Continuous evolving humanoid for advanced cellular models.

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

Preclinical Pharmacological Approaches in Drug Discovery for Chronic Pain.

Recent advances in generating induced pluripotent stem cells have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from somatic cells. However, a major obstacle to the use of iPSC for therapeutic applications is the potential genomic modifications resulted from viral insertion of transgenes in the cellular genome. Second, the culture of iPSCs and adult cells often requires the use of animal products, which hinder the generation of clinical-grade iPSCs. We report here the generation of iPSCs by an RNA Sendai virus vector that does not integrate transgenes into the cell's genome. In addition, reprogramming can be performed on a feeder-free or xeno-free condition without containing animal products. Generation of an integrant-free iPSCs in these conditions will facilitate the studies of iPSCs in cell-based therapies.

Establishment of a Robust Platform for Induced Pluripotent Stem Cell Research Using Maholo LabDroid.

Understanding features common to all forms of cancer is a vital component of treatment and research into malignant disease. All solid tumours share the ability to overcome contact inhibition of proliferation (CIP), the process by which intercellular contacts engage signalling to stop proliferation. Merlin, a tumour suppressor protein that is inactivated in a wide variety of cancers, plays a crucial role in CIP; merlin-deficient cells lose the ability to be contact-inhibited and subsequently form tumours. Although the role of merlin in cancer has been investigated extensively over the past 28 years, this protein has been notoriously difficult to study. To address this issue, we develop and utilize 2 split-luciferase biosensor systems that enable accurate quantification of merlin activity in real time. Merlin undergoes a conformational change that is functionally important in tumour suppression. Phosphorylation at a key C-terminal residue promotes transition of the protein from an open, active conformation to a closed, N-to-C terminal autoinhibited conformation. Therefore, monitoring this conformation change in real time could provide unique insights into merlin function. To do this, we apply NanoBiT split-luciferase technology to develop an intramolecular merlin biosensor (intra-Mer-BS). In brief, 2 split-luciferase components, LgBiT and SmBiT, are fused to the N- and C-terminus of merlin, respectively. Upon open-to-closed conformation change of merlin, LgBiT and SmBiT complement to reconstitute a functional luciferase and emit light. This enables accurate quantification of merlin’s functionally relevant conformation changes in real time. Importantly, cotransfection of the intra-Mer-BS alongside PAK1, an upstream merlin regulator that promotes transition to the closed conformation, significantly increases luminescent activity of the intra-Mer-BS, indicating that the biosensor faithfully reports on merlin’s conformation. Moreover, merlin has been shown to exert its tumour suppressive function through activation of LATS, the central mediator of the Hippo signalling pathway. In addition to the intra-Mer-BS, we develop and validate a NanoBiT biosensor to monitor the interaction between merlin and LATS (Mer-LATS-BS). This Mer-LATS-BS is used to quantify the effect of merlin activators and inhibitors on merlin/LATS tumour suppressive activity in cancer cells. In summary, we develop and validate 2 novel bioluminescent biosensors to monitor merlin’s conformation changes and activity in cancer cells. The intra-Mer-BS and Mer-LATS-BS provide real time information with high sensitivity and excellent reproducibility. Ultimately, these biosensors enable high throughput screening to discover novel upstream regulators of merlin in cancer and provide mechanistic insight into how contact inhibitive signalling is propagated through merlin and the Hippo pathway. Citation Format: Alexander J. Pipchuk, Xiaolong Yang. Development of ultrasensitive split luciferase biosensors monitoring activity of the merlin tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 858.

Flow cytometry is a mainstay technique in cell biology research, where it is used for phenotypic analysis of mixed cell populations. Quantitative approaches have unlocked a deeper value of flow cytometry in drug discovery research. As the number of drug modalities and druggable mechanisms increases, there is an increasing drive to identify meaningful biomarkers, evaluate the relationship between pharmacokinetics and pharmacodynamics (PK/PD), and translate these insights into the evaluation of patients enrolled in early clinical trials. In this review, we discuss emerging roles for flow cytometry in the translational setting that supports the transition and evaluation of novel compounds in the clinic.

The drug discovery and the drug development processes represent a continuum of recursive activities that range from initial drug target identification to final Food and Drug Administration approval and marketing of a new therapeutic. Drug discovery, as its name implies, is more exploratory and less focused in many cases, whereas drug development has a clinically defined endpoint and a specific disease goal. Academia has historically made major contributions to this process at the early discovery phases. However, current trends in the organization of the pharmaceutical industry suggest an expanded role for academia in the near future. Megamergers among major pharmaceutical corporations indicate their movement toward a focus on end-stage clinical trials, manufacturing, and marketing. There has been a parallel increase in outsourcing of intermediate steps to specialty small pharmaceutical, biotechnology, and contract service companies. The new paradigm suggests that academia will play an increasingly important role at the proof-of-principle stage of basic and clinical drug discovery research, in training the future skilled work force, and in close partnerships with small pharmaceutical and biotechnology companies. However, academic drug discovery research faces a set of barriers to progress, the relative importance of which varies with the home institution and the details of the research area. These barriers fall into four general categories: (1) the historical administrative structure and environment of academia; (2) the structure and emphasis of peer review panels that control research funding by government and private agencies; (3) the organization and operation of the academic infrastructure; and (4) the structure and availability of specialized resources and information management. Selected examples of barriers to drug discovery and drug development research and training in academia are presented, as are some specific recommendations designed to minimize or circumvent these barriers. In some cases, precedents established by other disease-focused areas may be relevant to Alzheimer disease and related disorders, but the overall impact of any changes requires adaptation at the top of the administrative structures in academia and funding agencies to support and encourage cooperative efforts among faculty investigators.

Simple SummaryThis review focuses on the advantages achieved by incorporating magnetic forces into culture platforms used to study cancer progression in the laboratory. Due to the complex interactions that occur between cancer cells and their environment throughout primary tumor growth and metastatic spread, benchtop techniques are essential for decoupling these factors at several stages of disease progression where traditional animal models would fail. Breakthroughs in our understanding of cancer biology and mechanics through these benchtop techniques can ultimately lead to better-designed precision medicine platforms and clinical therapeutics for patients.Worldwide, there are currently around 18.1 million new cancer cases and 9.6 million cancer deaths yearly. Although cancer diagnosis and treatment has improved greatly in the past several decades, a complete understanding of the complex interactions between cancer cells and the tumor microenvironment during primary tumor growth and metastatic expansion is still lacking. Several aspects of the metastatic cascade require in vitro investigation. This is because in vitro work allows for a reduced number of variables and an ability to gather real-time data of cell responses to precise stimuli, decoupling the complex environment surrounding in vivo experimentation. Breakthroughs in our understanding of cancer biology and mechanics through in vitro assays can lead to better-designed ex vivo precision medicine platforms and clinical therapeutics. Multiple techniques have been developed to imitate cancer cells in their primary or metastatic environments, such as spheroids in suspension, microfluidic systems, 3D bioprinting, and hydrogel embedding. Recently, magnetic-based in vitro platforms have been developed to improve the reproducibility of the cell geometries created, precisely move magnetized cell aggregates or fabricated scaffolding, and incorporate static or dynamic loading into the cell or its culture environment. Here, we will review the latest magnetic techniques utilized in these in vitro environments to improve our understanding of cancer cell interactions throughout the various stages of the metastatic cascade.

A graphene-based platform for induced pluripotent stem cells culture and differentiation

The induced pluripotent stem cell (iPSC) technology is instrumental in advancing the fields of disease modeling and cell transplantation. We herein discuss the various issues regarding disease modeling and cell transplantation presented in previous reports, and also describe new iPSC-based medicine including iPSC clinical trials. In such trials, iPSCs from patients can be used to predict drug responders/non-responders by analyzing the efficacy of the drug on iPSC-derived cells. They could also be used to stratify patients after actual clinical trials, including those with sporadic diseases, based on the drug responsiveness of each patient in the clinical trials. iPSC-derived cells can be used for the identification of response markers, leading to increased success rates in such trials. Since iPSCs can be used in micromedicine for drug discovery, and in macromedicine for actual clinical trials, their use would tightly connect both micro- and macromedicine. The use of iPSCs in disease modeling, cell transplantation, and clinical trials could therefore lead to significant changes in the future of medicine.

Research priorities in epilepsy for the next decade—A representative view of the European scientific community: Summary of the ILAE Epilepsy Research Workshop, Brussels, 17–18 January 2008

In the fight against cancer, immunotherapies are one of the largest growing therapeutics in development. Immunotherapeutics are designed to boost or harness the power of the immune system to prevent, control, or eliminate cancer; while many immune therapies have been found to be safe others have induced severe toxicities. For instance, even compounds that target the same molecule/antigen can have dramatically differing safety profiles. Current preclinical models evaluating these therapies are underequipped to assess the safety of these compounds: in vitro assays fail to predict systemic responses and traditional animal models often fail to correlate with human responses. To better meet the needs of assessing preclinical toxicity we developed a PBMC-humanized mouse model to test a variety of therapeutics, including both monoclonal and bispecific antibodies and induce human cytokine release responses which can manifest within hours or days later resulting in tissue damage and lethality of the mice. To date we have tested a variety of therapeutics, including Blinatumomab, Rituximab, EGFRxCD3 BiTE, CAR-T, and others in our platform while evaluating the ability of the therapeutic to induce human cytokines, bodyweight loss, clinical symptom assessment, and survival in the context of toxicity alone or along with the evaluation with efficacy. We found that many of the therapeutics tested in our platform showed similarities to clinical data in humans. For example, urelumab and utomilumab are both fully humanized monoclonal antibodies against 4-1BB (CD137). However, during clinical trials, urelumab was shown to induce severe liver toxicities while utomilumab was well tolerated. In our huPBMC mouse model, we likewise showed that huPBMC mice dosed with 10 mpk of urelumab experienced body weight loss, showed liver necrosis, and met the clinical criteria for early euthanasia compared to mice treated with 10 mpk utomilumab and PBS treated controls. Serum levels of enzymes associated with liver damage: AST, ALT and GLDH were significantly higher in urelumab treated mice and terminal serum cytokine analysis revealed similarities with those found to be increased in urelumab clinical trials, including elevated IFNγ, IP-10, MIG, and MIP-1α and MIP-1β. Further, HuPBMC mice are also capable of detecting variability among donors. We have screened well over 60 human PBMC donors in huPBMC mice treated with OKT3 and αCD28 and while we always see an increase in cytokines such as IFNγ - the range of induction varies greatly among donors. Further, we see PBMC-donor variability in body weight loss and survival rate after OKT3 and αCD28 treatments. We demonstrate that the PBMC humanized mouse model shows clinical relevance. The use of these models for preclinical safety assessments has the potential to become an important part of novel immunotherapeutic development for patient safety and reducing drug development costs. Citation Format: Destanie Rose, Won Lee, Guoxiang Yang, Jiwon Yang, Mingshan Cheng, Wenqian He, Bernard Buetow, Allison Vitsky, Maggie Liu, Bart Jessen, James Keck. The use of PBMC humanized mice to test the efficacy and safety of antibody and cell-based cancer immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB349.

In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.

Background: Regenerative periodontal surgical therapy faces significant challenges due to the limited ability of the body to regenerate damaged periodontal tissue. One of the primary goals in regenerative periodontal therapy is regaining periodontal tissue attachment after destruction by periodontal disease. Currently, stem cells, harnessing three pivotal components—cells, biomaterials, and growth factors—are widely used in periodontal regeneration. Stem cells can be obtained from various sources, either by isolating cells from bone marrow, teeth, and muscles or through the somatic cell programming method (reprogramming) known as induced pluripotent stem cells (iPSCs). Purpose: This review aims to describe the potential use of iPSCs in the treatment of periodontal defects. Review: Search strategies were developed using the PubMed, LILACS, Scielo, and Wiley online databases during the period of 2012–2022. Ten articles met the inclusion criteria. iPSCs were obtained by inducing somatic cells from both dental and non-dental sources with factors Oct3/4, Sox2, Klf4, and c-Myc. Periodontal tissue regeneration procedures can be augmented with iPSCs. Unlike tooth-based stem cells, iPSCs offer several advantages, such as unlimited cell sources and the capability to differentiate into any cell type, including periodontal tissue. The potential of iPSCs extends to correcting periodontal bone defects and forming new periodontal tissues, such as alveolar bone, cementum, and periodontal ligament. However, iPSCs do have limitations, including the need for clinical trials, cell programming production facilities, and optimization of differentiated-cell functionality. Conclusion: The combined use of iPSCs in cell-based tissue engineering holds vast potential for future periodontal treatment strategies.

Spinal cord injury (SCI) is a devastating trauma causing long-lasting disability. Although advances have occurred in the last decade in the medical, surgical and rehabilitative treatments of SCI, the therapeutic approaches are still not ideal. The use of cell transplantation as a therapeutic strategy for the treatment of SCI is promising, particularly since it can target cell replacement, neuroprotection and regeneration. Cell therapies for treating SCI are limited due to several translational roadblocks, including ethical and practical concerns regarding cell sources. The use of iPSCs has been particularly attractive, since they avoid the ethical and moral concerns that surround other stem cells. Furthermore, various cell types with potential for application in the treatment of SCI can be created from autologous sources using iPSCs. For applications in SCI, the iPSCs can be differentiated into neural precursor cells, neurons, oligodendrocytes, astrocytes, neural crest cells and mesenchymal stromal cells that can act by replacing lost cells or providing environmental support. Some methods, such as direct reprogramming, are being investigated to reduce tumorigenicity and improve reprogramming efficiencies, which have been some of the issues surrounding the use of iPSCs clinically to date. Recently, iPSCs have entered clinical trials for use in age-related macular degeneration, further supporting their promise for translation in other conditions, including SCI.