Ryan Hicks

Cell banking for pharmaceutical research

The provision of high-quality eukaryotic cells through robust cell banking processes is essential for the progression of drug discovery projects throughout the pharmaceutical research process. Numerous models exist to meet this aim, and this review describes many of the underlying principles, challenges and opportunities as well as detailing how these have been addressed within AstraZeneca. Crucial aspects discussed include cell line acquisition, cell bank generation, cryopreservation, storage, tracking and distribution. Because quality assurance underpins much of the process, quality control (QC) testing including mycoplasma screening and cell line authentication are also discussed in detail. Furthermore, because many of the underlying principles of cell banking are applicable in non-pharmaceutical settings, it is hoped that this review will prove a useful resource across the wider scientific community.

Human iPS-Derived Astroglia from a Stable Neural Precursor State Show Improved Functionality Compared with Conventional Astrocytic Models

Summary In vivo studies of human brain cellular function face challenging ethical and practical difficulties. Animal models are typically used but display distinct cellular differences. One specific example is astrocytes, recently recognized for contribution to neurological diseases and a link to the genetic risk factor apolipoprotein E (APOE). Current astrocytic in vitro models are questioned for lack of biological characterization. Here, we report human induced pluripotent stem cell (hiPSC)-derived astroglia (NES-Astro) developed under defined conditions through long-term neuroepithelial-like stem (ltNES) cells. We characterized NES-Astro and astrocytic models from primary sources, astrocytoma (CCF-STTG1), and hiPSCs through transcriptomics, proteomics, glutamate uptake, inflammatory competence, calcium signaling response, and APOE secretion. Finally, we assess modulation of astrocyte biology using APOE-annotated compounds, confirming hits of the cholesterol biosynthesis pathway in adult and hiPSC-derived astrocytes. Our data show large diversity among astrocytic models and emphasize a cellular context when studying astrocyte biology.

Extracellular vesicles from human pancreatic islets suppress human islet amyloid polypeptide amyloid formation

Significance Protein assembly into amyloid fibers underlies such neurodegenerative disorders as Alzheimer’s disease and Parkinson’s disease. Type 2 diabetes (T2D) also involves amyloid formation, although in the pancreas. Because there are no cures for amyloid diseases and T2D is on the rise due to an increasing prevalence of obesity, identifying involved mechanisms and control processes is of utmost importance. Extracellular vesicles (EVs) can mediate physiological and pathological communication both locally and at a distance. Here, we demonstrate that EVs secreted from healthy, but not from T2D, pancreatic cells slow amyloid formation of the major peptide found in amyloid deposits in T2D. We propose an EV-mediated process that tempers amyloid formation in the pancreas at normal conditions, which breaks down in T2D due to altered EV protein–lipid composition. Extracellular vesicles (EVs) are small vesicles released by cells to aid cell–cell communication and tissue homeostasis. Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in pancreatic islets of patients with type 2 diabetes (T2D). IAPP is secreted in conjunction with insulin from pancreatic β cells to regulate glucose metabolism. Here, using a combination of analytical and biophysical methods in vitro, we tested whether EVs isolated from pancreatic islets of healthy patients and patients with T2D modulate IAPP amyloid formation. We discovered that pancreatic EVs from healthy patients reduce IAPP amyloid formation by peptide scavenging, but T2D pancreatic and human serum EVs have no effect. In accordance with these differential effects, the insulin:C-peptide ratio and lipid composition differ between EVs from healthy pancreas and EVs from T2D pancreas and serum. It appears that healthy pancreatic EVs limit IAPP amyloid formation via direct binding as a tissue-specific control mechanism.

3D-Models of Insulin-Producing β-Cells: from Primary Islet Cells to Stem Cell-Derived Islets

There is a need for physiologically relevant assay platforms to provide functionally relevant models of diabetes, to accelerate the discovery of new treatment options and boost developments in drug discovery. In this review, we compare several 3D-strategies that have been used to increase the functional relevance of ex vivo human primary pancreatic islets and developments into the generation of stem cell derived pancreatic beta-cells (β-cells). Special attention will be given to recent approaches combining the use of extracellular matrix (ECM) scaffolds with pancreatic molecular memory, which can be used to improve yield and functionality of in vitro stem cell-derived pancreatic models. The ultimate goal is to develop scalable cell-based platforms for diabetes research and drug screening. This article will critically assess key aspects related to in vitro pancreatic 3D-ECM models and highlight the most promising approaches for future research.

Humanizing Miniature Hearts through 4-Flow Cannulation Perfusion Decellularization and Recellularization

Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases.

Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway

apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer’s disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.

Barrier properties and transcriptome expression in human iPSC-derived models of the blood-brain barrier

Cell‐based models of the blood–brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of interspecies differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC‐derived model of the BBB that includes endothelial cells in coculture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our coculture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non‐permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in coculture, including upregulation of tight junction proteins, such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF, and PI3K‐Akt pathways upon coculture. Our data suggest that coculture of iPSC‐derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in coculture can be used to further improve iPSC‐derived BBB models through selective pathway manipulation. Stem Cells 2018;36:1816–12

Diabetic Cardiomyopathy Modelling Using Induced Pluripotent Stem Cell Derived Cardiomyocytes: Recent Advances and Emerging Models

The global burden of diabetes has drastically increased over the past decades and in 2017 approximately 4 million deaths were caused by diabetes and cardiovascular complications. Diabetic cardiomyopathy is a common complication of diabetes with early manifestations of diastolic dysfunction and left ventricular hypertrophy with subsequent progression to systolic dysfunction and ultimately heart failure. An in vitro model accurately recapitulating key processes of diabetic cardiomyopathy would provide a useful tool for investigations of underlying disease mechanisms to further our understanding of the disease and thereby potentially advance treatment strategies for patients. With their proliferative capacity and differentiation potential, human induced pluripotent stem cells (iPSCs) represent an appealing cell source for such a model system and cardiomyocytes derived from induced pluripotent stem cells have been used to establish other cardiovascular related disease models. Here we review recently made advances and discuss challenges still to be overcome with regard to diabetic cardiomyopathy models, with a special focus on iPSC-based systems. Recent publications as well as preliminary data presented here demonstrate the feasibility of generating cardiomyocytes with a diabetic phenotype, displaying insulin resistance, impaired calcium handling and hypertrophy. However, capturing the full metabolic- and functional phenotype of the diabetic cardiomyocyte remains to be accomplished.

NKX6.1 induced pluripotent stem cell reporter lines for isolation and analysis of functionally relevant neuronal and pancreas populations

Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies.

Decoding non-random mutational signatures at Cas9 targeted sites

Abstract The mutation patterns at Cas9 targeted sites contain unique information regarding the nuclease activity and repair mechanisms in mammalian cells. However, analytical framework for extracting such information are lacking. Here, we present a novel computational platform called Rational InDel Meta-Analysis (RIMA) that enables an in-depth comprehensive analysis of Cas9-induced genetic alterations, especially InDels mutations. RIMA can be used to quantitate the contribution of classical microhomology-mediated end joining (c-MMEJ) pathway in the formation of mutations at Cas9 target sites. We used RIMA to compare mutational signatures at 15 independent Cas9 target sites in human A549 wildtype and A549-POLQ knockout cells to elucidate the role of DNA polymerase θ in c-MMEJ. Moreover, the single nucleotide insertions at the Cas9 target sites represent duplications of preceding nucleotides, suggesting that the flexibility of the Cas9 nuclease domains results in both blunt- and staggered-end cuts. Thymine at the fourth nucleotide before protospacer adjacent motif (PAM) results in a two-fold higher occurrence of single nucleotide InDels compared to guanine at the same position. This study provides a novel approach for the characterization of the Cas9 nucleases with improved accuracy in predicting genome editing outcomes and a potential strategy for homology-independent targeted genomic integration.