusmenetelmillä ei voida matkia kudosten moniulotteisia rakenteita eikä solujen ja niitä ympäröivän soluväliaineen vuorovaikutuksia, jotka ovat elintärkeitä kudosten, kuten maksan, toiminnan kannalta. Solukasvatusta on hyödynnetty tutkimuksessa jo yli 100 vuoden ajan, mutta useat käytössä olevat solukasvatusmallit ovat vielä puutteellisia, eivätkä kasvatetut solut usein vastaa toiminnaltaan kehon soluja. Erityisesti lääketutkimukseen kaivataan uusia, ihmisen maksaa paremmin jäljitteleviä solumalleja, joilla voitaisiin ennustaa kehitteillä olevien lääkeaineiden käyttäytymistä kehossa. Maksan solumalleissa käytettyjä hepatosyyttejä voidaan eristää suoraan maksasta, käyttää loputtomasti jakautuvia maksasolulinjoja tai erilaistaa hepatosyyttejä kantasoluista. Maksassa hepatosyytit ovat jatkuvassa vuorovaikutuksessa naapurisolujen ja soluja ympäröivän soluväliaineen kanssa. Perinteisiä solumalleja voidaan parantaa biomateriaaleilla, joilla soluille luodaan kudoksen kaltainen kasvuympäristö. Solumalleissa käytettävät biomateriaalit voivat olla peräisin luonnosta, synteettisiä tai näiden yhdistelmiä, hybridimateriaaleja. Tämän väitöskirjatyön tavoitteena oli löytää uusia kasvatusmateriaaleja maksasolujen erilaistamista varten. Työn ensimmäisessä osassa tutkittiin kolmiulotteisen (3D) maksasolumallin rakentamista ihmisen HepaRG-maksasolulinjalla ja kahdella biomateriaalilla: nanoselluloosahydrogeelillä sekä hyaluronihaposta ja gelatiinista valmistetulla hydrogeelillä. Molemmat tutkitut biomateriaalit soveltuivat solujen 3D-kasvatukseen, ja muodostuneet 3D-solurakenteet toimivat osittain kuten ihmisen maksakudos. Nanoselluloosassa kasvatetuissa soluissa vierasainemetabolia oli aktiivisempi kuin hyaluronihappogelatiinihydrogeelissä. Nanoselluloosa eroaa useimmista kaupallisista hydrogeeleistä siten, että sen sisällä kasvatetut kokonaiset 3D-solut on mahdollista irrottaa materiaalista solujen elävyyttä tai rakenteita vahingoittamatta. 3D-solurakenteiden morfologian tarkempaa tutkimista varten väitöskirjatyön seuraavassa osassa kehitettiin uusi analyysimenetelmä. Nanoselluloosahydrogeelissä muodostetut 3D-solut käsiteltiin piidioksidilla eli silikalla, joka muodosti erittäin ohuen ja kestävän kalvon solujen sisä- ja pintarakenteiden päälle. Muodostunut silikabioreplikaatti kuvattiin pyyhkäisyelektronimikroskoopilla, minkä ansiosta solujen hyvin pieniä rakenteita voidaan tarkastella yksityiskohtaisesti. Väitöskirjatyön toisessa osassa kehitettiin uusi kasvatusalusta ihmisen pluripotenteista eli monikykyisistä kantasoluista saatujen definitiivisen endodermin (DE) solujen erilaistamiseksi maksan hepatosyyteksi. Työn ensimmäisessä vaiheessa vahvistettiin hypoteesimme siitä, että maksan progenitorisolujen tuottama soluväliaine tukee DE-solujen erilaistumista hepatosyyttien kaltaisiksi soluiksi. Seuraavaksi identifioitiin tämän soluväliaineen koostumus ja käytettiin siitä löydettyjä proteiineja DE-solujen erilaistamisessa. Työn kolmannessa ja viimeisessä vaiheessa osoitettiin, että soluväliaineesta tunnistetuilla, geenitekniikalla valmistetuilla laminiini-511- ja -521-proteiineilla voitiin täysin korvata maksan progenitorisoluista saatu soluväliaine. Laminiinien avulla tuotetut hepatosyyttien kaltaiset solut eivät kuitenkaan olleet täysin kypsiä ja erilaistumista on yhä tutkittava. Yhteenvetona voidaan todeta, että tässä väitöskirjatyössä kuvattuja biomateriaaleja voidaan käyttää hepatosyyttien erilaistamiseen kantasoluista, 3D-solumallin rakentamiseen ja sen analysointiin, sekä työssä käytetyn kolmiportaisen mallin avulla voidaan löytää uusia materiaaleja solukasvatukseen

Lyhytterapiat opiskeluvalmennuksessa : Kognitiivis-analyyttisen ja ratkaisukeskeisen terapian soveltaminen

Toisen asteen opiskelijoilla opintojen keskeyttäminen on yleistä. Taustalla on usein monenlaisia psyykkisiä ja sosiaalisia ongelmia. Tämän vuoksi Kelan Opiskeluvalmennus-projektissa (2016–2020) kokeiltiin kahden lyhytpsykoterapeuttisen menetelmän soveltuvuutta kestoltaan rajatussa opiskelijoiden ohjauksessa. Menetelmät olivat kognitiivis-analyyttinen (KAT) ja ratkaisukeskeinen (RATKES). Tässä julkaisussa kuvataan lyhyesti näiden menetelmien perusperiaatteet ja työtavat sekä miten niitä sovellettiin opiskeluvalmennuksessa. Opiskeluvalmennuksen teoreettisena viitekehyksenä oli opiskelukykymalli. Opiskeluvalmennuksen tavoitteena oli vahvistaa ja tukea opiskelijan opiskelukykyä sekä ehkäistä opintojen keskeytymistä. Opiskelukyvyn tukemiseksi kummastakin menetelmästä valittiin työkaluja tukemaan, syventämään ja rakenteistamaan opiskeluvalmentajien työskentelyä. Projektissa työskennelleet kahdeksan opiskeluvalmentajaa koulutettiin jompaankumpaan menetelmään. He osallistuivat kuntoutuskokeilujen ajan myös opiskelemansa menetelmän työnohjaukseen. Projektissa opiskeluvalmennusta sai enintään 80 opiskelijaa ajalla 1.3.2018–30.3.2020. Opiskeluvalmennus suunniteltiin ennaltaehkäiseväksi matalan kynnyksen palveluksi. Ennakko-oletuksista poiketen kokeiluun ohjautui odotettua moniongelmaisempia opiskelijoista. Osa aloitetuista valmennusprosesseista keskeytyi, mutta saattoi silti olla keskeytyneenäkin hyödyksi opiskelijalle. Opiskeluvalmennus toimii ennaltaehkäisevänä, tukea antavana sekä palveluihin ohjaavana palveluna kustannustehokkaasti. Valmentajien ja valmennettavien kokemus lyhytterapeuttisten menetelmien sovellettavuudesta opiskeluvalmennuksessa oli myönteinen. Niiden käyttöä ja edelleen kehittämistä kannattaa jatkaa kuntoutuksessa ja ohjauksessa.nonPeerReviewedVertaisarvioimato

Differences in definitive endoderm induction approaches using growth factors and small molecules

Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (Activin A and Wnt-3a) and small molecules (sodium butyrate and IDE 1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapid DE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications. We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications.Peer reviewe

Multicellular dosimetric chain for molecular radiotherapy exemplified with dose simulations on 3D cell spheroids

Purpose: Absorbed radiation dose-response relationships are not clear in molecular radiotherapy (MRT). Here, we propose a voxel-based dose calculation system for multicellular dosimetry in MRT. We applied confocal microscope images of a spherical cell aggregate i.e. a spheroid, to examine the computation of dose distribution within a tissue from the distribution of radiopharmaceuticals. Methods: A confocal microscope Z-stack of a human hepatocellular carcinoma HepG2 spheroid was segmented using a support-vector machine algorithm and a watershed function. Heterogeneity in activity uptake was simulated by selecting a varying amount of the cell nuclei to contain In-111, I-125, or Lu-177. Absorbed dose simulations were carried out using vxlPen, a software application based on the Monte Carlo code PENELOPE. Results: We developed a schema for radiopharmaceutical dosimetry. The schema utilizes a partially supervised segmentation method for cell-level image data together with a novel main program for voxel-based radiation dose simulations. We observed that for 177Lu, radiation cross-fire enabled full dose coverage even if the radiopharmaceutical had accumulated to only 60% of the spheroid cells. This effect was not found with 111In and 125I. Using these Auger/internal conversion electron emitters seemed to guarantee that only the cells with a high enough activity uptake will accumulate a lethal amount of dose, while neighboring cells are spared. Conclusions: We computed absorbed radiation dose distributions in a 3D-cultured cell spheroid with a novel multicellular dosimetric chain. Combined with pharmacological studies in different tissue models, our cell-level dosimetric calculation method can clarify dose-response relationships for radiopharmaceuticals used in MRT. (C) 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix

Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs. (C) 2016 Elsevier Inc. All rights reserved.Peer reviewe

Laminin-511 and laminin-521-based matrices for efficient hepatic specification of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells. 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND licensePeer reviewe

Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture

Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy.Peer reviewe

Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.Peer reviewe

NF-E2-related factor 2 activation boosts antioxidant defenses and ameliorates inflammatory and amyloid properties in human Presenilin-1 mutated Alzheimer's disease astrocytes

Alzheimer's disease (AD) is a common dementia affecting a vast number of individuals and significantly impairing quality of life. Despite extensive research in animal models and numerous promising treatment trials, there is still no curative treatment for AD. Astrocytes, the most common cell type of the central nervous system, have been shown to play a role in the major AD pathologies, including accumulation of amyloid plaques, neuroinflammation, and oxidative stress. Here, we show that inflammatory stimulation leads to metabolic activation of human astrocytes and reduces amyloid secretion. On the other hand, the activation of oxidative metabolism leads to increased reactive oxygen species production especially in AD astrocytes. While healthy astrocytes increase glutathione (GSH) release to protect the cells, Presenilin‐1‐mutated AD patient astrocytes do not. Thus, chronic inflammation is likely to induce oxidative damage in AD astrocytes. Activation of NRF2, the major regulator of cellular antioxidant defenses, encoded by the NFE2L2 gene, poses several beneficial effects on AD astrocytes. We report here that the activation of NRF2 pathway reduces amyloid secretion, normalizes cytokine release, and increases GSH secretion in AD astrocytes. NRF2 induction also activates the metabolism of astrocytes and increases the utilization of glycolysis. Taken together, targeting NRF2 in astrocytes could be a potent therapeutic strategy in AD.</p

Early career researchers want Open Science : Comment

Creative Commons Attribution License (CC BY 4.0)Open Science is encouraged by the European Union and many other political and scientific institutions.However, scientific practice is proving slow to change.We propose, as early career researchers, that it is our task to change scientific research into open scientific research and commit to Open Science principles.Non peer reviewe