Induced Pluripotent Stem Cell-Derived Neural Culture Model of Alzheimer’s Disease: A 3D Organoid Approach

Alzheimer’s disease (AD) is a progressive neurologic disorder that impacts a diverse population of older adults. As three-dimensional (3D) models are powerful tools for advancing AD studies, the authors have been developed AD cortical organoids to enable the observation of AD pathology at the cellular, tissue, and organ levels. For creating the model, APPSwe/Ind (APP) and PSEN1 (PS1) mutant genes were transfected into mouse induced pluripotent stem cells (iPSCs) following which the iPSC lines that expressed mutant APP and PS1 proteins were obtained. Then, using modified serum-free suspended embryoid body culture, AD cerebral organoids were made successfully at various ages. The AD model can show AD’s biochemical and pathological alterations, such as overexpressions of Aβ40 and Aβ42 and a decrease of GABAergic interneurons. The proposed model has the potential for implementation in many biomedical applications, including AD drug screening, stem cell transplant, and neuronal tissue engineering.

Comprehensive analyses of the inotropic compound omecamtiv mecarbil in rat and human cardiac preparations

Omecamtiv mecarbil (OM), a myosin activator, was reported to induce complex concentration- and species-dependent effects on contractile function and clinical studies indicated a low therapeutic index with diastolic dysfunction at concentrations above 1 µM. To further characterize effects of OM in a human context and under different preload conditions, we constructed a setup that allows isometric contractility analyses of human induced pluripotent stem cell (hiPSC)-derived engineered heart tissues (EHTs). The results were compared to effects of OM on the very same EHTs measured under auxotonic conditions. OM induced a sustained, concentration-dependent increase in time-to-peak under all conditions (maximally 2-3 fold). Peak force, in contrast, was increased by OM only in human, but not rat EHTs and only under isometric conditions, varied between hiPSC lines and showed a biphasic concentration-dependency with maximal effects at 1 µM. Relaxation time tended to fall under auxotonic and strongly increase under isometric conditions, again with biphasic concentration-dependency. Diastolic tension concentration-dependently increased under all conditions. The latter was reduced by an inhibitor of the mitochondrial sodium calcium exchanger (CGP-37157). OM induced increases in mitochondrial oxidation in isolated cardiomyocytes, indicating that OM, an inotrope that does not increase intracellular and mitochondrial Ca2+, can induce mismatch between an increase in ATP and ROS production and unstimulated mitochondrial redox capacity. Taken together, we developed a novel setup well suitable for isometric measurements of EHTs. The effects of OM on contractility and diastolic tension are complex with concentration-, time-, species- and loading-dependent differences. Effects on mitochondrial function require further studies.

A Bioprinted Heart-on-a-Chip with Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Evaluation

In this work, we show that valve-based bioprinting induces no measurable detrimental effects on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The aim of the current study was three-fold: first, to assess the response of hiPSC-CMs to several hydrogel formulations by measuring electrophysiological function; second, to customise a new microvalve-based cell printing mechanism in order to deliver hiPSC-CMs suspensions, and third, to compare the traditional manual pipetting cell-culture method and cardiomyocytes dispensed with the bioprinter. To achieve the first and third objectives, iCell2 (Cellular Dynamics International) hiPSC-CMs were used. The effects of well-known drugs were tested on iCell2 cultured by manual pipetting and bioprinting. Despite the results showing that hydrogels and their cross-linkers significantly reduced the electrophysiological performance of the cells compared with those cultured on fibronectin, the bio-ink droplets containing a liquid suspension of live cardiomyocytes proved to be an alternative to standard manual handling and could reduce the number of cells required for drug testing, with no significant differences in drug-sensitivity between both approaches. These results provide a basis for the development of a novel bioprinter with nanolitre resolution to decrease the required number of cells and to automate the cell plating process.

Oxidative Stress Research on Huntington's Disease Neurons Using Genetically Encoded Biosensors

Many neurodegenerative diseases, including Huntington’s disease (HD), are associated with oxidative stress in the neurons of the brain. Genetically encoded biosensorsare useful for studying these processesin vitro. Human cell cultures expressing the biosensors can serve as a cell model for developing and testing effective agents that reduce oxidative stress. In this work, transgenes encoding biosensors of glutathione oxidative potential(Grx1-roGFP2) with cytoplasmic and mitochondrial localization were introduced into human induced pluripotent stem cells of a healthy donor and an HD patient using CRISPR/Cas9-mediated genome editing. The cells were subsequently differentiated into medium spiny neurons of the striatum. The expression of the biosensors was detected in the iPSCs, neuronal precursors and mature neurons.The obtained cells could be used to study the redox potential of glutathione in HD neurons and to screen for new drug compounds aimed at reducing oxidative stress. Keywords: genetically encoded biosensors, Huntington’s disease, induced pluripotent stem cells, medium spiny neurons, oxidative stress, glutathione, Grx1-roGFP2

Neuroprogenitor Cells From Patients With TBCK Encephalopathy Suggest Deregulation of Early Secretory Vesicle Transport

Biallelic pathogenic variants in TBCK cause encephaloneuropathy, infantile hypotonia with psychomotor retardation, and characteristic facies 3 (IHPRF3). The molecular mechanisms underlying its neuronal phenotype are largely unexplored. In this study, we reported two sisters, who harbored biallelic variants in TBCK and met diagnostic criteria for IHPRF3. We provided evidence that TBCK may play an important role in the early secretory pathway in neuroprogenitor cells (iNPC) differentiated from induced pluripotent stem cells (iPSC). Lack of functional TBCK protein in iNPC is associated with impaired endoplasmic reticulum-to-Golgi vesicle transport and autophagosome biogenesis, as well as altered cell cycle progression and severe impairment in the capacity of migration. Alteration in these processes, which are crucial for neurogenesis, neuronal migration, and cytoarchitecture organization, may represent an important causative mechanism of both neurodevelopmental and neurodegenerative phenotypes observed in IHPRF3. Whether reduced mechanistic target of rapamycin (mTOR) signaling is secondary to impaired TBCK function over other secretory transport regulators still needs further investigation.

Stem Cell Studies in Cardiovascular Biology and Medicine: A Possible Key Role of Macrophages

Stem cells are used in cardiovascular biology and biomedicine, and research in this field is expanding. Two types of stem cells have been used in research: induced pluripotent and somatic stem cells. Stem cell research in cardiovascular medicine has developed rapidly following the discovery of different types of stem cells. Induced pluripotent stem cells (iPSCs) possess potent differentiation ability, unlike somatic stem cells, and have been postulated for a long time. However, differentiating into adult-type mature and functional cardiac myocytes (CMs) remains difficult. Bone marrow stem/stromal cells (BMSCs), adipose-derived stem cells (ASCs), and cardiac stem cells (CSCs) are somatic stem cells used for cardiac regeneration. Among somatic stem cells, bone marrow stem/stromal cells (BMSCs) were the first to be discovered and are relatively well-characterized. BMSCs were once thought to have differentiation ability in infarcted areas of the heart, but it has been identified that paracrine cytokines and micro-RNAs derived from BMSCs contributed to that effect. Moreover, vesicles and exosomes from these cells have similar effects and are effective in cardiac repair. The molecular signature of exosomes can also be used for diagnostics because exosomes have the characteristics of their origin cells. Cardiac stem cells (CSCs) differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells, and supply cardiomyocytes during myocardial infarction by differentiating into newly formed cardiomyocytes. Stem cell niches and inflammatory cells play important roles in stem cell regulation and the recovery of damaged tissues. In particular, chemokines can contribute to the communication between inflammatory cells and stem cells. In this review, we present the current status of this exciting and promising research field.

Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells

The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (mTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage mT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus

The reprogramming of cells into induced neural stem cells (iNSCs), which are faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for fundamental and frontier research, as well as personalized cell-based therapies for neurological diseases. However, reprogramming cells with viral vectors increases the risk of tumor development due to vector and transgene integration in the host cell genome. To circumvent this issue, the Sendai virus (SeV) provides an alternative integration-free reprogramming method that removes the danger of genetic alterations and enhances the prospects of iNSCs from bench to bedside. Since pigs are among the most successful large animal models in biomedical research, porcine iNSCs (piNSCs) may serve as a disease model for both veterinary and human medicine. Here, we report the successful generation of piNSC lines from pig fibroblasts by employing the SeV. These piNSCs can be expanded for up to 40 passages in a monolayer culture and produce neurospheres in a suspension culture. These piNSCs express high levels of NSC markers (PAX6, SOX2, NESTIN, and VIMENTIN) and proliferation markers (KI67) using quantitative immunostaining and western blot analysis. Furthermore, piNSCs are multipotent, as they are capable of producing neurons and glia, as demonstrated by their expressions of TUJ1, MAP2, TH, MBP, and GFAP proteins. During the reprogramming of piNSCs with the SeV, no induced pluripotent stem cells developed, and the established piNSCs did not express OCT4, NANOG, and SSEA1. Hence, the use of the SeV can reprogram porcine somatic cells without first going through an intermediate pluripotent state. Our research produced piNSCs using SeV methods in novel, easily accessible large animal cell culture models for evaluating the efficacy of iNSC-based clinical translation in human medicine. Additionally, our piNSCs are potentially applicable in disease modeling in pigs and regenerative therapies in veterinary medicine.