Pluripotent Stem Cell Biology Research Articles

Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs.

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

Heterotrimeric G proteins regulate planarian regeneration and behavior.

G protein-coupled receptors play broad roles in development and stem cell biology, but few roles for G protein-coupled receptor signaling in complex tissue regeneration have been uncovered. Planarian flatworms robustly regenerate all tissues and provide a model with which to explore potential functions for G protein-coupled receptor signaling in somatic regeneration and pluripotent stem cell biology. As a first step toward exploring G protein-coupled receptor function in planarians, we investigated downstream signal transducers that work with G protein-coupled receptors, called heterotrimeric G proteins. Here, we characterized the complete heterotrimeric G protein complement in Schmidtea mediterranea for the first time and found that 7 heterotrimeric G protein subunits promote regeneration. We further characterized 2 subunits critical for regeneration, Gαq1 and Gβ1-4a, finding that they promote the late phase of anterior polarity reestablishment, likely through anterior pole-produced Follistatin. Incidentally, we also found that 5 G protein subunits modulate planarian behavior. We further identified a putative serotonin receptor, gcr052, that we propose works with Gαs2 and Gβx2 in planarian locomotion, demonstrating the utility of our strategy for identifying relevant G protein-coupled receptors. Our work provides foundational insight into roles of heterotrimeric G proteins in planarian biology and serves as a useful springboard toward broadening our understanding of G protein-coupled receptor signaling in adult tissue regeneration.

IDH mutation and cancer stem cell.

Cancer stem cells (CSCs) are a small population of cells in human malignancies that resemble the biology of human pluripotent stem cells. CSCs are closely related to the critical hallmarks in human cancers, ranging from oncogenesis to disease progression, therapeutic resistance, and overall outcome. Mutations in isocitrate dehydrogenase (IDH) were recently identified as founder mutations for human cancers. An increasing amount of evidence indicates that IDH mutations are closely related to the establishment and maintenance of CSCs. Biosynthesis of oncometabolite, metabolic reprogramming, and epigenetic shifts establish distinctive molecular signatures in IDH-mutated CSCs. Additionally, IDH mutation and IDH-related pathways could be valuable molecular targets to impact the CSC components in human cancers and to improve the disease outcome.

Contribution of Human Pluripotent Stem Cell-Based Models to Drug Discovery for Neurological Disorders.

One of the major obstacles to the identification of therapeutic interventions for central nervous system disorders has been the difficulty in studying the step-by-step progression of diseases in neuronal networks that are amenable to drug screening. Recent advances in the field of human pluripotent stem cell (PSC) biology offers the capability to create patient-specific human neurons with defined clinical profiles using reprogramming technology, which provides unprecedented opportunities for both the investigation of pathogenic mechanisms of brain disorders and the discovery of novel therapeutic strategies via drug screening. Many examples not only of the creation of human pluripotent stem cells as models of monogenic neurological disorders, but also of more challenging cases of complex multifactorial disorders now exist. Here, we review the state-of-the art brain cell types obtainable from PSCs and amenable to compound-screening formats. We then provide examples illustrating how these models contribute to the definition of new molecular or functional targets for drug discovery and to the design of novel pharmacological approaches for rare genetic disorders, as well as frequent neurodegenerative diseases and psychiatric disorders.

Exploration of Alternative Splicing Events in Mesenchymal Stem Cells from Human Induced Pluripotent Stem Cells.

Although comparative genome-wide transcriptomic analysis has provided insight into the biology of human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), the distinct alternative splicing (AS) signatures of iMSCs remain elusive. Here, we performed Illumina RNA sequencing analysis to characterize AS events in iMSCs compared with tissue-derived MSCs. A total of 4586 differentially expressed genes (|FC| > 2) were identified between iMSCs and umbilical cord blood-derived MSCs (UCB-MSCs), including 2169 upregulated and 2417 downregulated genes. Of these, 164 differentially spliced events (BF > 20) in 112 genes were identified between iMSCs and UCB-MSCs. The predominant type of AS found in iMSCs was skipped exons (43.3%), followed by retained introns (19.5%), alternative 3′ (15.2%) and 5′ (12.8%) splice sites, and mutually exclusive exons (9.1%). Functional enrichment analysis showed that the differentially spliced genes (|FC| > 2 and BF > 20) were mainly enriched in functions associated with focal adhesion, extracellular exosomes, extracellular matrix organization, cell adhesion, and actin binding. Splice isoforms of selected genes including TRPT1, CNN2, and AP1G2, identified in sashimi plots, were further validated by RT-PCR analysis. This study provides valuable insight into the biology of iMSCs and the translation of mechanistic understanding of iMSCs into therapeutic applications.

Interview with 2020 Hooke medal winner Ian Chambers

ABSTRACT Ian Chambers studied biochemistry at the University of Strathclyde in Glasgow, UK. He then did his PhD in the laboratory of Paul Harrison at the Beatson Institute for Cancer Research, also in Glasgow. Ian studied the control of gene expression during the differentiation of erythroid precursor cells, discovering that the amino acid selenocysteine is encoded by UGA, which until then was thought to work only as a termination codon. Ian did his post-doctoral work on the regulation of the human immunodeficiency virus (HIV) with Paul Berg at Stanford University in California, USA. In 1991, he returned to Scotland to work on stem cell regulation with Austin Smith at the Centre for Genome Research (later the Institute for Stem Cell Research) at the University of Edinburgh, UK. During that time, Ian identified the transcription factor Nanog, which directs efficient embryonic stem cell self-renewal. Ian started his research group in 2006 at the University of Edinburgh, where he is also a Professor of Pluripotent Stem Cell Biology. His laboratory tries to understand the regulatory networks and transcription factors that control the identity of pluripotent embryonic stem cells, and how these modulate cell fate decisions during the differentiation process. Ian is now the Head of the Institute for Stem Cell Research at University of Edinburgh, an EMBO member and a Fellow of the Royal Society of Edinburgh. Ian is the recipient of the 2020 Hooke Medal from the British Society for Cell Biology (BSCB).

When the Search for Stemness Genes Meets the Skin Substitute Bioengineering Field: KLF4 Transcription Factor under the Light.

The transcription factor “Kruppel-like factor 4” (KLF4) is a central player in the field of pluripotent stem cell biology. In particular, it was put under the spotlight as one of the four factors of the cocktail originally described for reprogramming into induced pluripotent stem cells (iPSCs). In contrast, its possible functions in native tissue stem cells remain largely unexplored. We recently published that KLF4 is a regulator of “stemness” in human keratinocytes. We show that reducing the level of expression of this transcription factor by RNA interference or pharmacological repression promotes the ex vivo amplification and regenerative capacity of two types of cells of interest for cutaneous cell therapy: native keratinocyte stem and progenitor cells from adult epidermis, which have been used for more than three decades in skin graft bioengineering, and keratinocytes generated by the lineage-oriented differentiation of embryonic stem cells (ESCs), which have potential for the development of skin bio-bandages. At the mechanistic level, KLF4 repression alters the expression of a large set of genes involved in TGF-β1 and WNT signaling pathways. Major regulators of TGF-β bioavailability and different TGF-β receptors were targeted, notably modulating the ALK1/Smad1/5/9 axis. At a functional level, KLF4 repression produced an antagonist effect on TGF-β1-induced keratinocyte differentiation.

PKD1-Dependent Renal Cystogenesis in Human Induced Pluripotent Stem Cell-Derived Ureteric Bud/Collecting Duct Organoids.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease leading to renal failure, wherein multiple cysts form in renal tubules and collecting ducts derived from distinct precursors: the nephron progenitor and ureteric bud (UB), respectively. Recent progress in induced pluripotent stem cell (iPSC) biology has enabled cyst formation in nephron progenitor-derived human kidney organoids in which PKD1 or PKD2, the major causative genes for ADPKD, are deleted. However, cysts have not been generated in UB organoids, despite the prevalence of collecting duct cysts in patients with ADPKD. CRISPR-Cas9 technology deleted PKD1 in human iPSCs and the cells induced to differentiate along pathways leading to formation of either nephron progenitor or UB organoids. Cyst formation was investigated in both types of kidney organoid derived from PKD1-deleted iPSCs and in UB organoids generated from iPSCs from a patient with ADPKD who had a missense mutation. Cysts formed in UB organoids with homozygous PKD1 mutations upon cAMP stimulation and, to a lesser extent, in heterozygous mutant organoids. Furthermore, UB organoids generated from iPSCs from a patient with ADPKD who had a heterozygous missense mutation developed cysts upon cAMP stimulation. Cysts form in PKD1 mutant UB organoids as well as in iPSCs derived from a patient with ADPKD. The organoids provide a robust model of the genesis of ADPKD.

Perspective on human pluripotent stem cell-derived cardiomyocytes in heart disease modeling and repair.

Heart diseases (HDs) are the leading cause of morbidity and mortality worldwide. Despite remarkable clinical progress made, current therapies cannot restore the lost myocardium, and the correlation of genotype to phenotype of many HDs is poorly modeled. In the past two decades, with the rapid developments of human pluripotent stem cell (hPSC) biology and technology that allow the efficient preparation of cardiomyocytes from individual patients, tremendous efforts have been made for using hPSC‐derived cardiomyocytes in preclinical and clinical cardiac therapy as well as in dissection of HD mechanisms to develop new methods for disease prediction and treatment. However, their applications have been hampered by several obstacles. Here, we discuss recent advances, remaining challenges, and the potential solutions to advance this field.

Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.

Our knowledge of pluripotent stem cell (PSC) biology has advanced to the point where we now can generate most cells of the human body in the laboratory. PSC-derived cardiomyocytes can be generated routinely with high yield and purity for disease research and drug development, and these cells are now gradually entering the clinical research phase for the testing of heart regeneration therapies. However, a major hurdle for their applications is the immature state of these cardiomyocytes. In this Review, we describe the structural and functional properties of cardiomyocytes and present the current approaches to mature PSC-derived cardiomyocytes. To date, the greatest success in maturation of PSC-derived cardiomyocytes has been with transplantation into the heart in animal models and the engineering of 3D heart tissues withelectromechanical conditioning. In conventional 2D cell culture, biophysical stimuli such as mechanical loading, electrical stimulation and nanotopology cues all induce substantial maturation, particularly of the contractile cytoskeleton. Metabolism has emerged as a potent means to control maturation with unexpected effects on electrical and mechanical function. Different interventions induce distinct facets of maturation, suggesting that activating multiple signalling networks might lead to increased maturation. Despite considerable progress, we are still far from being able to generate PSC-derived cardiomyocytes with adult-like phenotypes in vitro. Future progress will come from identifying the developmental drivers of maturation and leveraging them to create more mature cardiomyocytes for research and regenerative medicine.

Generation of human colonic organoids from human pluripotent stem cells.

Advances in human pluripotent stem cell (hPSC) biology now allow the generation of organoids that resemble different regions of the gastrointestinal tract. Generation of region-specific organoids has been facilitated by developmental biology studies carried out in model organisms such as mouse, frog and chick. By mimicking embryonic development, hPSC-derived human colonic organoids (HCOs) can be generated through a stepwise differentiation, first into definitive endoderm (DE), then into mid/hindgut spheroids which are then patterned into posterior gut tissue which gives rise to HCOs following prolonged in vitro culture. HCOs undergo transitions similar to those observed in the developing colon of model organisms and human embryos. HCOs develop into tissue that resembles fetal colon on the basis of morphology, gene expression and presence of differentiated cell types. Generation of HCOs without the proper training or expertise can be a daunting task. Here, we describe a detailed protocol for differentiating hPSCs into HCOs, we include suggestions for troubleshooting these differentiations, and we discuss experimental design considerations. We have also highlighted the key advantages and limitations of the system.

Insights Into Human Development and Disease From Human Pluripotent Stem Cell Derived Intestinal Organoids

In recent years, advances in human pluripotent stem cell (hPSC) biology have enabled the generation of gastrointestinal (GI) organoids which recapitulate aspects of normal organ development. HPSC derived gastrointestinal organoids are comprised of epithelium and mesenchyme and have a remarkable ability to self-organize and recapitulate early stages of human intestinal development. Furthermore, hPSC derived organoids can be transplanted into immunocompromised mice which allows further maturation of both the epithelium and mesenchyme. In this review, we will briefly summarize work from model systems which has elucidated mechanisms of GI patterning and how these insights have been used to guide the differentiation of hPSCs into organoids resembling small intestine and colon. We will succinctly discuss how developmental principles have been used to promote maturation of human intestinal organoids (HIOs) in vitro as well as to introduce an enteric nervous system into HIOs. We will then concisely review how organoids have been used to study human pathogens, how new genetic and bioengineering tools are being applied to organoid research, and how this integration has allowed researchers to elucidate mechanisms of human development and disease. Finally, we will briefly discuss remaining challenges in the field and how they can be addressed. HPSC derived organoids are promising new model systems which hold the potential of unlocking unknown mechanisms of human gastrointestinal development and disease.

Induced Pluripotent Stem Cell (iPSC)-Derived Exosomes for Precision Medicine in Heart Failure.

In the United States, heart failure (HF) is the leading cause of hospital admission and a major public health problem.1 Ischemic cardiomyopathy is the primary pathogenesis of advanced HF.1 More than 5 million people experience HF, with an annual incidence of 300 000 patients.1 Despite significant therapeutic advances during the past 3 decades, the 5-year survival remains at a dismal 50%.2 Clinical studies have confirmed that the high morbidity and mortality of HF are because of left ventricular arrhythmias and dilatation.1,2 Patients with a history of acute myocardial infarction and left ventricular dysfunction have a 6-month mortality of >10% of which one third is attributed to sudden cardiac death.3 Although implantable cardioverter defibrillators reduced mortality from left ventricular arrhythmia, patients with acute myocardial infarction do not qualify under the current guideline. An important trigger for ventricular arrhythmias and dilatation is the tissue heterogeneity in the peri-infarct region (PIR), which is independent of the actual infarct size.4–7 An effective targeted therapy to salvage the injured and vulnerable myocardium in the PIR is urgently needed to reduce the high mortality of patients with HF.7,8 Promising discoveries of induced pluripotent stem cell (iPSC) biology may restore the PIR.9,10 The initial theory for the regeneration of the myocardium hypothesized that the transplanted stem cells would differentiate into cardiomyocytes, engraft into the host myocardium, and augment the cardiac function through synchronized electromechanical integration. However, de novo cardiac differentiation of transplanted stem cells was rare and mostly led to teratoma.9,10 Similarly, ex vivo differentiated cardiac stem cells demonstrate transient in vivo engraftment and functional restoration.9,10 Other controversial studies examined the cardiac progenitor cells, a multipotent population of cardiac, vascular, and endothelial cells in the …

The Pleiotropic Effects of the Canonical Wnt Pathway in Early Development and Pluripotency.

The technology to derive embryonic and induced pluripotent stem cells from early embryonic stages and adult somatic cells, respectively, emerged as a powerful resource to enable the establishment of new in vitro models, which recapitulate early developmental processes and disease. Additionally, pluripotent stem cells (PSCs) represent an invaluable source of relevant differentiated cell types with immense potential for regenerative medicine and cell replacement therapies. Pluripotent stem cells support self-renewal, potency and proliferation for extensive periods of culture in vitro. However, the core pathways that rule each of these cellular features specific to PSCs only recently began to be clarified. The Wnt signaling pathway is pivotal during early embryogenesis and is central for the induction and maintenance of the pluripotency of PSCs. Signaling by the Wnt family of ligands is conveyed intracellularly by the stabilization of β-catenin in the cytoplasm and in the nucleus, where it elicits the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors. Interestingly, in PSCs, the Wnt/β-catenin–TCF/LEF axis has several unrelated and sometimes opposite cellular functions such as self-renewal, stemness, lineage commitment and cell cycle regulation. In addition, tight control of the Wnt signaling pathway enhances reprogramming of somatic cells to induced pluripotency. Several recent research efforts emphasize the pleiotropic functions of the Wnt signaling pathway in the pluripotent state. Nonetheless, conflicting results and unanswered questions still linger. In this review, we will focus on the diverse functions of the canonical Wnt signaling pathway on the developmental processes preceding embryo implantation, as well as on its roles in pluripotent stem cell biology such as self-renewal and cell cycle regulation and somatic cell reprogramming.

CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects.

Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using “molecular scissors.” Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine.

Thyroid Progenitors Are Robustly Derived from Embryonic Stem Cells through Transient, Developmental Stage-Specific Overexpression of Nkx2-1.

SummaryThe clinical importance of anterior foregut endoderm (AFE) derivatives, such as thyrocytes, has led to intense research efforts for their derivation through directed differentiation of pluripotent stem cells (PSCs). Here, we identify transient overexpression of the transcription factor (TF) NKX2-1 as a powerful inductive signal for the robust derivation of thyrocyte-like cells from mouse PSC-derived AFE. This effect is highly developmental stage specific and dependent on FOXA2 expression levels and precise modulation of BMP and FGF signaling. The majority of the resulting cells express thyroid TFs (Nkx2-1, Pax8, Foxe1, Hhex) and thyroid hormone synthesis-related genes (Tg, Tpo, Nis, Iyd) at levels similar to adult mouse thyroid and give rise to functional follicle-like epithelial structures in Matrigel culture. Our findings demonstrate that NKX2-1 overexpression converts AFE to thyroid epithelium in a developmental time-sensitive manner and suggest a general methodology for manipulation of cell-fate decisions of developmental intermediates.

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.

Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.

Prospects of Pluripotent and Adult Stem Cells for Rare Diseases.

Rare diseases are highly diverse and complex regarding molecular underpinning and clinical manifestation and afflict millions of patients worldwide. The lack of appropriate model systems with face and construct validity and the limited availability of live tissues and cells from patients has largely hampered the understanding of underlying disease mechanisms. As a consequence, there are no adequate treatment options available for the vast majority of rare diseases. Over the last decade, remarkable progress in pluripotent and adult stem cell biology and the advent of powerful genomic technologies opened up exciting new avenues for the investigation, diagnosis, and personalized therapy of intractable human diseases. Utilizing the entire range of available stem cell types will continue to cross-fertilize different research areas and leverage the investigation of rare diseases based on evidence-based medicine. Standardized cell engineering and manufacturing from inexhaustible stem cell sources should lay the foundation for next-generation drug discovery and cell therapies that are broadly applicable in regenerative medicine. In this chapter we discuss how patient- and disease-specific iPS cells as well as adult stem cells are changing the pace of biomedical research and the translational landscape.

Recreating the Cardiac Microenvironment in Pluripotent Stem Cell Models of Human Physiology and Disease.

The advent of human pluripotent stem cell (hPSC) biology has opened unprecedented opportunities for the use of tissue engineering to generate human cardiac tissue for in vitro study. Engineering cardiac constructs that recapitulate human development and disease requires faithful recreation of the cardiac niche in vitro. Here we discuss recent progress in translating the in vivo cardiac microenvironment into PSC models of the human heart. We review three key physiologic features required to recreate the cardiac niche and facilitate normal cardiac differentiation and maturation: the biochemical, biophysical, and bioelectrical signaling cues. Finally, we discuss key barriers that must be overcome to fulfill the promise of stem cell biology in preclinical applications and ultimately in clinical practice.

Human iPS Cell-Derived Cardiac Tissue Sheets: a Platform for Cardiac Regeneration.

Opinion statementStem cell therapy is a promising therapeutic option for severe cardiac diseases that are resistant to conventional therapies. To overcome the unsatisfactory results of most clinical researches on stem cell injections to an injured heart, we are developing bioengineered cardiac tissue grafts using pluripotent stem cell-derived cardiomyocytes and vascular cells. We have validated the functional benefits of mouse embryonic stem cell-derived and human induced pluripotent stem cell-derived cardiac tissue sheets (CTSs) in a rat myocardial infarction model. We further showed enhanced functional recovery and engraftment efficiency leading to de novo myocardium upon transplanting thick multi-layered CTSs that had gelatin hydrogel microspheres between the layers. We anticipate that the combination of pluripotent stem cell biology and tissue engineering will contribute to future stem cell therapies for severe heart diseases.