Induced Pluripotent Stem Cell-Derived Astroglia: A New Tool for Research Towards the Treatment of Alzheimer's Disease.

Despite over a century of research into Alzheimer's disease (AD), progress in understanding the complex aetiology has been hindered, in part, by a lack of human, disease relevant, cellular models, reflected in an inability to translate results from animal studies to successful human therapies. Induced pluripotent stem cell (iPSC) technology, in which somatic cells are reprogrammed to pluripotent stem cells, creates an ideal physiologically relevant model as they maintain the genetic identity of the donor. These iPSCs can self-renew indefinitely in vitro and have the capacity to differentiate into any cell type, opening up new discovery and therapeutic opportunities. Despite a plethora of publications indicating the generation and utility of iPSC-derived neurones for disease modelling to date, in comparison only a limited number of studies have described generation of enriched astroglia from patients with early- or late-stage onset of AD. We recently reported that iPSC-astroglia derived from these patients are capable of mimicking a wide variety of deficits in homeostatic molecular cascades, intimately associated with AD, that are routinely observed in vivo. This review examines the opportunities and limitations of this innovative technology in the context of AD modelling and uses for preclinical discovery to improve our success for an efficacious therapeutic outcome.

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.

BACKGROUND & AIMS: Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. METHODS: Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. RESULTS: HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. CONCLUSIONS: HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes.

Novel cell lines isolated from mouse embryonic stem cells exhibiting de novo methylation of the E-cadherin promoter.

Mouse embryonic stem cells (mESCs) and epiblast stem cells represent the naïve and primed pluripotent states, respectively. These cells self-renew via distinct signaling pathways and can transition between the two states in the presence of appropriate growth factors. Manipulation of signaling pathways has therefore allowed the isolation of novel pluripotent cell types such as Fibroblast growth factor, Activin and BIO-derived stem cells and IESCs. However, the effect of cell seeding density on pluripotency remains unexplored. In this study, we have examined whether mESCs can epigenetically regulate E-cadherin to enter a primed-like state in response to low cell seeding density. We show that low density seeding in the absence of leukaemia inhibitory factor (LIF) induces decreased apoptosis and maintenance of pluripotency via Activin/Nodal, concomitant with loss of E-cadherin, Signal transducer and activator of transcription phosphorylation, and chimera-forming ability. These cells, E-cadherin negative proliferating stem cells (ENPSCs) can be reverted to a naïve phenotype by addition of LIF or forced E-cadherin expression. However, prolonged culture of ENPSCs without LIF leads to methylation of the E-cadherin promoter (ENPSC(M)), which cannot be reversed by LIF supplementation, and increased histone H3K27 and decreased H3K4 trimethylation. Transcript analysis of ENPSC(M) revealed a primed-like phenotype and their differentiation leads to enrichment of neuroectoderm cells. The generation of ENPSCs is similar to tumorigenesis as ENPSCs exhibit transcript alterations associated with neoplasia, hyperplasia, carcinoma, and metastasis. We therefore describe a novel cell model to elucidate the role of E-cadherin in pluripotency and to investigate epigenetic regulation of this gene during mESC differentiation and tumor metastasis.

Generating an in vitro 3D cell culture model from zebrafish larvae for heart research.

We describe here a novel, fast and inexpensive method for producing a 3D 'heart' structure that forms spontaneously, in vitro, from larval zebrafish (ZF). We have named these 3D 'heart' structures 'zebrafish heart aggregate(s)' (ZFHAs) and have characterised their basic morphology and structural composition using histology, immunohistochemistry, electron microscopy and mass spectrometry. After 2 days in culture, the ZFHA spontaneously form and become a stable contractile syncytium consisting of cardiac tissue derived by in vitro maturation, which beats rhythmically and consistently for more than 8 days. We propose this model as a platform technology, which can be developed further to study in vitro cardiac maturation, regeneration, tissue engineering and safety pharmacological/toxicology testing.

E-cadherin and, in its absence, N-cadherin promotes Nanog expression in mouse embryonic stem cells via STAT3 phosphorylation.

We have recently shown that loss of E-cadherin in mouse embryonic stem cells (mESCs) results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways. Here, we show that E-cadherin promotes kruppel-like factor 4 (Klf4) and Nanog transcript and protein expression in mESCs via STAT3 phosphorylation and that ß-catenin, and its binding region in E-cadherin, is required for this function. To further investigate the role of E-cadherin in leukemia inhibitory factor (LIF)-dependent pluripotency, E-cadherin null (Ecad(-/-)) mESCs were cultured in LIF/bone morphogenetic protein supplemented medium. Under these conditions, Ecad(-/-) mESCs exhibited partial restoration of cell-cell contact and STAT3 phosphorylation and upregulated Klf4, Nanog, and N-cadherin transcripts and protein. Abrogation of N-cadherin using an inhibitory peptide caused loss of phospho STAT3, Klf4, and Nanog in these cells, demonstrating that N-cadherin supports LIF-dependent pluripotency in this context. We therefore identify a novel molecular mechanism linking E- and N-cadherin to the core circuitry of pluripotency in mESCs. This mechanism may explain the recently documented role of E-cadherin in efficient induced pluripotent stem cell reprogramming.

A Scalable 3D High-Content Imaging Protocol for Measuring a Drug Induced DNA Damage Response Using Immunofluorescent Subnuclear γH2AX Spots in Patient Derived Ovarian Cancer Organoids.

The high morbidity rate of ovarian cancer has remained unchanged during the past four decades, partly due to a lack of understanding of disease mechanisms and difficulties in developing new targeted therapies. Defective DNA damage detection and repair is one of the hallmarks of cancer cells and is a defining characteristic of ovarian cancer. Most in vitro studies to date involve viability measurements at scale using relevant cancer cell lines; however, the translation to the clinic is often lacking. The use of patient derived organoids is closing that translational gap, yet the 3D nature of organoid cultures presents challenges for assay measurements beyond viability measurements. In particular, high-content imaging has the potential for screening at scale, providing a better understanding of the mechanism of action of drugs or genetic perturbagens. In this study we report a semiautomated and scalable immunofluorescence imaging assay utilizing the development of a 384-well plate based subnuclear staining and clearing protocol and optimization of 3D confocal image analysis for studying DNA damage dose response in human ovarian cancer organoids. The assay was validated in four organoid models and demonstrated a predictable response to etoposide drug treatment with the lowest efficacy observed in the clinically most resistant model. This imaging and analysis method can be applied to other 3D organoid and spheroid models for use in high content screening.

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad(-/-)), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad(-/-) ES cells is dependent on the beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad(-/-) ES cells is unlikely to be via beta-catenin signaling. Exposure of Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor beta family signaling in the self-renewal of Ecad(-/-) ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

Recommended Guidelines for Developing, Qualifying, and Implementing Complex In Vitro Models (CIVMs) for Drug Discovery.

The pharmaceutical industry is continuing to face high research and development (R&D) costs and low overall success rates of clinical compounds during drug development. There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery, thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower. There needs to be a paradigm shift in the early drug discovery phase (which is lengthy and costly), away from simplistic cellular models that show an inability to effectively and efficiently reproduce healthy or human disease-relevant states to steer target and compound selection for safety, pharmacology, and efficacy questions. This perspective article covers the various stages of early drug discovery from target identification (ID) and validation to the hit/lead discovery phase, lead optimization, and preclinical safety. We outline key aspects that should be considered when developing, qualifying, and implementing complex in vitro models (CIVMs) during these phases, because criteria such as cell types (e.g., cell lines, primary cells, stem cells, and tissue), platform (e.g., spheroids, scaffolds or hydrogels, organoids, microphysiological systems, and bioprinting), throughput, automation, and single and multiplexing endpoints will vary. The article emphasizes the need to adequately qualify these CIVMs such that they are suitable for various applications (e.g., context of use) of drug discovery and translational research. The article ends looking to the future, in which there is an increase in combining computational modeling, artificial intelligence and machine learning (AI/ML), and CIVMs.

High throughput cryopreservation of cells by rapid freezing of sub-µl drops using inkjet printing--cryoprinting.

We have successfully used inkjet printing to cryopreserve 3T3 mouse fibroblast cells and human neuroprogenitor cells (NPCs) derived from human embryonic stem cells (hESCs). Sessile drops of volume 114 nl were formed by printing cell suspensions containing dimethyl sulphoxide (DMSO) as a cryoprotection agent (CPA) at rates in the region 100 Hz-20 kHz, from individual droplets of 380 pl. After printing and a freeze/thaw cycle (with a minimum 24 hours hold period at liquid N2 temperature), 3T3 cells showed an average viability of >90% with CPA concentration <0.8 M at all drop deposition rates. This is a significantly lower CPA concentration than normally used with conventional cryopreservation methods. Cell viability shows a small variation with the polymer substrates used, with the best results obtained using a polyimide substrate. The viability of 3T3 cells after 2 months storage at liquid nitrogen temperature was slightly reduced compared to the cells held for 24 hours but there was no significant further deterioration after 4 or 6 months storage. The viability of NPCs after an identical freeze/thaw cycle were only 55% but this is comparable with conventional cryopreservation methods that use much higher CPA concentrations. A parallel series of experiments printing cells onto substrates held at 195 K or directly into liquid N2 showed considerable variation in cell survival rate with drop deposition rate. Cell suspensions required higher levels of CPA than when printing followed by freezing. At low deposition rates a combination of DMSO and polyethylene glycol (PEG) was needed to allow cell viability after freezing. These results show that inkjet printing provides a practical high throughput method for the cryopreservation of cells with lower CPA concentrations than are required for current low volume cryopreservation methods.

Endostatin expression by MDA-MB-435 breast cancer cells effectively inhibits tumor growth.

Tumors must induce the formation of new blood vessels in order to grow and metastasize. Endostatin, a cleaved product of collagen XVIII, inhibits endothelial cell proliferation and suppresses tumor growth and metastases. Several recent reports have questioned the efficacy of endostatin as a tumor suppressor in experimental animals. Our objective was to determine whether endostatin expression in breast cancer cells inhibits neovascularization and tumor growth in nude mice. MDA-MB-435 cells were transfected with an endostatin expression vector while control cells were transfected with an empty vector. Endostatin expression and secretion were confirmed by RT-PCR and a dot blot assay. No differences were observed in the growth rates of the endostatin-expressing and control clones in vitro. When injected into male and female nude mice, tumors from the control clones increased in size 10-15 fold over 8-10 weeks. In contrast, the endostatin clones formed small tumors which did not increase in size after the first 3 weeks. The endostatinderived tumors had a significantly higher apoptotic index (5.6%) compared to controls (2.0%) and showed a marked reduction in vascularization. In conclusion, expression of endostatin in MDA-MB-435 breast cancer cells effectively suppressed breast tumor growth by inhibiting angiogenesis and increasing apoptosis.

Familial Alzheimer's disease modelling using induced pluripotent stem cell technology.

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which patients exhibit gradual loss of memory that impairs their ability to learn or carry out daily tasks. Diagnosis of AD is difficult, particularly in early stages of the disease, and largely consists of cognitive assessments, with only one in four patients being correctly diagnosed. Development of novel therapeutics for the treatment of AD has proved to be a lengthy, costly and relatively unproductive process with attrition rates of > 90%. As a result, there are no cures for AD and few treatment options available for patients. Therefore, there is a pressing need for drug discovery platforms that can accurately and reproducibly mimic the AD phenotype and be amenable to high content screening applications. Here, we discuss the use of induced pluripotent stem cells (iPSCs), which can be derived from adult cells, as a method of recapitulation of AD phenotype in vitro. We assess their potential use in high content screening assays and the barriers that exist to realising their full potential in predictive efficacy, toxicology and disease modelling. At present, a number of limitations need to be addressed before the use of iPSC technology can be fully realised in AD therapeutic applications. However, whilst the use of AD-derived iPSCs in drug discovery remains a fledgling field, it is one with immense potential that is likely to reach fruition within the next few years.

Loss of function of e-cadherin in embryonic stem cells and the relevance to models of tumorigenesis.

E-cadherin is the primary cell adhesion molecule within the epithelium, and loss of this protein is associated with a more aggressive tumour phenotype and poorer patient prognosis in many cancers. Loss of E-cadherin is a defining characteristic of epithelial-mesenchymal transition (EMT), a process associated with tumour cell metastasis. We have previously demonstrated an EMT event during embryonic stem (ES) cell differentiation, and that loss of E-cadherin in these cells results in altered growth factor response and changes in cell surface localisation of promigratory molecules. We discuss the implication of loss of E-cadherin in ES cells within the context of cancer stem cells and current models of tumorigenesis. We propose that aberrant E-cadherin expression is a critical contributing factor to neoplasia and the early stages of tumorigenesis in the absence of EMT by altering growth factor response of the cells, resulting in increased proliferation, decreased apoptosis, and acquisition of a stem cell-like phenotype.

E-cadherin acts as a regulator of transcripts associated with a wide range of cellular processes in mouse embryonic stem cells.

BACKGROUND: We have recently shown that expression of the cell adhesion molecule E-cadherin is required for LIF-dependent pluripotency of mouse embryonic stem (ES) cells. METHODOLOGY: In this study, we have assessed global transcript expression in E-cadherin null (Ecad-/-) ES cells cultured in either the presence or absence of LIF and compared these to the parental cell line wtD3. RESULTS: We show that LIF has little effect on the transcript profile of Ecad-/- ES cells, with statistically significant transcript alterations observed only for Sp8 and Stat3. Comparison of Ecad-/- and wtD3 ES cells cultured in LIF demonstrated significant alterations in the transcript profile, with effects not only confined to cell adhesion and motility but also affecting, for example, primary metabolic processes, catabolism and genes associated with apoptosis. Ecad-/- ES cells share similar, although not identical, gene expression profiles to epiblast-derived pluripotent stem cells, suggesting that E-cadherin expression may inhibit inner cell mass to epiblast transition. We further show that Ecad-/- ES cells maintain a functional ß-catenin pool that is able to induce ß-catenin/TCF-mediated transactivation but, contrary to previous findings, do not display endogenous ß-catenin/TCF-mediated transactivation. We conclude that loss of E-cadherin in mouse ES cells leads to significant transcript alterations independently of ß-catenin/TCF transactivation.

Abrogation of E-cadherin-mediated cellular aggregation allows proliferation of pluripotent mouse embryonic stem cells in shake flask bioreactors.

BACKGROUND: A fundamental requirement for the exploitation of embryonic stem (ES) cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralising antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.

Prolactin overexpression by MDA-MB-435 human breast cancer cells accelerates tumor growth.

Prolactin (PRL) is an important hormone in mammary tumorigenesis in rodents but its involvement in human breast cancer has been controversial. A role for locally produced PRL in breast carcinogenesis is suggested by its mitogenic action on breast cancer cells and the expression of both PRL and its receptor (PRL-R) in breast carcinomas. Our objective was to examine whether PRL, overexpressed by breast cancer cells, forms an autocrine/paracrine loop that confers a growth advantage for tumors. MDA-MB-435 breast cancer cells overexpressing 23K human PRL were generated, and PRL production and secretion by the clones were confirmed by RT-PCR, western blotting, and the Nb2 bioassay; control clones contain vector only. In vitro the 23K PRL clones proliferated faster and expressed higher levels of the PRL-R protein than controls only when incubated in charcoal-stripped serum (CSS) devoid of lactogenic hormones. When injected into the mammary fatpad of female nude mice or subcutaneously into males, the PRL-overexpressing clones formed tumors that grew 2-4-fold faster than tumors derived from control clones or wild type MDA-MB-435 cells. Western analysis demonstrated significantly higher PRL, PRL-R, and bcl-2 levels in the tumors overexpressing PRL compared to control tumors. These data support a role for breast PRL as a growth/anti-apoptotic factor and suggest that it may serve as a novel therapeutic target for the treatment of breast cancer.