Single-cell epigenomic reconstruction of developmental trajectories from pluripotency in human neural organoid systems.

Cell fate progression of pluripotent progenitors is strictly regulated, resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study, we use human brain and retina organoid models and present single-cell profiling of H3K27ac, H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage, providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction, resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination.

Efficient Vascular and Neural Engraftment of Stem Cell-Derived Islets.

Pluripotent stem cell-derived islets (SC-islets) have emerged as a new source for ß-cell replacement therapy. The function of human islet transplants is hampered by excessive cell death posttransplantation; contributing factors include inflammatory reactions, insufficient revascularization, and islet amyloid formation. However, there is a gap in knowledge of the engraftment process of SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at 3 months posttransplantation and observed that cell apoptosis rates were lower but vascular density was similar in SC-islets compared with human islets. Whereas the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. Oxygenation in the SC-islet grafts was twice as high as that in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, reinnervation of the SC-islets was four- to fivefold higher than that of the human islets. Both SC-islets and human islets contained amyloid at 1 and 3 months posttransplantation. We conclude that the vascular and neural engraftment of SC-islets are superior to those of human islets, but grafts of both origins develop amyloid, with potential long-term consequences.

Regenerative capacity of human pluripotent stem cell-derived articular chondrocytes in vitro.

The ideal cell source for articular cartilage repair remains elusive. Using developmentally inspired differentiation protocols, we induced human pluripotent stem cells (hPSCs) toward articular chondrocytes capable of joint cartilage repair in rodent models, which were distinct from growth plate chondrocytes, fated to be replaced by bone in vivo. Working toward clinical translation, we demonstrated controlled differentiation into chondrocytes by comprehensive gene expression analysis at each step of the differentiation. Articular chondrocytes derived from hPSCs could be expanded several passages in vitro without losing chondrogenic potential. Furthermore, chondrocytes isolated from these articular cartilage tissues had the potential to serially regenerate new articular and growth plate cartilage tissues. Finally, the ability to cryopreserve articular chondrocytes with the desired phenotype is critical for clinical translation and here we report no loss in cell viability or regenerative potential following cryopreservation. These results support the immense potential of hPSC-derived articular chondrocytes as a cell-based therapy for cartilage repair.

Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

INTRODUCTION: Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCM assay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). METHODS: Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. RESULTS: Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). CONCLUSION: These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Reproducible Differentiation of Human Pluripotent Stem Cells into Two-Dimensional Cortical Neuron Cultures with Checkpoints for Success.

The patterning of excitatory cortical neurons from human pluripotent stem cells (hPSCs) is a desired technique for the study of neurodevelopmental disorders, as neurons can be created and compared from control hPSC lines, hPSC lines generated from patients, and CRISPR-modified hPSC lines. Therefore, this technique allows for the examination of disease phenotypes and assists in the development of potential new therapeutics for neurodevelopmental disorders. Many protocols, however, are optimized for use with specific hPSC lines or within a single laboratory, and they often provide insufficient guidance on how to identify positive stages in the differentiation or how to troubleshoot. Here, we present an efficient and reproducible directed differentiation protocol to generate two-dimensional cultures of hPSC-derived excitatory cortical neurons without intermediary embryoid body formation. This novel protocol is supported by our data generated with five independent hPSC lines and in two independent laboratories. Importantly, as neuronal differentiations follow a long time course to reach maturity, we provide extensive guidance regarding morphological and flow cytometry checkpoints allowing for early indications of successful differentiation. We also include extensive troubleshooting tips and support protocols to assist the operator. The goal of this protocol is to assist others in the successful differentiation of excitatory cortical neurons from hPSCs. © 2023 Wiley Periodicals LLC. Basic Protocol: Directed differentiation of hPSCs into excitatory cortical neurons Support Protocol 1: Harvesting and fixing cells for flow cytometry analyses Support Protocol 2: Performing flow cytometry analyses Support Protocol 3: Thawing NPCs from a cryopreserved stock Alternate Protocol 1: Continuing Expansion of NPCs Alternate Protocol 2: Treatment of neurons with Ara-C to ablate radial glia Support Protocol 4: Experimental methods for validation of excitatory cortical neurons.

Neural Cells for Neurodegenerative Diseases in Clinical Trials.

Neurodegenerative diseases (ND) are an entire spectrum of clinical conditions that affect the central and peripheral nervous system. There is no cure currently, with treatment focusing mainly on slowing down progression or symptomatic relief. Cellular therapies with various cell types from different sources are being conducted as clinical trials for several ND diseases. They include neural, mesenchymal and hemopoietic stem cells, and neural cells derived from embryonic stem cells and induced pluripotent stem cells. In this review, we present the list of cellular therapies for ND comprising 33 trials that used neural stem progenitors, 8 that used differentiated neural cells ,and 109 trials that involved non-neural cells in the 7 ND. Encouraging results have been shown in a few early-phase clinical trials that require further investigations in a randomized setting. However, such definitive trials may not be possible given the relative cost of the trials, and in the setting of rare diseases.

Context matters: hPSC-derived microglia thrive in a humanized brain environment in vivo.

It remains challenging to create a physiologically relevant human-brain-like environment that would support maturation of human pluripotent stem cell (hPSC)-derived microglia (hMGs). Schafer et al.1 (Cell, 2023) now develop an in vivo neuroimmune organoid model with mature homeostatic hMGs for the study of brain development and disease.

Transplantation Strategies to Enhance Maturity and Cellular Complexity in Brain Organoids.

Human brain organoids are 3-dimensional cell aggregates that are generated from pluripotent stem cells and recapitulate features of the early developing human brain. Brain organoids mainly consist of cells from the neural lineage, such as neural progenitor cells, neurons, and astrocytes. However, current brain organoid systems lack functional vasculature as well as other non-neuronal cells that are indispensable for oxygen and nutrient supply to the organoids, causing cell stress and formation of a necrotic center. Attempts to utilize intracerebral transplantation approaches have demonstrated successful vascularization of brain organoids and robust neurodifferentiation. In this review, we summarize recent progress and discuss ethical considerations in the field of brain organoid transplantation.

Structural and functional integration of human forebrain organoids with the injured adult rat visual system.

Brain organoids created from human pluripotent stem cells represent a promising approach for brain repair. They acquire many structural features of the brain and raise the possibility of patient-matched repair. Whether these entities can integrate with host brain networks in the context of the injured adult mammalian brain is not well established. Here, we provide structural and functional evidence that human brain organoids successfully integrate with the adult rat visual system after transplantation into large injury cavities in the visual cortex. Virus-based trans-synaptic tracing reveals a polysynaptic pathway between organoid neurons and the host retina and reciprocal connectivity between the graft and other regions of the visual system. Visual stimulation of host animals elicits responses in organoid neurons, including orientation selectivity. These results demonstrate the ability of human brain organoids to adopt sophisticated function after insertion into large injury cavities, suggesting a translational strategy to restore function after cortical damage.

Embryonic origins of adult pluripotent stem cells.

Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.

Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells.

Precise gene editing in human pluripotent stem cells (hPSCs) holds great promise for studying and potentially treating human diseases. Both prime editing and base editing avoid introducing double strand breaks, but low editing efficiencies make those techniques still an arduous process in hPSCs. Here we report that co-delivering of p53DD, a dominant negative fragment of p53, can greatly enhance prime editing and cytosine base editing efficiencies in generating precise mutations in hPSCs. We further apply PE3 in combination with p53DD to efficiently create multiple isogenic hPSC lines, including lines carrying GBA or LRRK2 mutations associated with Parkinson disease and a LMNA mutation linked to Hutchinson-Gilford progeria syndrome. We also correct GBA and LMNA mutations in the patient-specific iPSCs. Our data show that p53DD improves PE3 efficiency without compromising the genome-wide safety, making it feasible for safe and routine generation of isogenic hPSC lines for disease modeling.

Contractile Force of Transplanted Cardiomyocytes Actively Supports Heart Function After Injury.

BACKGROUND: Transplantation of pluripotent stem cell-derived cardiomyocytes represents a promising therapeutic strategy for cardiac regeneration, and the first clinical studies in patients with heart failure have commenced. Yet, little is known about the mechanism of action underlying graft-induced benefits. Here, we explored whether transplanted cardiomyocytes actively contribute to heart function. METHODS: We injected cardiomyocytes with an optogenetic off-on switch in a guinea pig cardiac injury model. RESULTS: Light-induced inhibition of engrafted cardiomyocyte contractility resulted in a rapid decrease of left ventricular function in ≈50% (7/13) animals that was fully reversible with the offset of photostimulation. CONCLUSIONS: Our optogenetic approach demonstrates that transplanted cardiomyocytes can actively participate in heart function, supporting the hypothesis that the delivery of new force-generating myocardium can serve as a regenerative therapeutic strategy.

Human organoids in basic research and clinical applications.

Organoids are three-dimensional (3D) miniature structures cultured in vitro produced from either human pluripotent stem cells (hPSCs) or adult stem cells (AdSCs) derived from healthy individuals or patients that recapitulate the cellular heterogeneity, structure, and functions of human organs. The advent of human 3D organoid systems is now possible to allow remarkably detailed observation of stem cell morphogens, maintenance and differentiation resemble primary tissues, enhancing the potential to study both human physiology and developmental stage. As they are similar to their original organs and carry human genetic information, organoids derived from patient hold great promise for biomedical research and preclinical drug testing and is currently used for personalized, regenerative medicine, gene repair and transplantation therapy. In recent decades, researchers have succeeded in generating various types of organoids mimicking in vivo organs. Herein, we provide an update on current in vitro differentiation technologies of brain, retinal, kidney, liver, lung, gastrointestinal, cardiac, vascularized and multi-lineage organoids, discuss the differences between PSC- and AdSC-derived organoids, summarize the potential applications of stem cell-derived organoids systems in the laboratory and clinic, and outline the current challenges for the application of organoids, which would deepen the understanding of mechanisms of human development and enhance further utility of organoids in basic research and clinical studies.

From engineered heart tissue to cardiac organoid.

The advent of human pluripotent stem cells (hPSCs) presented a new paradigm to employ hPSC-derived cardiomyocytes (hPSC-CMs) in drug screening and disease modeling. However, hPSC-CMs differentiated in conventional two-dimensional systems are structurally and functionally immature. Moreover, these differentiation systems generate predominantly one type of cell. Since the heart includes not only CMs but other cell types, such monolayer cultures have limitations in simulating the native heart. Accordingly, three-dimensional (3D) cardiac tissues have been developed as a better platform by including various cardiac cell types and extracellular matrices. Two advances were made for 3D cardiac tissue generation. One type is engineered heart tissues (EHTs), which are constructed by 3D cell culture of cardiac cells using an engineering technology. This system provides a convenient real-time analysis of cardiac function, as well as a precise control of the input/output flow and mechanical/electrical stimulation. The other type is cardiac organoids, which are formed through self-organization of differentiating cardiac lineage cells from hPSCs. While mature cardiac organoids are more desirable, at present only primitive forms of organoids are available. In this review, we discuss various models of hEHTs and cardiac organoids emulating the human heart, focusing on their unique features, utility, and limitations.

Artificial Oocyte: Development and Potential Application.

Millions of people around the world suffer from infertility, with the number of infertile couples and individuals increasing every year. Assisted reproductive technologies (ART) have been widely developed in recent years; however, some patients are unable to benefit from these technologies due to their lack of functional germ cells. Therefore, the development of alternative methods seems necessary. One of these methods is to create artificial oocytes. Oocytes can be generated in vitro from the ovary, fetal gonad, germline stem cells (GSCs), ovarian stem cells, or pluripotent stem cells (PSCs). This approach has raised new hopes in both basic research and medical applications. In this article, we looked at the principle of oocyte development, the landmark studies that enhanced our understanding of the cellular and molecular mechanisms that govern oogenesis in vivo, as well as the mechanisms underlying in vitro generation of functional oocytes from different sources of mouse and human stem cells. In addition, we introduced next-generation ART using somatic cells with artificial oocytes. Finally, we provided an overview of the reproductive application of in vitro oogenesis and its use in human fertility.

Perspectives for Future Use of Cardiac Microtissues from Human Pluripotent Stem Cells.

Cardiovascular disorders remain a critical health issue worldwide. While animals have been used extensively as experimental models to investigate heart disease mechanisms and develop drugs, their inherent drawbacks have shifted focus to more human-relevant alternatives. Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs, collectively called hPSCs) have been identified as a source of different cardiac cells, but to date, they have rarely offered functional and structural maturity of the adult human heart. However, the combination of patient derived hPSCs with microphysiological tissue engineering approaches has presented new opportunities to study heart development and disease and identify drug targets. These models often closely mimic specific aspects of the native heart tissue including intercellular crosstalk and microenvironmental cues such that maturation occurs and relevant disease phenotypes are revealed. Most recently, organ-on-chip technology based on microfluidic devices has been combined with stem cell derived organoids and microtissues to create vascularized structures that can be subjected to fluidic flow and to which immune cells can be added to mimic inflammation of tissue postinjury. Similarly, the integration of nerve cells in these models can provide insight into how the cardiac nervous system affects heart pathology, for example, after myocardial infarction. Here, we consider these models and approaches in the context of cardiovascular disease together with their applications and readouts. We reflect on perspectives for their future implementation in understanding disease mechanisms and the drug discovery pipeline.

Generation of the organotypic kidney structure by integrating pluripotent stem cell-derived renal stroma.

Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions.

Kinase inhibitor-induced cardiotoxicity assessed in vitro with human pluripotent stem cell derived cardiomyocytes.

Many small molecule kinase inhibitors (SMKIs), used predominantly in cancer therapy, have been implicated in serious clinical cardiac adverse events, which means that traditional preclinical drug development assays were not sufficient for identifying these cardiac liabilities. To improve clinical cardiac safety predictions, the effects of SMKIs targeting many different signaling pathways were studied using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) in combined assays designed for the detection of both electrophysiological (proarrhythmic) and non-electrophysiological (non-proarrhythmic) drug-induced cardiotoxicity. Several microplate-based assays were used to quantitate cell death, apoptosis, mitochondrial damage, energy depletion, and oxidative stress as mechanism-based non-electrophysiological cardiomyocyte toxicities. Microelectrode arrays (MEA) were used to quantitate in vitro arrhythmic events (iAEs), field potential duration (FPD) prolongation, and spike amplitude suppression (SAS) as electrophysiological effects. To enhance the clinical relevance, SMKI-induced cardiotoxicities were compared by converting drug concentrations into multiples of reported clinical maximum therapeutic plasma concentration, "FoldCmax", for each assay. The results support the conclusion that the combination of the hPSC-CM based electrophysiological and non-electrophysiological assays have significantly more predictive value than either assay alone and significantly more than the current FDA-recommended hERG assay. In addition, the combination of these assays provided mechanistic information relevant to cardiomyocyte toxicities, thus providing valuable information on potential drug-induced cardiotoxicities early in drug development prior to animal and clinical testing. We believe that this early information will be helpful to guide the development of safer and more cost-effective drugs.

Recapitulating human myogenesis ex vivo using human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) provide a human model for developmental myogenesis, disease modeling and development of therapeutics. Differentiation of hPSCs into muscle stem cells has the potential to provide a cell-based therapy for many skeletal muscle wasting diseases. This review describes the current state of hPSCs towards recapitulating human myogenesis ex vivo, considerations of stem cell and progenitor cell state as well as function for future use of hPSC-derived muscle cells in regenerative medicine.