Undesired Cell Research Articles

Single-cell transcriptomics reveals the cell fate transitions of human dopaminergic progenitors derived from hESCs

BackgroundMidbrain dopaminergic (DA) progenitors derived from human pluripotent stem cells are considered to be a promising treatment for Parkinson’s disease (PD). However, the differentiation process produces undesired cell types, which influence the in vivo evaluation of DA cells. In this paper, we analyze the cell fate choice during differentiation and provide valuable information on cell preparation.MethodsHuman embryonic stem cells were differentiated into DA progenitors. We applied single-cell RNA sequencing (scRNA-seq) of the differentiation cells at different time points and investigated the gene expression profiles. Based on the differentially expressed genes between DA and non-DA cells, we investigated the impact of LGI1 (DA enriched) overexpression on DA differentiation and the enrichment effect of CD99 (non-DA enriched) sorting.ResultsTranscriptome analyses revealed the DA differentiation trajectory as well as non-DA populations and three key lineage branch points. Using genetic gain- and loss-of-function approaches, we found that overexpression of LGI1, which is specific to EN1+ early DA progenitors, can promote the generation of TH+ neurons. We also found that choroid plexus epithelial cells and DA progenitors are major components of the final product (day 25), and CD99 was a specific surface marker of choroid plexus epithelial cells. Sorting of CD99− cells eliminated major contaminant cells and improved the purity of DA progenitors.ConclusionsOur study provides the single-cell transcriptional landscape of in vitro DA differentiation, which can guide future improvements in DA preparation and quality control for PD cell therapy.

PEG Spacer Length Substantially Affects Antibody-Based Nanocarrier Targeting of Dendritic Cell Subsets.

Successful cell targeting depends on the controlled positioning of cell-type-specific antibodies on the nanocarrier’s (NC) surface. Uncontrolled antibody immobilization results in unintended cell uptake due to Fc-mediated cell interaction. Consequently, precise immobilization of the Fc region towards the nanocarrier surface is needed with the Fab regions staying freely accessible for antigen binding. Moreover, the antibody needs to be a certain distance from the nanocarrier surface, influencing the targeting performance after formation of the biomolecular corona. This can be achieved by using PEG linker molecules. Here we demonstrate cell type-specific targeting for dendritic cells (DC) as cellular key regulators of immune responses. However, to date, dendritic cell targeting experiments using different linker lengths still need to be conducted. Consequently, we focused on the surface modification of nanocarriers with different molecular weight PEG linkers (0.65, 2, and 5 kDa), and their ability to reduce undesired cell uptake, while achieving efficient DC targeting via covalently immobilized antibodies (stealth targeting). Our findings demonstrate that the PEG linker length significantly affects active dendritic cell targeting from cell lines (DC2.4) to primary cells (BMDCs, splenocytic conventional DCs type 1 (cDC1)). While antibody-functionalized nanocarriers with a shorter PEG length (0.65 kDa) showed the best targeting in DC2.4, a longer PEG length (5 kDa) was required to specifically accumulate in BMDCs and splenocytic cDC1. Our study highlights that these crucial aspects must be considered when targeting dendritic cell subsets, which are of great importance in the fields of cancer immunotherapy and vaccine development.

Emerging Role of Non-Coding RNAs in Senescence.

Senescence is defined as a gradual weakening of functional features of a living organism. Cellular senescence is a process that is principally aimed to remove undesirable cells by prompting tissue remodeling. This process is also regarded as a defense mechanism induced by cellular damage. In the course of oncogenesis, senescence can limit tumor progression. However, senescence participates in the pathoetiology of several disorders such as fibrotic disorders, vascular disorders, diabetes, renal disorders and sarcopenia. Recent studies have revealed contribution of different classes of non-coding RNAs in the cellular senescence. Long non-coding RNAs, microRNAs and circular RNAs are three classes of these transcripts whose contributions in this process have been more investigated. In the current review, we summarize the available literature on the impact of these transcripts in the cellular senescence.

Abstract 2824: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance anti-GPC3 CAR T cells to drive complete responses in solid epithelial tumor models

Abstract CAR T cell therapy has demonstrated clinical efficacy against hematological malignancies. However, prominent barriers including poor T cell effector function, lack of proliferation, and limited CAR T cell persistence have prevented CAR T cell therapies from reaching their full curative potential, especially in solid tumors. Interleukin-2 (IL-2) is a potent stimulator of T cell proliferation, survival, and cytotoxic function, thereby making it an attractive cytokine to support CAR T cell therapy. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations, including non-tumor reactive T cells and NK cells. To facilitate selective ex vivo and in vivo expansion of engineered T cells we have developed a human orthogonal (ortho) ligand/receptor system consisting of a IL-2 mutein (STK-009) that does not significantly stimulate cells expressing wild type IL-2 receptor and a mutated IL-2 Receptor Beta (orthoIL-2Rβ) that responds to STK-009 but not wild type IL-2. This system enables in vivo IL-2 signaling in engineered cells that express the orthoIL-2Rβ while avoiding stimulation of native T cells and NK cells. Previously, we demonstrated the ability of the STK-009/orthoIL-2Rβ receptor pair to selectively enhance the anti-tumor efficacy of orthoIL-2Rβ (hoRb) expressing CD19 CAR T cells (SYNCAR-001) in preclinical lymphoma mouse models. We also demonstrated that STK-009 is selective for the orthoIL-2Rβ expressing cells and therefore in a non-human primate model does not stimulate native T or NK cells. Here, we demonstrate the ability of the STK-009/hoRb system to enhance the anti-tumor activity and persistence of anti-glypican 3 (GPC3) CAR T cells. GPC3 overexpression is associated with various malignancies such as hepatocellular carcinoma (HCC), pediatric sarcomas, and non small cell lung carcinoma (NSCLC). Clinical trials of GPC3 CAR T therapy are ongoing, but early data suggests a need to boost CAR T cell function and persistence to achieve significant clinical responses. We incorporated the hoRb downstream of an anti-GPC3_28z CAR via a T2A cleavage peptide (SYNCAR-002). In vivo, STK-009 administration enhanced the anti-tumor efficacy of SYNCAR-002 in highly aggressive subcutaneous and intraperitoneal HCC models. STK-009 treatment resulted in significant expansion of SYNCAR-002 and drove infiltration of SYNCAR-002 into tumors. STK-009 treatment also induced intratumoral granzyme B+ and IFN-γ+ production by SYNCAR-002 indicating activation of effector T cell function. These findings validate that the orthogonal IL-2 platform has the potential to improve the efficacy and durability of CAR T therapy for solid tumor targets such as GPC3 by selectively expanding CAR-T cells in vivo, driving CAR-T cells into the tumor, and activating CAR-T cells in the tumor microenvironment. Citation Format: Paul-Joseph Aspuria, Marie Semana, Sandro Vivona, Mahalaksmi Ramadass, Navneet Ratti, Romina Riener, Michele Bauer, Mohammed Ali, Deepti Rokkam, Rob A. Kastelein, Patrick J. Lupardus, Martin Oft. Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance anti-GPC3 CAR T cells to drive complete responses in solid epithelial tumor models [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 2824.

Abstract 5819: Tumor nutrient conditions confer drug resistance in pancreatic cancer

Abstract Drug resistance is a major obstacle to the treatment of pancreatic ductal adenocarcinoma (PDAC). Current standard of care includes combination chemotherapies; however, the majority of patients fail to respond to these treatments. Despite significant advances in our understanding of PDAC biology, it remains unclear what biological processes drives the drug resistance observed in most PDAC cases. Contributing to our limited understanding of this problem, in vitro models of PDAC fail to recapitulate in vivo drug resistance, suggesting the tumor microenvironment is a critical regulator.A defining feature of PDAC tumors is an abnormal nutrient environment, which may carry important biological cues driving many of the undesirable cell behaviors related to disease progression. Using a novel medium formulation that mimics the nutrient conditions observed within PDAC tumors (TIFM), we find that PDAC cells cultured in TIFM become highly resistant to common chemotherapeutic agents, better recapitulating the in vivo drug resistance phenotype than standard cultures. Several of these agents engage with their intended target in TIFM cultured cells, discouraging the idea that drug resistance is primarily mediated by drug efflux. However, TIFM cultured cells display reduced apoptotic priming, enabling their survival to drug exposure. Drug resistance enabled by TIFM nutrients is not immediately reversible with standard culture and can be inherited over multiple cell generations. Consistent with this, we find that TIFM nutrient conditions results in significant alterations to the epigenetic landscape of pancreatic cancer cells, rewiring expression of genes that may function as critical regulators of cell survival in PDAC. Ultimately, the work from this project will help elucidate how poor nutrient perfusion within solid tumors regulates cell survival processes and may lead towards better treatment options for PDAC patients beyond combination chemotherapy. Citation Format: Colin Sheehan, James Martin, Grace Croley, Joshua McKeever, Anika Thomas-Toth, Patrick Jonker, James LaBelle, Andrew Koh, Alexander Muir. Tumor nutrient conditions confer drug resistance in pancreatic cancer [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 5819.

Abstract 2948: A multimodal, label-free tool for gentle and efficient cell separation

Abstract LevitasBio has developed the LeviCell™ platform, a powerful new technology for cell separation and characterization that utilizes magnetic fields to levitate cells. Unlike other methods, cell separation via magnetic levitation does not require dyes, antibodies or specific markers, and the cells are not modified or perturbed in any fashion. However, magnetic levitation also does not preclude the use of these tools to further enhance separation of cells. In its simplest form, magnetic levitation requires only the addition of an inert paramagnetic compound to the media in which the cells are suspended. The cells are subsequently introduced into an externally applied magnetic field while contained within a single-use cartridge, which causes the cells to levitate in solution. The cells’ intrinsic properties, including density and magnetic susceptibility, determine the height to which the cells levitate. As different cell types often have different such properties, they may often levitate to different heights, facilitating their separation. Continuous imaging allows for complete sample analysis and characterization with the benefit of real-time control over the cell separation. The simplicity of magnetic levitation enables cells to be treated gently without use of high pressure or other perturbations that commonly lead to increased cellular stress responses, specific cell type activation, or even cell death. The direct and gentle flow path leads to high yields of live cells even with very low input cell numbers. This novel cell separation technology enables a variety of applications, including: the enrichment of viable cells from dead/dying cells and debris; separation of different cell types possessing distinct densities; and, in cases where the intrinsic density changes, the separation of differentiated from undifferentiated cells. Given that the levitation chamber is flanked by two magnets on the top and bottom, the levitation technology also inherently allows for the use of magnetic particles to deplete undesired cells within a cell population. Here we illustrate the performance of anti-CD45 antibody-conjugated magnetic particles for CD45+ cell depletion with the LeviCell system. CD45+ depletion rates of greater than 99% are readily obtained across a wide range of total CD45+ input cell numbers, from 105 to 107 total cells. Furthermore, CD45+ cell depletion is performed simultaneously with live cell enrichment, allowing for the collection of a high viability CD45- live cell population. Citation Format: Kevin Travers, Chris Stevens, Susana Jett, Suzanne Weaver, Teddy de Groot, Stefan Ha, Geoff Facer, Kathy Lazaruk. A multimodal, label-free tool for gentle and efficient cell separation [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 2948.

Immune-vascular mural cell interactions: consequences for immune cell trafficking, cerebral blood flow, and the blood-brain barrier.

.Brain barriers are crucial sites for cerebral energy supply, waste removal, immune cell migration, and solute exchange, all of which maintain an appropriate environment for neuronal activity. At the capillary level, where the largest area of brain–vascular interface occurs, pericytes adjust cerebral blood flow (CBF) by regulating capillary diameter and maintain the blood–brain barrier (BBB) by suppressing endothelial cell (EC) transcytosis and inducing tight junction expression between ECs. Pericytes also limit the infiltration of circulating leukocytes into the brain where resident microglia confine brain injury and provide the first line of defence against invading pathogens. Brain “waste” is cleared across the BBB into the blood, phagocytosed by microglia and astrocytes, or removed by the flow of cerebrospinal fluid (CSF) through perivascular routes—a process driven by respiratory motion and the pulsation of the heart, arteriolar smooth muscle, and possibly pericytes. “Dirty” CSF exits the brain and is probably drained around olfactory nerve rootlets and via the dural meningeal lymphatic vessels and possibly the skull bone marrow. The brain is widely regarded as an immune-privileged organ because it is accessible to few antigen-primed leukocytes. Leukocytes enter the brain via the meninges, the BBB, and the blood-CSF barrier. Advances in genetic and imaging tools have revealed that neurological diseases significantly alter immune–brain barrier interactions in at least three ways: (1) the brain’s immune-privileged status is compromised when pericytes are lost or lymphatic vessels are dysregulated; (2) immune cells release vasoactive molecules to regulate CBF, modulate arteriole stiffness, and can plug and eliminate capillaries which impairs CBF and possibly waste clearance; and (3) immune–vascular interactions can make the BBB leaky via multiple mechanisms, thus aggravating the influx of undesirable substances and cells. Here, we review developments in these three areas and briefly discuss potential therapeutic avenues for restoring brain barrier functions.

Suppressing lithium dendrite growth on lithium-ion/metal batteries by a tortuously porous γ-alumina separator

The challenges of dendrite propagation limit the operational safety and long-term cycling stability of lithium-ion batteries and development of high energy density lithium-metal batteries. Solid state electrolytes may eliminate the propagation of dendrites, but they fail to achieve high Li-ion conductivity and charge transfer rate at room temperature resulting in undesired cell performance. Here we report the usage of electrode-coated plate-structured γ-alumina separators with liquid electrolyte for high performance, safe lithium-ion batteries. The γ-alumina separators, made of γ-alumina plates of high aspect ratio, are mechanically strong and tortuously porous, and therefore effective in preventing passage of dendrites. Nickel-manganese-cobalt-oxide/lithium cells with a plate-structured γ-alumina separator show stable cycle performance without passage of dendrites up to 3 C-rate of charging and discharging. Same cells with commercial polypropylene (PP-2500) separator or spherical-structured α-alumina separator of similar pore size to γ-alumina separator, could not achieve the above-mentioned dendrite free cycling even at much lower C-rates (0.2 C and 1.0 C respectively). A comparison of the three cells with different separators clearly shows that the hardness and high tortuosity of the separator are effective in preventing dendrite propagation. This work results in the development of a lithium-ion-battery with improved safety and shows a new direction in the design and fabrication of high performance and safe lithium-metal batteries.

Analysis of Mitochondria by Single-Organelle Resolution.

Cellular organelles are highly specialized compartments with distinct functions. With the increasing resolution of detection methods, it is becoming clearer that same organelles may have different functions or properties not only within different cell populations of a tissue but also within the same cell. Dysfunction or altered function affects the organelle itself and may also lead to malignancies or undesirable cell death. To understand cellular function or dysfunction, it is therefore necessary to analyze cellular components at the single-organelle level. Here, we review the recent advances in analyzing cellular function at single-organelle resolution using high-parameter flow cytometry or multicolor confocal microscopy. We focus on the analysis of mitochondria, as they are organelles at the crossroads of various cellular signaling pathways and functions. However, most of the applied methods/technologies are transferable to any other organelle, such as the endoplasmic reticulum, lysosomes, or peroxisomes.

Effects of microRNA-298 on APP and BACE1 translation differ according to cell type and 3\u2032-UTR variation

Alzheimer’s disease (AD) is marked by neurofibrillary tangles and senile plaques composed of amyloid β (Aβ) peptides. However, specific contributions of different cell types to Aβ deposition remain unknown. Non-coding microRNAs (miRNA) play important roles in AD by regulating translation of major associated proteins, such as Aβ precursor protein (APP) and β-site APP-cleaving enzyme (BACE1), two key proteins associated with Aβ biogenesis. MiRNAs typically silence protein expression via binding specific sites in mRNAs’ 3′-untranslated regions (3′-UTR). MiRNAs regulate protein levels in a cell-type specific manner; however, mechanisms of the variation of miRNA activity remain unknown. We report that miR-298 treatment reduced native APP and BACE1 protein levels in an astrocytic but not in a neuron-like cell line. From miR-298’s effects on APP-3′-UTR activity and native protein levels, we infer that differences in APP 3′-UTR length could explain differential miR-298 activity. Such varied or truncated, but natural, 3′-UTR specific to a given cell type provides an opportunity to regulate native protein levels by particular miRNA. Thus, miRNA’s effect tailoring to a specific cell type, bypassing another undesired cell type with a truncated 3′-UTR would potentially advance clinically-relevant translational research.

BH3-Only Proteins Noxa and Puma Are Key Regulators of Induced Apoptosis.

Apoptosis is an evolutionarily conserved and tightly regulated cell death pathway. Physiological cell death is important for maintaining homeostasis and optimal biological conditions by continuous elimination of undesired or superfluous cells. The BH3-only pro-apoptotic members are strong inducers of apoptosis. The pro-apoptotic BH3-only protein Noxa activates multiple death pathways by inhibiting the anti-apoptotic Bcl-2 family protein, Mcl-1, and other protein members leading to Bax and Bak activation and MOMP. On the other hand, Puma is induced by p53-dependent and p53-independent apoptotic stimuli in several cancer cell lines. Moreover, this protein is involved in several physiological and pathological processes, such as immunity, cancer, and neurodegenerative diseases. Future heat shock research could disclose the effect of hyperthermia on both Noxa and BH3-only proteins. This suggests post-transcriptional mechanisms controlling the translation of both Puma and Noxa mRNA in heat-shocked cells. This study was also the chance to recapitulate the different reactional mechanisms investigated for caspases.

Design of foldable, responsively drug-eluting polyacrylic intraocular lens bulk materials for prevention of postoperative complications.

Posterior capsular opacification (PCO), resulting from undesired intracapsular cell proliferation, is the most common complication of intraocular lens (IOL) implantation after cataract surgery. In recent years, IOLs have been developed into a drug delivery platform. Compared with traditional eye drops, drug-loaded IOLs have the characteristics of independent patient compliance and no other operation except surgical implantation. In this work, a series of poly(glycidyl methacrylate-co-2-(2-ethoxyethoxy)ethyl acrylate) (PGE) acrylic intraocular lens materials were synthesized as drug delivery platforms. The PGE synthesized with 10% crosslinking agent has excellent optical, foldable, and thermomechanical properties. An aldehyde group was subsequently introduced into the PGE chains, and an antiproliferative drug (doxorubicin) was immobilized onto the PGE chains via an H+-sensitive imine bond. The IOL exhibits H+-dependent Dox release behavior in a simulated pathological environment. The in vitro and in vivo systematical evaluations indicate that such a responsively drug-eluting PGE IOL can effectively prevent PCO.

The Effect of Photodynamic Therapy on Oral-Premalignant Lesions: A Systematic Review.

BackgroundDentists now have access to a wide range of unique treatment methods as a result of substantial scientific and technological breakthroughs in the field of dentistry. Photodynamic therapy (PDT) is a non-invasive treatment procedure that use photosensitizers, a specific wavelength of light, and the production of singlet oxygen and reactive oxygen species (ROS) to kill undesired eukaryotic cells (such as oral tumors) and harmful microbes. In several disciplines of dentistry, it is seen as a valid therapeutic option. The purpose of this study was to examine the effectiveness and side effects of PDT in the treatment of oral premalignant lesions. Material and MethodsThree search engines (PubMed, ISI Web of Science, and the Cochrane Library) were used to conduct a systematic review using the phrases photodynamic therapy and PDT in combination with other terms. To define our study eligibility criteria, we used the Population, Intervention and Comparison, Outcomes, and Study design technique. ResultsInitial results were 33. Definitely, 18 studies met our selection criteria. ConclusionsOur analysis suggests ALA- PDT as a promising therapeutic modality for OEL lesions which should be treated first with the topical ALA-PDT using either the LED or laser light for successful clinical outcome for OEL lesions. Key words:Photodynamic Therapy, Photosensitizer, Aminolevulinic Acid (ALA), Methylene Blue (MB), Toludine Blue, Oral Leukoplakia, Oral Erythroplakia, Oral Verrucous hyperplasia, Oral Lichen Planus.

The Emerging Scenario of the Gut-Brain Axis: The Therapeutic Actions of the New Actor Kefir against Neurodegenerative Diseases.

The fact that millions of people worldwide suffer from Alzheimer’s disease (AD) or Parkinson’s disease (PD), the two most prevalent neurodegenerative diseases (NDs), has been a permanent challenge to science. New tools were developed over the past two decades and were immediately incorporated into routines in many laboratories, but the most valuable scientific contribution was the “waking up” of the gut microbiota. Disturbances in the gut microbiota, such as an imbalance in the beneficial/pathogenic effects and a decrease in diversity, can result in the passage of undesired chemicals and cells to the systemic circulation. Recently, the potential effect of probiotics on restoring/preserving the microbiota was also evaluated regarding important metabolite and vitamin production, pathogen exclusion, immune system maturation, and intestinal mucosal barrier integrity. Therefore, the focus of the present review is to discuss the available data and conclude what has been accomplished over the past two decades. This perspective fosters program development of the next steps that are necessary to obtain confirmation through clinical trials on the magnitude of the effects of kefir in large samples.

Tumorigenesis and cell competition in Drosophila in the absence of polyhomeotic function

Cell competition is a homeostatic process that eliminates by apoptosis unfit or undesirable cells from animal tissues, including tumor cells that appear during the life of the organism. In Drosophila there is evidence that many types of oncogenic cells are eliminated by cell competition. One exception is cells mutant for polyhomeotic (ph), a member of the Polycomb family of genes; most of the isolated mutant ph clones survive and develop tumorous overgrowths in imaginal discs. To characterize the tumorigenic effect of the lack of ph, we first studied the growth of different regions of the wing disc deficient in ph activity and found that the effect is restricted to the proximal appendage. Moreover, we found that ph-deficient tissue is partially refractory to apoptosis. Second, we analyzed the behavior of clones lacking ph function and found that many suffer cell competition but are not completely eliminated. Unexpectedly, we found that nonmutant cells also undergo cell competition when surrounded by ph-deficient cells, indicating that within the same tissue cell competition may operate in opposite directions. We suggest two reasons for the incompleteness of cell competition in ph mutant cells: 1) These cells are partially refractory to apoptosis, and 2) the loss of ph function alters the identity of imaginal cells and subsequently their cell affinities. It compromises the winner/loser interaction, a prerequisite for cell competition.

The effects of chemotherapeutic drugs on PD-L1 gene expression in breast cancer cell lines.

Breast cancer is the most common cancer among women in terms of prevalence and mortality, and chemotherapy is one of the most effective treatments at higher stages. However, resistance to chemotherapy is the main obstacle in the treatment of this cancer. Accumulated evidence identified the PD-L1 protein as an essential protein in the development of different cancers. Abnormal expression of this protein in various tumor cells is linked to cancer development and inhibiting the function of immune cells, which correlated with reduced beneficial effects of chemotherapy drugs. In the present study, the effects of common chemotherapy drugs including doxorubicin, paclitaxel, and docetaxel on the expression of the PD-L1 gene were investigated by qRT-PCR before and after the treatment with these drugs in MD231, MD468, SKBR3 breast cancer cell lines. Also, the MTT test was applied to examine the effects of drugs on the growth and proliferation of cancer cells considering PD-L1 expression. The expression of the PD-L1 gene increased after 24 and 48h of treatment with chemotherapy drugs. The obtained results indicate the enhancing effects of chemotherapy drugs on PD-L1 gene expression, which have a suppressive effect on the immune system against breast cancer. The use of these drugs as the first line of chemotherapy in triple-negative breast cancer is not recommended. However, there is still a need for further experimental and clinical research on the exact effects of these drugs on undesired immune cells exhaustion in breast cancer therapy.

Senolytic Cocktail Dasatinib Plus Quercetin Enhances the Antitumor Effect of Senescence-Inducing Radiotherapy in a Preclinical Model of Melanoma

Senolytic Cocktail Dasatinib Plus Quercetin Enhances the Antitumor Effect of Senescence-Inducing Radiotherapy in a Preclinical Model of Melanoma

Downstream bioprocessing of human pluripotent stem cell-derived therapeutics.

With the advancement in lineage-specific differentiation from human pluripotent stem cells (hPSCs), downstream cell separation has now become a critical step to produce hPSC-derived products. Since differentiation procedures usually result in a heterogeneous cell population, cell separation needs to be performed either to enrich the desired cell population or remove the undesired cell population. This article summarizes recent advances in separation processes for hPSC-derived cells, including the standard separation technologies, such as magnetic-activated cell sorting, as well as the novel separation strategies, such as those based on adhesion strength and metabolic flux. Specifically, the downstream bioprocessing flow and the identification of surface markers for various cell lineages are discussed. While challenges remain for large-scale downstream bioprocessing of hPSC-derived cells, the rational quality-by-design approach should be implemented to enhance the understanding of the relationship between process and the product and to ensure the safety of the produced cells.

Induced Complementary Resistive Switching in Forming-Free TiOx/TiO2/TiOx Memristors.

The undesirable sneak current path is one of the key challenges in high-density memory integration for the emerging cross-bar memristor arrays. This work demonstrates a new heterojunction design of oxide multilayer stacking with different oxygen vacancy contents to manipulate the oxidation state. We show that the bipolar resistive switching (BRS) behavior of the Pt/TiOx/Pt cross-bar structure can be changed to complementary resistive switching (CRS) by introducing a thin TiO2 layer in the middle of the TiOx layer to obtain a Pt/TiOx/TiO2/TiOx/Pt device architecture with a double-junction active matrix. In contrast to the BRS in a single-layer TiOx matrix, the device with a double-junction matrix remains in a high-resistance state in the voltage range below the SET voltage, which makes it an efficient structure to overcome the sneak path constraints of undesired half-selected cells that lead to incorrect output reading. This architecture is capable of eliminating these half-selected cells between the nearby cross-bar cells in a smaller programming voltage range. A simplified model for the switching mechanism can be used to account for the observed high-quality switching performance with excellent endurance and current retention properties.

Panorama of the Intracellular Molecular Concert Orchestrated by Actinoporins, Pore-Forming Toxins from Sea Anemones

Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs’ intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.