There are a number of pressing issues that the creation of a biomimetic liver culture may be able to solve including catching hepatic mal interactions that are currently missed in preclinical drug screening and offering an alternative solution in the shortage of organs for liver transplant. There are multiple hepatic models that have been created to overcome these issues. However, of the many considerations that need to be taken in the creation of a biomimetic liver model, many fail to capture the functional zone-patterning that is found in vivo. Here is detailed the creation of a hepatic assembloid model that incorporates zone-specific human pluripotent stem cell derived hepatocytes for the recapitulation of zone-patterning in the liver tissues. Use of our lab’s z-wire plate and PGS z-wire scaffold allows for the formation of elongated 3D tissues that resembles the overall morphology of the liver acinus and facilitate the spontaneous development of an aligned vascular-like network. Sustained hepatocyte-specific function in these tissues are promising indicators for application of the hepatic models in drug screening, disease modelling, and regenerative medicine. / Thesis / Master of Applied Science (MASc)

Indiana University-Purdue University Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) have the ability to self renew indefinitely while maintaining their pluripotency, allowing for the study of virtually any human cell type in a dish. The focus of the current study was the differentiation of hPSCs to retinal ganglion cells (RGCs), the primary cell type affected in optic neuropathies. hPSCs were induced to become retinal cells using a stepwise differentiation protocol that allowed for formation of optic vesicle (OV)-like structures. Enrichment of OV like structures allowed for the definitive identification of RGCs. RGCs displayed the proper temporal, spatial, and phenotypic characteristics of RGCs developing in vivo. To test the ability of hPSC-RGCs to serve as a disease model, lines were generated from a patient with an E50K mutation in the Optineurin gene, causative for normal tension primary open angle glaucoma. E50K RGCs displayed significantly higher levels of apoptosis compared to a control lines. Apoptosis was reduced with exposure to neuroprotective factors. Lastly, hPSC-derived RGCs were studied for their ability to develop functional features possessed by mature in vivo RGCs. hPSC-derived RGCs displayed a few immature functional features and as such, strategies in which to expedite synaptogenesis using hPSC-derived astrocytes were explored. Astrocyte and RGG co-cultures displayed expedited synaptic and functional maturation, more closely resembling mature in vivo RGCs. Taken together, the results of this study have important implications for the study of RGC development and by extension, the advancement of translational therapies for optic neuropathies.

Diabetes mellitus is a group of metabolic disorders that are characterized by chronic hyperglycaemia. There are currently over 460 million people living with diabetes and the incidence rate for the most common types is sharply on the rise. The hyperglycaemia and subsequently the majority of diabetic side-effects can be cured by ß-cell transplantation. There is a severe lack of donor tissue for clinical transplantations, hence ß cells derived from human pluripotent stem cells (hPSCs) offer an attractive option for obtaining the necessary cells for a ß-cell therapy. The current ß-cell differentiation protocols are lengthy, expensive and relatively inefficient. Therefore, it remains unrealistic to scale up production to obtain the necessary 1 billion cells per patient to achieve normoglycaemia. The exponential expansion of an intermediate pancreatic progenitor (PP) population would allow for significant reduction of the cost and time necessary for the production of ß-cells in vitro and, more importantly, it would allow to relatively easily obtain the number of cells necessary for clinical applications. Thus far, the reproducible expansion of suitable hPSC-derived PPs in a chemically-defined, feeder-free culture condition has not been reported. Our lab has found that a previously reported medium suitable for the temporary expansion of reprogrammed fibroblast-derived PPs could occasionally, in 20% of the cases, mediate expansion of PSC derived PPs. To elucidate the requirements for reproducible expansion, I analysed transcriptomic data from PSC-derived PPs before and following expansion. Several regulated signalling pathways were identified and from these data, I formulated nine candidate expansion conditions (C0-C8) to test. Five of these conditions were able to reproducibly expand hPSC-derived PPs. Of those five conditions, C6 was found to mediate the fastest expansion with a doubling time of 2.2 days, maintained a stable expression of the crucial bipotent trunk progenitor transcription factor (TF) markers PDX1, NKX6.1, SOX9 and FOXA2 and minimized the expression of the hepatic and intestinal markers AFP and CDX2. The C6 expanded PPs were able to further differentiate into pancreatic endocrine progenitors (PEPs) in three-dimensional (3-D) islet-sized clusters formed in micropatterned wells. Micropatterned wells offer the additional advantage of size-controlled, uniform clusters and low culture volumes compared to suspension culture bioreactors proposed for the large-scale production of such clusters, but the use of micropatterned wells has not been reported for such an application thus far. These PEPs showed strong upregulation of the crucial endocrine specific TF NGN3 and around 90% were positive for NEUROD1, a downstream effector of NGN3, as determined by flow cytometry analysis. The PEPs were subsequently differentiated into insulin (INS)-producing ß-cells at around 20% efficiency. The ß-cells were also strongly expressing functionality genes such as zinc transporter 8 (ZnT8), glucokinase (GCK), prohormone convertase 1/3 (PC1/3) and sulfonylurea receptor 1 (SUR1, a subunit of KATP channel) and around 10% of them were expressing the crucial maturation marker MAFA, as determined by flow cytometry analysis of ß-cells derived from the H1INS1-GFP/MAFA-mCHERRY double reporter line generated in the lab. In summary, I have established a feeder-free, chemically defined and ‘good manufacturing practice’ (GMP)-compatible culture condition for the exponential expansion of hPSC-derived PPs that allows for the production of ß-cells at a fraction of the cost of conventional differentiation protocols. Ongoing work is being done to further optimise the expansion conditions to completely eliminate hepatic and intestinal markers and to optimise the subsequent differentiation steps in micropatterned wells to achieve a high-efficiency differentiation towards functional ß-cells. / Diabetes Mellitus ist eine Gruppe metabolischer Krankheiten, die sich durch chronische Hyperglykämie charakterisiert. Es gibt zurzeit über 460 Millionen Menschen, die mit Diabetes leben und die Inzidenzrate der häufigsten Formen steigt dramatisch. Die Hyperglykämie und die Mehrheit der daraus resultierenden diabetischen Begleiterscheinungen können durch ß Zell Transplantation geheilt werden. Da es einen enormen Gewebespendermangel für klinische Transplantationen gibt, bieten von humanen pluripotenten Stammzellen (hPSCs) stammende ß Zellen eine attraktive Option, um die notwendigen Zellen für eine ß Zell Therapie zu erhalten. Die momentanen ß Zell Differenzierungsprotokolle sind langwierig, teuer und relativ ineffizient. Deshalb bleibt eine Produktionsvergrößerung zur Gewinnung der notwenigen 1 Milliarde Zellen pro Patient zum Erreichen einer Normoglykämie unrealistisch. Die exponentielle Expansion einer intermediären pankreatischen Vorläuferpopulation (PP) würde eine signifikante Kosten und Zeitreduktion zur Produktion von ß Zellen in vitro erlauben und, noch wichtiger, würde relativ einfach ermöglichen, die notwendigen Zellzahlen für klinische Anwendungen zu erzielen. Bisher wurde von einer reproduzierbaren Expansion geeigneter, von hPSC stammenden PPs in einer chemisch definierten Zellkulturbedingung, die frei von Feeder Zellen ist, nicht berichtet. Unser Labor fand heraus, dass ein im Vorfeld veröffentlichtes Medium, welches für die vorrübergehende Expansion von reprogrammierten, von Fibroblasten stammenden PPs geeignet ist, in 20% der Fälle die Expansion von aus hPSC gewonnenen PPs herbeiführen konnte. Um die Anforderungen für eine reproduzierbare Expansion zu eruieren, analysierte ich transkriptomische Daten von aus PSC gewonnenen PPs vor und nach erfolgter Expansion. Mehrere regulierte Signalwege wurden identifiziert und von diesen Daten formulierte ich neun zu testende Kandidatenexpansionsbedingungen (C0-C8). Fünf dieser Bedingungen ermöglichten es von PSC stammende PPs reproduzierbar zu expandieren. Von diesen fünf Bedingungen war C6 die, welche die schnellste Expansion mit einer Dopplungszeit von 2.2 Tagen herbeiführte, eine stabile Expression der essenziellen Marker von bipotenten Stammvorläuferzellen, PDX1, NKX6.1, SOX9 und FOXA2, aufrechterhielt und die Expression von den hepatischen und intestinalen Markern AFP und CDX2 minimierte. Den C6 expandierten PPs war es möglich, sich in pankreatisch endokrine Vorläufer (PEP) in dreidimensionalen (3 D), inselgroßen Kluster, welche in mikrogemusterten Wells geformt wurden, zu differenzieren. Mikrogemusterte Wells bieten den zusätzlichen Vorteil von größenkontrollierten, uniformen Kluster und niedrigen Kulturvolumina im Vergleich zu Suspensionskulturbioreaktoren, welche für die Großmengenproduktion solcher Kluster vorgeschlagen wurden. Jedoch wurde bisher über die Benutzung von mikrogemusterten Wells für solch eine Anwendung nicht berichtet. Diese PEPs zeigten eine starke Hochregulation des essenziellen, endokrin spezifischen Transkriptionsfaktors (TF) NGN3 und 90% waren positiv für NEUROD1, einem nachgelagerten Effektor von NGN3, was anhand der Analyse der Durchflusszytometrie bestimmt wurde. Darauffolgend wurden die PEPs mit einer Effizienz von etwa 20% in Insulin (INS) produzierende ß Zellen differenziert. Diese ß Zellen zeigten ebenfalls eine starke Expression von Funktionalitätsgenen wie ZnT8, GCK, PC1/3 und SUR1 und etwa 10% von ihnen exprimierten den essenziellen Maturationsmarker MAFA, wie es mittels Analyse der Durchflusszytometrie von aus einer INS-GFP/MAFA-mCHERRY Doppelreporterlinie gewonnenen ß Zellen bestimmt wurde. Zusammenfassend habe ich eine Kulturbedingung für die exponentielle Expansion von aus hPSC gewonnenen PPs etabliert, die frei von Feeder Zellen, chemisch definiert und kompatibel mit der Guten Herstellungspraxis ist, welche die Produktion von ß Zellen zu einem Bruchteil der Kosten konventioneller Differenzierungsprotokollen ermöglicht. Es wird weiterhin daran gearbeitet, die Expansionsbedingungen zu optimieren, um die hepatischen und intestinalen Marker komplett zu eliminieren und die darauffolgenden Differenziationsschritte in mikrogemusterten Wells zu verbessern, um letztendlich eine hocheffiziente Differenzierung zu funktionellen ß-Zellen zu erreichen.

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