Fibroblast growth factor signalling in the development and functions of the choroid plexus

The choroid plexuses (CPs) are secretory organs that lie within the brain ventricles. Mature CPs produce cerebrospinal fluid (CSF) which surrounds the central nervous system (CNS). During embryogenesis, prior to terminal differentiation of neurons and glia, CPs are morphologically distinct and have secretory capacity, suggesting that CPs may influence the extracellular environment of the developing CNS. Although the roles of fibroblast growth factors (FGFs) as morphogenic and mitogenic signals during CNS development are well characterised, they have not been addressed in embryonic CPs. In this study, I investigated the FGF receptor (FGFR) family in relation to CP development and function. I found that several FGFRs are differentially expressed in the CPs during murine development. This finding was important because many individuals with the human disorder, syndromic craniosynostosis (SCS), have constitutively active FGFRs, and frequently suffer from raised intracranial pressure. This led to the hypothesis that increased FGFR signalling alters the function of the CPs. In order to elucidate the functional role of FGF signalling in the embryonic CP, I used several approaches. Firstly, I cultured CP epithelial cells (CPe) in Matrigel, and demonstrated that CPe in this matrix formed hollow vesicles, maintained their ultrastructural characteristics, and had secretory activity. I found that the CPe marker, transthyretin, was expressed in culture. Treatment of CPe with FGF2 significantly increased vesicle diameter, but did not seem to alter secretion, indicating that the FGF2 effect was on vesicle formation. Secondly, I cultured CPe monolayers, and established that at least one effect of FGF2 was to stimulate CPe cell division. An analysis of proteins in CSF of children with SCS showed no significant change in the concentration of transthyretin secreted by the CPs. These data indicate that FGF signalling is involved in CP development, and that FGFs may function to stimulate CP proliferation, rather than affecting CSF constituents or CP secretion rates during embryonic development

Molecular mechanisms of drug resistance, invasion and metastasis in pancreatic carcinoma

The research outlined in this thesis aims to further our knowledge of the biological characteristics underlying the aggressive behaviour by which pancreatic cancer invades and metastasises at an early stage and is refractory to most chemotherapeutic drugs. Investigation into drug resistance in pancreatic cancer involved pulse-selection of three cell lines with epirubicin, taxotere and gemcitabine. The drug-selected cell lines, in general, displayed low changes in sensitivity to their respective selecting drugs and no obvious cross-resistance profile. However, drug treatment modulated the invasive potential of the pulse-selected cell lines. Expression of multi-drug resistant proteins (P-gp and MRP-1) was also determined by IHC in 45 pancreatic tumour specimens, results suggested a contributory role for P-gp expression in pancreatic cancer. An in vitro pancreatic invasion model was established by single cell cloning of MiaPaCa-2, yielding sub-clones displaying diverse invasive properties. The malignant phenotypes of MiaPaCa-2, Clone #3 (highly invasive) and Clone #8 (poorly invasive) were characterised, comparative proteomic profiling by 2-D DIGE, MALDI-TOF MS and RNAi revealed three targets, ALDH1A1, VIM and STIP1 with functional involvement in the invasion process. ALDH1A1 appeared to play a role in the aggressive invasive phenotype; expression was associated with increased invasion, drug resistance and decreased adhesion. VIM and STIP expression was also linked to increased invasion and decreased adhesion, with STIP1 displaying a role in proliferation, which has not previously been described. Factors secreted into conditioned media of Clone #3 and Clone #8 was also examined by proteomics. Targets, GSN and ALDH1A1 were chosen for functional analysis and their potential involvement in cancer cell invasion assessed. EGFR/HER2 expression was analysed in 5 pancreatic cancer cell lines, and two cell lines which co-expressed EGFR and HER2 were tested with lapatinib. Lapatinib, a dual EGFR/HER2 inhibitor in combination with chemotherapeutic drugs showed additive interactions, suggesting the possibility of a therapeutic role for lapatinib in combination with chemotherapy in pancreatic cancer patients co-expressing EGFR/HER2

Role of extracellular matrix in vascular smooth muscle cell behaviour and the identification of novel markers of cell phenotype.

Increased synthesis and deposition of extracellular matrix proteins in the blood vessel wall are implicated in vascular disorders such as atherosclerosis, restenosis and hypertension. (Liau and Chan 1989). The increase in extracellular matrix proteins can be mostly attributed to smooth muscle cells (SMC’s) within vascular lesions (Mecham et al 1987). It is presumed that modulation of SMC’s from their normally quiescent, contractile phenotype to a proliferative synthetic phenotype results in increased synthesis of extracellular matrix proteins. Studies have also demonstrated that signals elicited from the extracellular matrix (ECM) may play a role in the regulation of this SMC phenotypic modulation (Yamamoto et al 1993; Hedin et al 1988). The mechanism by which ECM can alter the phenotypic state of SMC’s is not well understood but clearly involves the induction of intracellular signals as a consequence of ECM ligand - cell surface integrin binding. These signals must subsequently exert downstream molecular events altering gene expression and ultimately cell phenotype. The research project presented in this thesis examined the influence various extracellular matrix substrates have on vascular SMC behaviour in vitro. Initial observations demonstrated that SMC’s cultured on different matrix substrates exhibit distinct morphological growth patterns. Functional differences in SMC proliferation and migration rates were also observed in response to seeding on different ECM surfaces.Analysis of the expression levels of known SMC phenotypic protein markers between SMC’s cultured on different matrix substrates did not reveal any significant differences in protein expression. Slight upregulation of Myosin Light Chain Kinase (MLCK)-210 kd isoform was observed in SMC’s cultured on cellular fibronectin, Collagen III and Vitronectin substrates. The Meta-vinculin protein was upregulated in SMC’s cultured on fibronectin coated substrates.In order to identify altered gene expression patterns induced by ECM adhesion, SMC populations cultured on fibronectin coated plastic and SMC’s grown on uncoated plastic were selected for analysis by the differential display technique. Differential display is a recently developed PCR based technique that allows the identification of differentially expressed genes between related cell populations (Liang and Pardee 1992). Much effort was spent optimising the differential display procedure to overcome such limitations as primer redundancy and high false positive selection rates. However, as a result of the persistence of false positives only one gene, meta-vinculin was confirmed as being differentially expressed between SMC populations cultured on fibronectin coated and uncoated tissue culture plastic surfaces.It was concluded that the failure to identify a significant number of fibronectin modulated genes was probably a result of limitations in the differential display procedure as carried out in this study, and may also possibly be due to the existence of only very minor differences in phenotype between SMC’s grown on plastic with or without fibronectin precoating

Novel Avenues to Model and Investigate Alzheimer’s Disease In Vitro

Alzheimer's’ disease (AD) is a complex neurodegenerative disease which presents with a number of neuropathological features, most noticeably include gross brain atrophy, neuroinflammation, insoluble parenchymal amyloid-β (Aβ) deposits and intracellular neurofibrillary tangles containing hyperphosphorylated tau. Ab42-related neurotoxicity has been an evolving topic of debate within the field over many decades. The lack of molecular tools to modulate specific aspects of the amyloid aggregation pathway, a neuron-centric exploration of AD and use of in vitro models with poor face validity have all contributed to the lack of successful drug discovery within AD. This thesis sought to investigate and model AD through a number of novel avenues. These included: 1) investigating the ability of novel Ab42 monomer-sequestering drugs to protect against Ab42-induced neurotoxicity in vitro, 2) characterising the morphological and inflammatory phenotype of induced pluripotent stem cell (iPSC)-derived astrocytes and microglia from patients harboring a previously uncharacterised PSEN2 (N141I) mutation, and 3) developing a novel tuneable and bioprintable 3D culture system which supports the function of iPSC-derived CNS cells. In chapter 2, we showed that diverting the monomeric form of the Ab42 peptide away from the amyloidogenic pathway using novel Ab42 monomer-sequestering triazole-linked macrocycles prevented formation of pre-fibrillar species that are toxic to differentiated SH-SY5Y cells. In chapter 3, PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a ‘primed’ phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aβ42 production and phagocytosis. In chapter 4, we reported the optimisation of a novel CNS-mimetic 3D hydrogel compatible with medium-high throughput 3D bioprinting. We showed that the 3D bioprinted cell-containing hydrogels enhanced neuronal differentiation and supported the viable culture of iPSC-derived astrocytes and neural progenitors. Additionally, we showed that iPSC-derived neurons from fAD patients harboring a PSEN2 (N141I) mutation exhibited alterations in spontaneous neuronal activity

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

Engineering a microwell duct-on-chip technology to translate exocrine pancreatic organoids to a cancer model

Das duktale Adenokarzinom der Bauchspeicheldrüse (PDAC) ist eine der tödlichsten Erkrankungen der exokrinen Bauchspeicheldrüse, für die uns relevante Frühdiagnosemarker fehlen. Um PDAC-Marker zu identifizieren, werden in vitro kultivierte exokrine Pankreasmodelle aus dem frühestmöglichen, präkanzerösen Stadium benötigt. Die Übertragung der Pankreasgang-Differenzierung von humanen pluripotenten Stammzellen (hiPSCs) in in vitro-Krankheitsmodelle erfordert ein umfassendes Verständnis der Entwicklungsbahnen von pankreasspezifischen Zelltypen. In dieser Arbeit wurde eine Microwell-Chip-Technologie mit definierten mikrostrukturierten Strukturen entwickelt, um aus hiPSC differenzierte Vorläuferzellen des Pankreas (PP) in einer 3-dimensionalen Zellkultur zu assemblieren. Die Vorteile der Chip-Plattform sind i) die parallele Bildung von Hunderten gleichgroßer 3D-Zellaggregate, ii) eine Matrigel-freie Mikroumgebung, iii) die Kompatibilität mit hochauflösender Bildgebung, iv) die einfache Anwendbarkeit für verschiedene nachfolgende Analysen mit minimaler Störung und v) die Möglichkeit, Ko-Kulturen zu etablieren. Der Chip wurde verwendet, um in weniger als 6 Stunden tausende von 3D-Zellaggregaten aus etwa 600 PPs zu bilden. In den folgenden 14 Tagen wurden die 3D-PP-Kulturen mit einem definierten Wachstumsfaktorprotokoll in pankreatische dukt-ähnliche Organoide differenziert. Zeitaufgelöste Einzelzell-Transkriptionsprofile und Immunfluoreszenz von gereinigten dukt-ähnlichen Organoiden der Bauchspeicheldrüse zeigten die Entstehung von zwei Arten von duktalen Vorläufern, Zwischenstufen, und reifen duktalen Zellen und wenigen nicht-duktalen Zelltypen. Entsprechende dynamische Transkriptionsstadien wiesen auf definierte Differenzierungsrouten der duktalen Zellen hin, die in zwei entweder CFTR+ oder Mucin+ Subpopulationen resultieren. Diese Subpopulationen wurden bereits in primären Einzelzelltranskriptomen des Pankreas gefunden[4]. Die Integration unseres Einzelzelldatensatzes mit drei primären Pankreasdatensätzen[4-6] zeigte, dass unsere dukt-ähnlichen Zellen zusammen mit primären duktalen Zellen zu den beiden Subpopulationen clustern. Außerdem konnten die Marker der Subpopulationen in einem reanalysierten Primärdatensatz[5] erneut identifiziert und in menschlichem Primärgewebe angefärbt werden. Darüber hinaus wurde die Duct-on-Chip-Plattform genutzt, um Organoid-Ko-Kulturen mit humanen Stellat-Zellen zu etablieren. Als zusätzliche Anwendung ermöglichte die Matrigel-freie Chip-Technologie die Entnahme des Sekretoms und Proteoms der Organoide. In Verbindung mit dem Einzelzell-Transkriptom und der klinischen Validierung ermöglichten uns diese Sekretomstudien die Entdeckung eines beispielhaften frühen PDAC-Marker namens FLNB, welcher sowohl in Biopsien als auch im peripheren Blut von Patienten im Frühstadium nachweisbar ist. Zusammenfassend zeigt diese Arbeit die erfolgreiche Herstellung von Pankreas dukt-ähnlichen Organoiden aus hiPSCs, die ein Reifestadium aufweisen, welches mit dem des fötalen Pankreas vergleichbar ist. Durch die Kombination von zeitaufgelöster Einzelzelltranskriptomik mit verschiedenen Analysemethoden, Sekretomstudien, Proteomstudien und klinischer Validierung auf unserem Microwell-Chip wurde ein patientenspezifisches Duktmodell und ein potenzielles Krebsdiagnoseinstrument entwickelt.Pancreatic ductal adenocarcinoma (PDAC) is one of the most severe diseases of the exocrine pancreas, for which relevant early diagnostic markers are still missing. To identify PDAC biomarkers, experimental models employing in vitro cultivation of exocrine pancreas models require as early as possible precancerous stages. The translation of pancreatic ductal differentiation of human pluripotent stem cells (hiPSCs) into in vitro disease models requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. In this study, a microwell chip technology exhibiting defined microstructured patterns to assemble hiPSC-derived pancreatic progenitor cells (PP) into a 3-dimensional cell culture was developed. The advantages of the chip platform are i) the parallel formation of hundreds of equally sized 3D cell aggregates, ii) a Matrigel-free microenvironment, iii) the compatibility with high-resolution imaging, iv) simple applicability for several downstream analyses with minimal perturbation, and v) the possibility to establish co-cultures. The chip was used to generate thousands of 3D cell aggregates from approximately 600 PPs, in less than six hours. For the following 14 days, the 3D PP cultures were differentiated towards pancreatic ductal-like organoids by employing defined growth factor protocols. Time-resolved single-cell transcriptional profiling and immunofluorescence of cleared pancreatic duct-like organoids revealed the emergence of two types of ductal progenitors, intermediates, mature duct-like cells, and a few non-ductal cell types. Corresponding dynamic transcriptional stages indicated defined differentiation routes of duct-like cells, cumulating in two either CFTR+ or mucin+ subpopulations, which have been found before in primary single-cell transcriptomes of the pancreas[4]. The integration of the PDLO single-cell dataset into three primary pancreas datasets[4-6] showed that the duct-like cells clustered together with primary ductal cells into the two subpopulations. Furthermore, the markers of the subpopulations could be reidentified in a reanalyzed primary dataset[5] and subjected to confirmation by immunofluorescence in primary human tissue. Additionally, the duct-on-chip platform was exploited to establish organoid co-cultures with stellate cells. As an additional application, the Matrigel-free chip technology allowed the characterization of secretome and proteome. Together with the single-cell transcriptome and clinical validation, these secretome studies revealed an exemplary early PDAC marker, called FLNB, which is detectable in biopsies and early-stage patients' peripheral blood. In conclusion, this study reports the successful engineering of pancreatic duct-like organoids from hiPSCs, which show a maturation stage comparable to the fetal pancreas. By combining time-resolved single-cell transcriptomics with different analysis methods, secretome, proteome and clinical validation on our microwell chip, a patient-specific duct model and a potential cancer diagnostic tool was developed

THE ROLE OF PHLPP IN PANCREATIC CANCER

Medicine has come a long way in recent years with reliable treatments for many cancers. Pancreatic ductal adenocarcinoma (PDAC) has very few treatment options available. PDAC has a dismal 5 year survival rate of 4% and a median survival span of 6 months from point of diagnosis; with a high rate of chemotherapy and radiation resistance. A better understanding of the molecular events leading to cancer progression is needed in order to improve the treatment and prognosis of PDAC patients. We begin to elucidate the functional importance of PHLPP on suppressing progression and metastasis of PDAC. PHLPP belongs to a novel family of Ser/Thr protein phosphatases. Our previously published studies have demonstrated that PHLPP plays a tumor suppressor role in colon cancer by negatively regulating Akt and inhibiting cell proliferation. To determine the effect of PHLPP on cell migration and invasion, stable cells were generated to knock down or overexpress PHLPP in PDAC cells. The ability of cells to migrate and invade was examined using Transwell assays. We found that increased PHLPP expression significantly reduced the rate of migration and invasion in PDAC cells whereas knockdown of PHLPP had the opposite effect. To begin to elucidate the molecular mechanism underlying PHLPP-mediated inhibition of migration and invasion in PDAC cells, we discovered that the expression level of β4 Integrin was decreased in PHLPP overexpressing cells and increased in PHLPP knockdown cells. The increased expression of β4 Integrin has been shown to promote PDAC development and metastasis, although the mechanism leading to β4 Integrin upregulation is less clear. Interestingly, we found that the expression of β4 Integrin was highly sensitive to PI3K/Akt/mTOR activity in cells in which inhibition of PI3K/Akt/mTOR signaling significantly decreased the expression of β4 Integrin. Moreover, the quantitative real-time RT-PCR analysis revealed that the mRNA expression of β4 Integrin was not altered by changes in PHLPP expression or PI3K/Akt/mTOR activity, thus suggesting a post-transcriptional mechanism. Taken together, these results identify a tumor suppressor role of PHLPP in PDAC. Mechanistically, PHLPP suppresses PDAC cell migration and invasion by negatively controlling β4 Integrin expression through its ability to inhibit PI3K/Akt/mTOR signaling