Search

With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy. [ABSTRACT FROM AUTHOR]

To accurately study human organ function and disease 'in the dish', it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver, which is the largest solid organ in the body. The liver is a multifunctional and highly regenerative organ; however, severe liver damage can have dire consequences for human health. A common cause of liver damage is adverse reactions to prescription drugs. Therefore, the development of predictive liver models that capture human drug metabolism patterns is required to optimise the drug development process. In our study, we aimed to identify compounds that could improve the metabolic function of stem cell-derived liver tissue. Therefore, we screened a compound library to identify additives that improved the maturity of in vitro-engineered human tissue, with the rationale that by taking such an approach, we would be able to fine-tune neonatal and adult cytochrome P450 metabolic function in stem cell-derived liver tissue. [ABSTRACT FROM AUTHOR]

Dystonia is a movement disorder with an estimated prevalence of 1.2% and is characterised by involuntary muscle contractions leading to abnormal postures and pain. Only symptomatic treatments are available with no disease-modifying or curative therapy, in large part due to the limited understanding of the underlying pathophysiology. However, the inherited monogenic forms of dystonia provide an opportunity for the development of disease models to examine these mechanisms. Myoclonus Dystonia, caused by SGCE mutations encoding the ε-sarcoglycan protein, represents one of now >50 monogenic forms. Previous research has implicated the involvement of the basal ganglia–cerebello-thalamo-cortical circuit in dystonia pathogenesis, but further work is needed to understand the specific molecular and cellular mechanisms. Pluripotent stem cell technology enables a patient-derived disease modelling platform harbouring disease-causing mutations. In this review, we discuss the current understanding of the aetiology of Myoclonus Dystonia, recent advances in producing distinct neuronal types from pluripotent stem cells, and their application in modelling Myoclonus Dystonia in vitro. Future research employing pluripotent stem cell-derived cellular models is crucial to elucidate how distinct neuronal types may contribute to dystonia and how disruption to neuronal function can give rise to dystonic disorders. [ABSTRACT FROM AUTHOR]

Recent advancements in vascular organoid (VO) and vessel-on-chip (VoC) technologies have revolutionized our approach to studying human diseases, offering unprecedented insights through more physiologically relevant models. VOs generated from human pluripotent stem cells exhibit remarkable self-organization capabilities, forming complex three-dimensional structures that closely mimic human blood vessel architecture and function, while VoCs are engineered with microfluidic systems that meticulously recreate the physical and functional attributes of blood vessels. These innovative constructs serve as powerful tools for investigating vascular development, disease progression, and therapeutic efficacy. By enabling the creation of patient-specific VOs and VoCs, they pave the way for personalized medicine approaches, allowing researchers to delve into genetic variations, intricate cellular interactions, and dynamic processes with exceptional resolution. The synergy between VOs and VoCs with newly developed cutting-edge technologies has further amplified their potential, unveiling novel mechanisms underlying human pathologies and identifying promising therapeutic targets. Herein, we summarize different types of VOs and VoCs and present an extensive overview on the generation and applications of VOs and VoCs. We will also highlight clinical and translational challenges and future perspectives around VOs and VoCs. [ABSTRACT FROM AUTHOR]

Recent advancements in vascular organoid (VO) and vessel-on-chip (VoC) technologies have revolutionized our approach to studying human diseases, offering unprecedented insights through more physiologically relevant models. VOs generated from human pluripotent stem cells exhibit remarkable self-organization capabilities, forming complex three-dimensional structures that closely mimic human blood vessel architecture and function, while VoCs are engineered with microfluidic systems that meticulously recreate the physical and functional attributes of blood vessels. These innovative constructs serve as powerful tools for investigating vascular development, disease progression, and therapeutic efficacy. By enabling the creation of patient-specific VOs and VoCs, they pave the way for personalized medicine approaches, allowing researchers to delve into genetic variations, intricate cellular interactions, and dynamic processes with exceptional resolution. The synergy between VOs and VoCs with newly developed cutting-edge technologies has further amplified their potential, unveiling novel mechanisms underlying human pathologies and identifying promising therapeutic targets. Herein, we summarize different types of VOs and VoCs and present an extensive overview on the generation and applications of VOs and VoCs. We will also highlight clinical and translational challenges and future perspectives around VOs and VoCs.

Symmetry breaking leading to axis formation and spatial patterning is crucial for achieving more accurate recapitulation of human development in organoids. While these processes can occur spontaneously by self-organizing capabilities of pluripotent stem cells, they can often result in variation in structure and composition of cell types within organoids. To address this limitation, bioengineering techniques that utilize geometric, topological and stiffness factors are increasingly employed to enhance control and consistency. Here, we review how spontaneous manners and engineering tools such as micropattern, microfluidics, biomaterials, etc. can facilitate the process of symmetry breaking leading to germ layer patterning and the formation of anteroposterior and dorsoventral axes in blastoids, gastruloids, neuruloids and neural organoids. Furthermore, brain assembloids, which are composed of multiple brain regions through fusion processes are discussed. The overview of organoid polarization in terms of patterning tools can offer valuable insights for enhancing the physiological relevance of organoid system.

BACKGROUND: The self-renewal and multi-directional differentiation of pluripotent stem cells possess the potential to revolutionize people’s understanding of biology, medicine, development, and disease. Stem cells play an important role in the early stage of embryonic development, and the study of them could be beneficial to understanding of the basic principles of biological development and tissue or organ formation, exploring the potential mechanisms of various diseases, studying the repair and regeneration of damaged tissues or organs, and promoting drug discovery and personalized treatment. OBJECTIVE: To review the research progress of pluripotent stem cells, summarize and categorize the fundamental types of pluripotent stem cells, and elucidate the lineage situations of various types of pluripotent stem cells in common mammals. METHODS: PubMed, Web of Science, CNKI, and WanFang databases were searched systematically, with the keywords “pluripotent stem cells; embryonic stem cells; induced pluripotent stem cells; expanded potential stem cells; livestock pluripotent stem cells” in English and Chinese. The 99 articles related to mammalian pluripotent stem cells were systematically screened according to inclusion and exclusion criteria, and then reviewed. RESULTS AND CONCLUSION: (1) According to classical theory in mouse embryonic stem cell research, the pluripotent state of stem cells is divided into two forms: naïve and primed. Naïve state corresponds to the inner cell mass of pre-implantation embryos before attachment to the uterine wall, while primed state corresponds to the epiblast after implantation. These two states exhibit significant differences in epigenetic features, transcriptional activity, external signal dependency, and metabolic phenotype. It is later discovered that there is an intermediate state between naïve and primed called formative pluripotency. Therefore, the pluripotency of pluripotent stem cells is a continuous developmental process rather than a unique cell state. (2) In addition to obtaining pluripotent stem cells from the inner cell mass, there are various methods and lineages for acquiring pluripotent stem cells, including embryonic germ cells established using primitive germ cells from mouse embryos, induced pluripotent stem cells created by the dedifferentiation of adult mouse and human fibroblasts with four factors-Oct3/4, Sox2, c-Myc, and Klf4; embryonic stem cell-like cell lines cultured from somatic cell nuclear transfer, parthenogenesis, neonatal or adult testicular or ovarian tissue, very small embryonic-like stem cells derived from various adult tissues and expanded pluripotent stem cells derived from pre-implantation stages. These pluripotent stem cells all share the common characteristics of continuous self-renewal, expressing core pluripotency factors and possessing the ability to differentiate into the three primary germ layers. (3) Currently, pluripotent stem cells are being used for disease modeling to study the mechanisms of various diseases and develop new drugs. Simultaneously, scientists are attempting to use pluripotent stem cells to cultivate various tissues and organs, offering new possibilities for regenerative medicine and transplantation. However, the clinical application of pluripotent stem cells faces safety challenges, including issues of cell mutations and immune rejection. Continual improvement in the methods of generating pluripotent stem cells will make them safer and more efficient for clinical applications. (4) Based on the methods of obtaining and lineage establishment of pluripotent stem cells in mice and humans, various types of pluripotent stem cells have been established in livestock, including embryonic stem cells, induced pluripotent stem cells, germ lineages of pluripotent stem cells, and expanded potential stem cells. Research on livestock pluripotent stem cells opens up new avenues for animal reproduction, breeding, genetic engineering, disease modeling, drug screening, and the conservation of endangered wildlife. [ABSTRACT FROM AUTHOR]

Many strands of research by different groups, starting from teratocarcinomas in the laboratory mouse, later moving the corresponding human tumors, contributed to the isolation and description of human pluripotent stem cells (PSCs). In this review, I highlight the contributions from my own research, particularly at the Wistar Institute during the 1980s, when with my colleagues we characterized one of the first clonal lines of pluripotent human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, and identified key features including cell surface antigen markers that have since found a place in the study and exploitation of human PSC. Much of this research depended upon close teamwork with colleagues, many in other laboratories, who contributed different expertise and experience. It was also often driven by circumstance and chance rather than pursuit of a grand design. [ABSTRACT FROM AUTHOR]

Allogeneic natural killer (NK) cell therapy has been effective in treating cancer. Many studies have tested NK cell therapy using human pluripotent stem cells (hPSCs). However, the impacts of the origin of PSC-NK cells on competence are unclear. In this study, several types of hPSCs, including human-induced PSCs (hiPSCs) generated from CD34+, CD3−CD56+, and CD56− cells in umbilical cord blood (UCB), three lines of human embryonic stem cells (hESCs, ES-1. ES-2 and ES-3) and MHC I knockout (B2M-KO)-ESCs were used to differentiate into NK cells and their capacities were analyzed. All PSC types could differentiate into NK cells. Among the iPSC-derived NK cells (iPSC-NKs) and ESC-derived NK cells (ES-NKs), 34+ iPSCs and ES-3 had a higher growth rate and cytotoxicity, respectively, ES-3 also showed better efficacy than 34+ iPSCs. B2M-KO was similar to the wild type. These results suggest that the screening for differentiation of PSCs into NK cells prior to selecting the PSC lines for the development of NK cell immunotherapy is an essential process for universal allotransplantation, including the chimeric antigen receptor (CAR). [ABSTRACT FROM AUTHOR]

Today, human pluripotent stem cell technologies find widespread application across biomedical research, as models for early human development, as platforms for functional human genomics, as tools for the study of disease, drug screening and toxicology, and as a renewable source of cellular therapeutics for a range of intractable diseases. The foundations of this human pluripotent stem cell revolution rest on advances in a wide range of disciplines, including cancer biology, assisted reproduction, cell culture and organoid technology, somatic cell nuclear transfer, primate embryology, single‐cell biology, and gene editing. This review surveys the slow emergence of the study of human pluripotency and the exponential growth of the field during the past several decades. [ABSTRACT FROM AUTHOR]

BACKGROUND: Myocardial patches are used as an effective way to repair damaged myocardium, and there is controversy over which cells to use to make myocardial patches and how to maximize the therapeutic effect of myocardial patches in vivo. OBJECTIVE: To find out the best way to make myocardial patches by overviewing the cellular sources of myocardial patches and strategies for perfecting them. METHODS: The first author searched PubMed and Web of Science databases by using “cell sheet, cell patch, cardiomyocytes, cardiac progenitor cells, fibroblasts, embryonic stem cell, mesenchymal stem cells” as English search terms, and searched CNKI and Wanfang databases by using “myocardial patch, biological 3D printing, myocardial” as Chinese search terms. After enrollment screening, 94 articles were ultimately included in the result analysis. RESULTS AND CONCLUSION: (1) The cellular sources of myocardial patches are mainly divided into three categories: somatic cells, monoenergetic stem cells, and pluripotent stem cells, respectively. There are rich sources of cells for myocardial patches, but not all of them are suitable for making myocardial patches, e.g., myocardial patches made from fibroblasts and skeletal myoblasts carry a risk of arrhythmogenicity, and mesenchymal stem cells have a short in vivo duration of action and ethical concerns. With the discovery of induced multifunctional stem cells, a reliable source of cells for making myocardial patches is available. (2) There are two methods of making myocardial patches. One is using cell sheet technology. The other is using biological 3D printing technology. Cell sheet technology can preserve the extracellular matrix components intact and can maximally mimic the cell growth ring in vivo. However, it is still difficult to obtain myocardial patches with three-dimensional structure by cell sheet technology. Biologicasl 3D printing technology, however, can be used to obtain myocardial patches with three-dimensional structures through computerized personalized design. (3) The strategies for perfecting myocardial patches mainly include: making myocardial patches after co-cultivation of multiple cells, improving the ink formulation and scaffold composition in biological 3D printing technology, improving the therapeutic effect of myocardial patches, suppressing immune rejection after transplantation, and perfecting the differentiation and cultivation protocols of stem cells. (4) There is no optimal cell source or method for making myocardial patches, and myocardial patches obtained from a particular cell or technique alone often do not achieve the desired therapeutic effect. Therefore, researchers need to choose the appropriate strategy for making myocardial patches based on the desired therapeutic effect before making them. [ABSTRACT FROM AUTHOR]

Analysis of single cell transcriptomics (scRNA-seq) data is typically performed after subsetting to highly variable genes (HVGs). Here, we show that Entropy Sorting provides an alternative mathematical framework for feature selection. On synthetic datasets, continuous Entropy Sort Feature Weighting (cESFW) outperforms HVG selection in distinguishing cell-state-specific genes. We apply cESFW to six merged scRNA-seq datasets spanning human early embryo development. Without smoothing or augmenting the raw counts matrices, cESFW generates a high-resolution embedding displaying coherent developmental progression from eight-cell to postimplantation stages and delineating 15 distinct cell states. The embedding highlights sequential lineage decisions during blastocyst development, while unsupervised clustering identifies branch point populations obscured in previous analyses. The first branching region, where morula cells become specified for inner cell mass or trophectoderm, includes cells previously asserted to lack a developmental trajectory. We quantify the relatedness of different pluripotent stem cell cultures to distinct embryo cell types and identify marker genes of naïve and primed pluripotency. Finally, by revealing genes with dynamic lineage-specific expression, we provide markers for staging progression from morula to blastocyst. [ABSTRACT FROM AUTHOR]

The lung is regarded as having limited regenerative capacity, and there are few treatment options for refractory lung diseases, such as interstitial pneumonia. Lung transplantation is the final option available in some scenarios. Research in this area has been slow owing to the complex structure of the lung for efficient gas exchange between the alveolar spaces and capillaries as well as the difficulty in obtaining specimens from patients with progressive lung disease. However, basic research over the past decade in the field of mouse and human embryology using genetic lineage tracing techniques and stem cell biology using primary and pluripotent stem cell-derived alveolar organoids has begun to clarify the tissue response in various intractable lung diseases and the mechanisms underlying remodeling. Advancement in this area may expand potential therapeutic targets for alveolar regeneration, providing alternatives to lung transplantation, and contribute to the development of effective therapeutic methods that activate or repopulate stem cells in the lung. In this review, we cover research focused on alveolar epithelial cells and discuss methods expected to regenerate lungs that are damaged by diseases. [ABSTRACT FROM AUTHOR]

Despite growing descriptions of wild-type Huntingtin (wt-HTT) roles in both adult brain function and, more recently, development, several clinical trials are exploring HTT-lowering approaches that target both wt-HTT and the mutant isoform (mut-HTT) responsible for Huntington's disease (HD). This non-selective targeting is based on the autosomal dominant inheritance of HD, supporting the idea that mut-HTT exerts its harmful effects through a toxic gain-of-function or a dominant-negative mechanism. However, the precise amount of wt-HTT needed for healthy neurons in adults and during development remains unclear. In this study, we address this question by examining how wt-HTT loss affects human neuronal network formation, synaptic maturation, and homeostasis in vitro. Our findings establish a role of wt-HTT in the maturation of dendritic arborization and the acquisition of network-wide synchronized activity by human cortical neuronal networks modeled in vitro. Interestingly, the network synchronization defects only became apparent when more than two-thirds of the wt-HTT protein was depleted. Our study underscores the critical need to precisely understand wt-HTT role in neuronal health. It also emphasizes the potential risks of excessive wt-HTT loss associated with non-selective therapeutic approaches targeting both wt- and mut-HTT isoforms in HD patients.

Pluripotent stem cells (PSCs), characterized by self-renewal and capacity of differentiating into three germ layers, are the programmable building blocks of life. PSC-derived cells and multicellular systems, particularly organoids, exhibit great potential for regenerative medicine. However, this field is still in its infancy, partly due to limited strategies to robustly and precisely control stem cell behaviors, which are tightly regulated by inner gene regulatory networks in response to stimuli from the extracellular environment. Synthetic receptors and genetic circuits are powerful tools to customize the cellular sense-and-response process, suggesting their underlying roles in precise control of cell fate decision and function reconstruction. Herein, we review the progress and challenges needed to be overcome in the fields of PSC-based cell therapy and multicellular system generation, respectively. Furthermore, we summarize several well-established synthetic biology tools and their applications in PSC engineering. Finally, we highlight the challenges and perspectives of harnessing synthetic biology to PSC engineering for regenerative medicine.

Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance. [ABSTRACT FROM AUTHOR]

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor β (TGFβ) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFβ-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

Dystonia is a movement disorder with an estimated prevalence of 1.2% and is characterised by involuntary muscle contractions leading to abnormal postures and pain. Only symptomatic treatments are available with no disease-modifying or curative therapy, in large part due to the limited understanding of the underlying pathophysiology. However, the inherited monogenic forms of dystonia provide an opportunity for the development of disease models to examine these mechanisms. Myoclonus Dystonia, caused by SGCE mutations encoding the ε-sarcoglycan protein, represents one of now >50 monogenic forms. Previous research has implicated the involvement of the basal ganglia–cerebello-thalamo-cortical circuit in dystonia pathogenesis, but further work is needed to understand the specific molecular and cellular mechanisms. Pluripotent stem cell technology enables a patient-derived disease modelling platform harbouring disease-causing mutations. In this review, we discuss the current understanding of the aetiology of Myoclonus Dystonia, recent advances in producing distinct neuronal types from pluripotent stem cells, and their application in modelling Myoclonus Dystonia in vitro. Future research employing pluripotent stem cell-derived cellular models is crucial to elucidate how distinct neuronal types may contribute to dystonia and how disruption to neuronal function can give rise to dystonic disorders.

To accurately study human organ function and disease ‘in the dish’, it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver, which is the largest solid organ in the body. The liver is a multifunctional and highly regenerative organ; however, severe liver damage can have dire consequences for human health. A common cause of liver damage is adverse reactions to prescription drugs. Therefore, the development of predictive liver models that capture human drug metabolism patterns is required to optimise the drug development process. In our study, we aimed to identify compounds that could improve the metabolic function of stem cell-derived liver tissue. Therefore, we screened a compound library to identify additives that improved the maturity of in vitro-engineered human tissue, with the rationale that by taking such an approach, we would be able to fine-tune neonatal and adult cytochrome P450 metabolic function in stem cell-derived liver tissue.

Acute Respiratory Distress Syndrome (ARDS) is a sudden onset of lung injury characterized by bilateral pulmonary edema, diffuse inflammation, hypoxemia, and a low P/F ratio. Epithelial injury and endothelial injury are notable in the development of ARDS, which is more severe under mechanical stress. This review explains the role of alveolar epithelial cells and endothelial cells under physiological and pathological conditions during the progression of ARDS. Mechanical injury not only causes ARDS but is also a side effect of ventilator-supporting treatment, which is difficult to model both in vitro and in vivo. The development of lung organoids has seen rapid progress in recent years, with numerous promising achievements made. Multiple types of cells and construction strategies are emerging in the lung organoid culture system. Additionally, the lung-on-a-chip system presents a new idea for simulating lung diseases. This review summarizes the basic features and critical problems in the research on ARDS, as well as the progress in lung organoids, particularly in the rapidly developing microfluidic system-based organoids. Overall, this review provides valuable insights into the three major factors that promote the progression of ARDS and how advances in lung organoid technology can be used to further understand ARDS. [ABSTRACT FROM AUTHOR]

Recent studies have reported the differentiation of pluripotent cells into oocytes in vitro. However, the developmental competence of in vitro‐generated oocytes remains low. Here, we perform a comprehensive comparison of mouse germ cell development in vitro over all culture steps versus in vivo with the goal to understand mechanisms underlying poor oocyte quality. We show that the in vitro differentiation of primordial germ cells to growing oocytes and subsequent follicle growth is critical for competence for preimplantation development. Systematic transcriptome analysis of single oocytes that were subjected to different culture steps identifies genes that are normally upregulated during oocyte growth to be susceptible for misregulation during in vitro oogenesis. Many misregulated genes are Polycomb targets. Deregulation of Polycomb repression is therefore a key cause and the earliest defect known in in vitro oocyte differentiation. Conversely, structurally normal in vitro‐derived oocytes fail at zygotic genome activation and show abnormal acquisition of 5‐hydroxymethylcytosine on maternal chromosomes. Our data identify epigenetic regulation at an early stage of oogenesis limiting developmental competence and suggest opportunities for future improvements. Synopsis: Generation of female germ cells from pluripotent stem cells (PSCs) has extensive potential, but these oocytes often show low developmental competence. This study that reveals deregulation of Polycomb repression during the early oogenesis is a key cause of defects in in vitro‐generated mouse oocytes. Stage‐specific single‐cell transcriptomics and functional assessment of PSC‐derived oocytes provides a comprehensive resource for comparisons to oogenesis in vivo.Culture steps for growth and differentiation of reconstituted follicles are critical for defining embryonic competence of in vitro‐generated oocytes.Failure of zygotic genome activation and epigenetic impairment are hallmarks of in vitro‐generated oocytes that fail to develop after fertilization or parthenogenetic activation.Computational analysis of gene expression and chromatin modification changes identifies Polycomb‐mediated repression as a key vulnerability during in vitro oocyte development. [ABSTRACT FROM AUTHOR]

Regenerative medicine mainly relies on heterologous transplantation, often hindered by sample availability and ethical issues. Furthermore, patients are required to take immunosuppressive medications to prevent adverse side effects. Stem cell-derived 3D-organoid culture has provided new alternatives for transplantation and regenerative medicine. Scholars have combined organoids with tissue engineering technology to improve reproducibility, the accuracy of constitution and throughput, and genetic correction to achieve a more personalized therapy. Here, we review the available applications of organoids in regenerative medicine and the current challenges concerning this field. Graphical Abstract [ABSTRACT FROM AUTHOR]

Diabetes mellitus (D.M.) is a common metabolic disorder caused mainly by combining two primary factors, which are (1) defects in insulin production by the pancreatic β-cells and (2) responsiveness of insulin-sensitive tissues towards insulin. Despite the rapid advancement in medicine to suppress elevated blood glucose levels (hyperglycemia) and insulin resistance associated with this hazard, a demand has undoubtedly emerged to find more effective and curative dimensions in therapeutic approaches against D.M. The administration of diabetes treatment that emphasizes insulin production and sensitivity may result in unfavorable side effects, reduced adherence, and potential treatment ineffectiveness. Recent progressions in genome editing technologies, for instance, in zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR-Cas)-associated nucleases, have greatly influenced the gene editing technology from concepts to clinical practices. Improvements in genome editing technologies have also opened up the possibility to target and modify specific genome sequences in a cell directly. CRISPR/Cas9 has proven effective in utilizing ex vivo gene editing in embryonic stem cells and stem cells derived from patients. This application has facilitated the exploration of pancreatic beta-cell development and function. Furthermore, CRISPR/Cas9 enables the creation of innovative animal models for diabetes and assesses the effectiveness of different therapeutic strategies in treating the condition. We, therefore, present a critical review of the therapeutic approaches of the genome editing tool CRISPR-Cas9 in treating D.M., discussing the challenges and limitations of implementing this technology. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells (hPSCs) can self-renew indefinitely or can be induced to differentiate. We previously showed that exogenous glutamine (Gln) withdrawal biased hPSC differentiation toward ectoderm and away from mesoderm. We revealed that, although all three germ lineages are capable of de novo Gln synthesis, only ectoderm generates sufficient Gln to sustain cell viability and differentiation, and this finding clarifies lineage fate restrictions under Gln withdrawal. Furthermore, we found that Gln acts as a signaling molecule for ectoderm that supersedes lineage-specifying cytokine induction. In contrast, Gln in mesoderm and endoderm is the preferred precursor of α-ketoglutarate without a direct signaling role. Our work raises a question about whether the nutrient environment functions directly in cell differentiation during development. Interestingly, transcriptome analysis of a gastrulation-stage human embryo shows that unique Gln enzyme-encoding gene expression patterns may also distinguish germ lineages in vivo. Together, our study suggests that intracellular Gln may help coordinate differentiation of the three germ layers.

Abstract Background Human mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype. Methods hiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer. Results Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential. Conclusion Overall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.

Allogeneic natural killer (NK) cell therapy has been effective in treating cancer. Many studies have tested NK cell therapy using human pluripotent stem cells (hPSCs). However, the impacts of the origin of PSC-NK cells on competence are unclear. In this study, several types of hPSCs, including human-induced PSCs (hiPSCs) generated from CD34+, CD3−CD56+, and CD56− cells in umbilical cord blood (UCB), three lines of human embryonic stem cells (hESCs, ES-1. ES-2 and ES-3) and MHC I knockout (B2M-KO)-ESCs were used to differentiate into NK cells and their capacities were analyzed. All PSC types could differentiate into NK cells. Among the iPSC-derived NK cells (iPSC-NKs) and ESC-derived NK cells (ES-NKs), 34+ iPSCs and ES-3 had a higher growth rate and cytotoxicity, respectively, ES-3 also showed better efficacy than 34+ iPSCs. B2M-KO was similar to the wild type. These results suggest that the screening for differentiation of PSCs into NK cells prior to selecting the PSC lines for the development of NK cell immunotherapy is an essential process for universal allotransplantation, including the chimeric antigen receptor (CAR).

Pluripotent stem cells model certain features of early mammalian development ex vivo. Medium-supplied nutrients can influence self-renewal, lineage specification, and earliest differentiation of pluripotent stem cells. However, which specific nutrients support these distinct outcomes, and their mechanisms of action, remain under active investigation. Here, we evaluate the available data on nutrients and their metabolic conversion that influence pluripotent stem cell fates. We also discuss key questions open for investigation in this rapidly expanding area of increasing fundamental and practical importance.

The specification of inhibitory neurons has been described for the mouse and human brain, and many studies have shown that pluripotent stem cells (PSCs) can be used to create interneurons in vitro. It is unclear whether in vitro methods to produce human interneurons generate all the subtypes found in brain, and how similar in vitro and in vivo interneurons are. We applied single-nuclei and single-cell transcriptomics to model interneuron development from human cortex and interneurons derived from PSCs. We provide a direct comparison of various in vitro interneuron derivation methods to determine the homogeneity achieved. We find that PSC-derived interneurons capture stages of development prior to mid-gestation, and represent a minority of potential subtypes found in brain. Comparison with those found in fetal or adult brain highlighted decreased expression of synapse-related genes. These analyses highlight the potential to tailor the method of generation to drive formation of particular subtypes.

The transcription factor NKX2-5 is a highly conserved master regulator of heart development which is widely expressed in cardiac progenitors and cardiomyocytes. Fluorescent reporters of NKX2-5 that minimally perturb normal protein expression can enable the identification, quantification and isolation of NKX2-5-expressing cells in a normal physiological state. Here we report the generation of two new hESC lines with eGFP inserted upstream (5′) or downstream (3′) of NKX2-5, linked by a cleavable T2A peptide. These complementary reporters produce a robust fluorescent signal in cardiac cells and have wide utility particularly for research on developmental biology and disease modelling.

Background: Human mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype. Methods: hiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer. Results: Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential. Conclusion: Overall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations. [ABSTRACT FROM AUTHOR]

The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.

Abstract Preclinical models that can accurately predict the toxicity and efficacy of candidate drugs to human liver tissue are in urgent need. Human liver organoid (HLO) derived from human pluripotent stem cells offers a possible solution. Herein, we generated HLOs, and demonstrated the utility of these HLOs in modeling a diversity of phenotypes associated with drug-induced liver injury (DILI), including steatosis, fibrosis, and immune responses. Phenotypic changes in HLOs after treatment with tool compounds such as acetaminophen, fialuridine, methotrexate, or TAK-875 showed high concordance with human clinical data in drug safety testings. Moreover, HLOs were able to model liver fibrogenesis induced by TGFβ or LPS treatment. We further devised a high-content analysis system, and established a high-throughput anti-fibrosis drug screening system using HLOs. SD208 and Imatinib were identified that can significantly suppress fibrogenesis induced by TGFβ, LPS, or methotrexate. Taken together, our studies demonstrated the potential applications of HLOs in drug safety testing and anti-fibrotic drug screening.

Patient-derived autologous chimeric antigen receptor (CAR)-T cell therapy is a revolutionary breakthrough in immunotherapy and has made impressive progress in both preclinical and clinical studies. However, autologous CAR-T cells still have notable drawbacks in clinical manufacture, such as long production time, variable cell potency and possible manufacturing failures. Allogeneic CAR-T cell therapy is significantly superior to autologous CAR-T cell therapy in these aspects. The use of allogeneic CAR-T cell therapy may provide simplified manufacturing process and allow the creation of ‘off-theshelf’ products, facilitating the treatments of various types of tumors at less delivery time. Nevertheless, severe graft-versus-host disease (GvHD) or hostmediated allorejection may occur in the allogeneic setting, implying that addressing these two critical issues is urgent for the clinical application of allogeneic CAR-T cell therapy. In this review, we summarize the current approaches to overcome GvHD and host rejection, which empower allogeneic CAR-T cell therapy with a broader future. [ABSTRACT FROM AUTHOR]

Applying programmable nucleases in gene editing has greatly shaped current research in basic biology and clinical translation. Gene editing in human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), is highly relevant to clinical cell therapy and thus should be examined with particular caution. First, since all mutations in PSCs will be carried to all their progenies, off-target edits of editors will be amplified. Second, due to the hypersensitivity of PSCs to DNA damage, double-strand breaks (DSBs) made by gene editing could lead to low editing efficiency and the enrichment of cell populations with defective genomic safeguards. In this regard, DSB-independent gene editing tools, such as base editors and prime editors, are favored due to their nature to avoid these consequences. With more understanding of the microbial world, new systems, such as Cas-related nucleases, transposons, and recombinases, are also expanding the toolbox for gene editing. In this review, we discuss current applications of programmable nucleases in PSCs for gene editing, the efforts researchers have made to optimize these systems, as well as new tools that can be potentially employed for differentiation modeling and therapeutic applications. [ABSTRACT FROM AUTHOR]

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD. [ABSTRACT FROM AUTHOR]

Initiating from multipotent progenitors, the lineages extrapolated from hematopoietic stem cells are determined by transcription factors specific to each of them. The commitment factors assist in the differentiation of progenitor cells into terminally differentiated cells. B lymphocytes constitute a population of cells that expresses clonally diverse cell surface immunoglobulin (Ig) receptors specific to antigenic epitopes. B cells are a significant facet of the adaptive immune system. The secreted antibodies corresponding to the B cell recognize the antigens via the B cell receptor (BCR). Following antigen recognition, the B cell is activated and thereafter undergoes clonal expansion and proliferation to become memory B cells. The essence of'cellular reprogramming' has aided in reliably altering the cells to desired tissue type. The potential of reprogramming has been harnessed to decipher and find solutions for various genetically inherited diseases and degenerative disorders. B lymphocytes can be reprogrammed to their initial naive state from where they get differentiated into any lineage or cell type similar to a pluripotent stem cell which can be accomplished by the deletion of master regulators of the B cell lineage. B cells can be reprogrammed into pluripotent stem cells and also can undergo transdifferentiation at the midway of cell differentiation to other cell types. Mandated expression of C/EBP in specialized B cells corresponds to their fast and effective reprogramming into macrophages, reversing the cell fate of these lymphocytes and allowing them to differentiate freshly into other types of cells. The co-expression of C/EBPα and OKSM (Oct4, Sox2, Klf4, c-Myc) amplified the reprogramming efficiency of B lymphocytes. Various human somatic cells including the immune cells are compliant to reprogramming which paves a path for opportunities like autologous tissue grafts, blood transfusion, and cancer immunotherapy. The ability to reprogram B cells offers an unprecedented opportunity for developing a therapeutic approach for several human diseases. Here, we will focus on all the proteins and transcription factors responsible for the developmental commitment of B lymphocytes and how it is harnessed in various applications. [ABSTRACT FROM AUTHOR]

Objective: To outline progress in induced pluripotent stem cells (iPSCs) in generating iPSC-derived cardiovascular cell lineages, and cardiac tissue engineering. Materials and methods: A data search for original articles was conducted on PubMed, using keywords: "induced pluripotent stem cells" OR "iPSCs" AND "myocardial infarction" OR "heart failure" AND "cardiomyocyte" on December 18, 2021. Search results were filtered and selected by titles and abstract to get relevant articles, which contained data on iPSC-related tissue engineering for heart tissue repair after myocardial infarction. Data were grouped and synthesize as text. Results and Discussion: we got 35 articles that provide information on cell based therapy, iPSCs, advancement in creating efficient techniques for generating iPSC-derived cardiovascular cell lineages, and recent cardiac tissue engineering advances in myocardial infarction animal models. iPSCs can be derived from skin fibroblasts by expressing four transcription factors that were found to be critical for cellular reprogramming into inner cell mass derived ESC-like cells, namely Oct3/4, c-Myc, Sox2 and Klf4. Cardiomyocytes can be produced from iPSC using special medium and supplements. Tissue engineering can be made possible by 3D printing technique and showed promising results upon transplantation. Conclusion: Tissue engineering using patient-derived human iPSC-cardiomyocytes holds great potential in medicine because it might avoid the problem of possible immune rejection. [ABSTRACT FROM AUTHOR]

Stirred suspension culture is becoming a popular method for expanding human pluripotent stem cells (hPSCs). While stirring generates adequate fluid motions to lift the cells and facilitates mass transfers (of nutrients, dissolved gases, and metabolic wastes), excessive stirring could impose hydrodynamic forces deleterious for the growth of the cells. In this study, computational fluid dynamics (CFD) simulations were performed to first investigate hydrodynamic characteristics of fluid flows in a spinner flask, a common stirred suspension culture vessel used in laboratories. Flow patterns and distributions of shear stresses and the Kolmogorov length scales at varying impeller speeds were obtained. Comparison of the Kolmogorov length scales and sizes of hPSC aggregates, measured in the authors’ previous experimental study, showed a strong correlation between the two. In addition to the spinner flask which generated complex and transient turbulent flows, this study investigated a newly developed rotary wall vessel that had been designed to produce laminar, circular Couette flows in order to control shear stress. CFD simulations revealed significantly more uniform and homogeneous flows compared to those in the spinner flask, suggesting that the rotary wall vessel is a suitable culture vessel to investigate roles of shear stress on hPSCs in suspension.

Stirred suspension culture is becoming a popular method for expanding human pluripotent stem cells (hPSCs). While stirring generates adequate fluid motions to lift the cells and facilitates mass transfers (of nutrients, dissolved gases, and metabolic wastes), excessive stirring could impose hydrodynamic forces deleterious for the growth of the cells. In this study, computational fluid dynamics (CFD) simulations were performed to first investigate hydrodynamic characteristics of fluid flows in a spinner flask, a common stirred suspension culture vessel used in laboratories. Flow patterns and distributions of shear stresses and the Kolmogorov length scales at varying impeller speeds were obtained. Comparison of the Kolmogorov length scales and sizes of hPSC aggregates, measured in the authors’ previous experimental study, showed a strong correlation between the two. In addition to the spinner flask which generated complex and transient turbulent flows, this study investigated a newly developed rotary wall vessel that had been designed to produce laminar, circular Couette flows in order to control shear stress. CFD simulations revealed significantly more uniform and homogeneous flows compared to those in the spinner flask, suggesting that the rotary wall vessel is a suitable culture vessel to investigate roles of shear stress on hPSCs in suspension.

Abstract 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.