ABSTRACT
Reprogramming of somatic cells achieved by combination of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has very low efficiency. To increase the reprogramming efficiency and better understand the process, we sought to identify factors that mediate reprogramming with higher efficiency. We established an assay to screen nuclear fractions from extracts of pluripotent mouse cells based on Oct4 reactivation. Using proteomics, we identified components of the ATP-dependent BAF chromatin-remodeling complex, which significantly increases reprogramming efficiency when used together with the four factors. The reprogrammed cells could transmit to the germline and exhibited pluripotency. Reprogramming remained highly efficient when c-Myc was not present but BAF components were overexpressed. BAF complex components mediate this effect by facilitating enhanced Oct4 binding to target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells.
Subject(s)
Cellular Reprogramming , Chromatin Assembly and Disassembly , Animals , Cell Line , Chromatin/metabolism , DNA Helicases/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Kruppel-Like Factor 4 , Mice , Nuclear Proteins/metabolism , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Transcription Factors/metabolismABSTRACT
Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.
Subject(s)
Germ Layers/cytology , Mice/embryology , Stem Cells/cytology , Animals , Female , Gene Expression Profiling , Male , Mice, 129 Strain , Mice, Inbred C57BL , Octamer Transcription Factor-3/analysis , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolismABSTRACT
Achieving pharmacological control over cardiomyocyte proliferation represents a prime goal in therapeutic cardiovascular research. Here, we identify a novel chemical tool compound for the expansion of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. The forkhead box O (FOXO) inhibitor AS1842856 was identified as a significant hit from an unbiased proliferation screen in early, immature hiPSC- cardiomyocytes (eCMs). The mitogenic effects of AS1842856 turned out to be robust, dose-dependent, sustained, and reversible. eCM numbers increased >30-fold as induced by AS1842856 over three passages. Phenotypically as well as by marker gene expression, the compound interestingly appeared to counteract cellular maturation both in immature hiPSC-CMs as well as in more advanced ones. Thus, FOXO inhibitor AS1842856 presents a novel proliferation inducer for the chemically defined, xeno-free expansion of hiPSC-derived CMs, while its de-differentiation effect might as well bear potential in regenerative medicine.
Subject(s)
Induced Pluripotent Stem Cells , Cell Differentiation , Cell Proliferation , Cells, Cultured , Extracellular Matrix , Humans , Induced Pluripotent Stem Cells/metabolism , Myocytes, CardiacABSTRACT
Human-induced pluripotent stem cells (h-iPSCs) are a unique in vitro model for cardiovascular research. To realize the potential applications of h-iPSCs-derived cardiomyocytes (CMs) for drug testing or regenerative medicine and disease modeling, characterization of the metabolic features is critical. Here, we show the transcriptional profile during stages of cardiomyogenesis of h-iPSCs-derived CMs. CM differentiation was not only characterized by the expression of mature structural components (MLC2v, MYH7) but also accompanied by a significant increase in mature metabolic gene expression and activity. Our data revealed a distinct substrate switch from glucose to fatty acids utilization for ATP production. Basal respiration and respiratory capacity in 9 days h-iPSCs-derived CMs were glycolysis-dependent with a shift towards a more oxidative metabolic phenotype at 14 and 28 day old CMs. Furthermore, mitochondrial analysis characterized the early and mature forms of mitochondria during cardiomyogenesis. These results suggest that changes in cellular metabolic phenotype are accompanied by increased O2 consumption and ATP synthesis to fulfill the metabolic needs of mature CMs activity. To further determine functionality, the physiological response of h-iPSCs-derived CMs to ß-adrenergic stimulation was tested. These data provide a unique in vitro human heart model for the understanding of CM physiology and metabolic function which may provide useful insight into metabolic diseases as well as novel therapeutic options.
Subject(s)
Cell Differentiation/physiology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Cells, Cultured , HumansABSTRACT
During vertebrate embryogenesis, vascular endothelial cells (ECs) and primitive erythrocytes become specified within close proximity in the posterior lateral plate mesoderm (LPM) from a common progenitor. However, the signaling cascades regulating the specification into either lineage remain largely elusive. Here, we analyze the contribution of ß-catenin dependent Wnt signaling to EC and erythrocyte specification during zebrafish embryogenesis. We generated novel ß-catenin dependent Wnt signaling reporters which, by using destabilized fluorophores (Venus-Pest, dGFP), specifically allow us to detect Wnt signaling responses in narrow time windows as well as in spatially restricted domains, defined by Cre recombinase expression (Tg(axin2BAC:Venus-Pest)mu288; Tg(14TCF:loxP-STOP-loxP-dGFP)mu202). We therefore can detect ß-catenin dependent Wnt signaling activity in a subset of the Fli1a-positive progenitor population. Additionally, we show that mesodermal Wnt3a-mediated signaling via the transcription factor Lef1 positively regulates EC specification (defined by kdrl expression) at the expense of primitive erythrocyte specification (defined by gata1 expression) in zebrafish embryos. Using mesoderm derived from human embryonic stem cells, we identified the same principle of Wnt signaling dependent EC specification in conjunction with auto-upregulation of LEF1. Our data indicate a novel role of ß-catenin dependent Wnt signaling in regulating EC specification during vasculogenesis.
Subject(s)
Cell Lineage , Endothelial Cells/cytology , Endothelial Cells/metabolism , Transcription Factors/metabolism , Wnt Signaling Pathway , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Cell Count , Cell Differentiation , Cell Line , Erythrocytes/cytology , Erythrocytes/metabolism , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Mesoderm/cytology , Mesoderm/metabolism , Models, Biological , Organogenesis , Somites/embryology , Somites/metabolism , Wnt3A Protein/metabolism , beta Catenin/metabolismABSTRACT
Human embryonic stem cells (hESCs) present a fascinating and powerful system for generating specialized cell types of the human body. Culture and directed differentiation of these cells however requires an understanding of the pluripotent ground state and of how cell lineage decisions in this system are made. In this review, we highlight both these aspects in light of recent findings and technical progress. Hence, advances in culturing the human preimplantation embryo beyond the implantation barrier and in analyzing it at the single-cell level shed new light on the hESC tissue of origin. We argue that these findings have important implications for our view of hESC identity and we critically discuss recent efforts in converting these cells to a more primitive state. With an emphasis on the roles played by major signaling pathways, we furthermore attempt to infer key principles underlying cell fate control in hESCs from recently published work. This integrated model combines defined signaling pathway manipulation with the regulation of core hESC genes, to aid in controlling cell lineage allocation in a rational manner. Stem Cells 2017;35:277-283.
Subject(s)
Cell Lineage , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Signal Transduction , Embryo, Mammalian/cytology , Humans , Models, BiologicalABSTRACT
The possibility to generate induced pluripotent stem cells (iPSC) opens the way to generate virtually all cell types of our human body. In combination with modern gene editing techniques like CRISPR/CAS, a new set of powerful tools becomes available for life science. Scientific fields like genotype and cell type-specific pharmacology, disease modeling, stem cell biology, and developmental biology have been dramatically fostered and their faces have been changed. However, as golden as the age of iPSC-derived cells and their manipulation has started, the shine begins to tarnish. Researchers face more and more practical problems intrinsic to the system. These problems are related to the specific culturing conditions which are not yet sufficient to mimic the natural environment of native stem cells differentiating towards adult cells. However, researchers work hard to uncover these factors. Here, we review a common standard approach to generate iPSCs and transduce these to iPSC cardiomyocytes. Further, we review recent achievements and discuss their current limitations and future perspectives. We are on track, but the road is still under construction.
Subject(s)
Cell Differentiation/genetics , Gene Editing , Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/metabolism , Animals , Cells, Cultured , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , HumansABSTRACT
Jervell and Lange-Nielsen syndrome (JLNS) is one of the most severe life-threatening cardiac arrhythmias. Patients display delayed cardiac repolarization, associated high risk of sudden death due to ventricular tachycardia, and congenital bilateral deafness. In contrast to the autosomal dominant forms of long QT syndrome, JLNS is a recessive trait, resulting from homozygous (or compound heterozygous) mutations in KCNQ1 or KCNE1. These genes encode the α and ß subunits, respectively, of the ion channel conducting the slow component of the delayed rectifier K(+) current, IKs. We used complementary approaches, reprogramming patient cells and genetic engineering, to generate human induced pluripotent stem cell (hiPSC) models of JLNS, covering splice site (c.478-2A>T) and missense (c.1781G>A) mutations, the two major classes of JLNS-causing defects in KCNQ1. Electrophysiological comparison of hiPSC-derived cardiomyocytes (CMs) from homozygous JLNS, heterozygous, and wild-type lines recapitulated the typical and severe features of JLNS, including pronounced action and field potential prolongation and severe reduction or absence of IKs. We show that this phenotype had distinct underlying molecular mechanisms in the two sets of cell lines: the previously unidentified c.478-2A>T mutation was amorphic and gave rise to a strictly recessive phenotype in JLNS-CMs, whereas the missense c.1781G>A lesion caused a gene dosage-dependent channel reduction at the cell membrane. Moreover, adrenergic stimulation caused action potential prolongation specifically in JLNS-CMs. Furthermore, sensitivity to proarrhythmic drugs was strongly enhanced in JLNS-CMs but could be pharmacologically corrected. Our data provide mechanistic insight into distinct classes of JLNS-causing mutations and demonstrate the potential of hiPSC-CMs in drug evaluation.
Subject(s)
Induced Pluripotent Stem Cells/physiology , Jervell-Lange Nielsen Syndrome/drug therapy , Jervell-Lange Nielsen Syndrome/genetics , Jervell-Lange Nielsen Syndrome/physiopathology , KCNQ1 Potassium Channel/genetics , Models, Biological , Phenotype , Action Potentials/physiology , Analysis of Variance , Base Sequence , Cell Line , Genes, Recessive/genetics , Genetic Engineering , Humans , In Vitro Techniques , KCNQ1 Potassium Channel/chemistry , Models, Molecular , Molecular Sequence Data , Mutation, Missense/genetics , Myocytes, Cardiac/physiology , Sequence Analysis, DNAABSTRACT
BACKGROUND/AIMS: Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. METHODS: Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. RESULTS: We show that currents carried by Kv7.1 (EC50 = 1.48 µM), Kv7.1/KCNE1 (EC50 = 4.9 µM), and Kv7.4 (EC50 = 0.148 µM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1. CONCLUSION: Rottlerin or an optimized derivative holds a potential as QT interval correcting drug.
Subject(s)
Acetophenones/pharmacology , Benzopyrans/pharmacology , Biological Products/pharmacology , Ion Channel Gating/drug effects , KCNQ1 Potassium Channel/metabolism , Acetophenones/chemistry , Animals , Benzopyrans/chemistry , Biological Products/chemistry , Computer Simulation , Humans , Induced Pluripotent Stem Cells/cytology , KCNQ1 Potassium Channel/chemistry , Membrane Potentials/drug effects , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Protein Domains , Protein Multimerization/drug effects , Xenopus laevisABSTRACT
Long QT syndrome is a potentially life-threatening disease characterized by delayed repolarization of cardiomyocytes, QT interval prolongation in the electrocardiogram, and a high risk for sudden cardiac death caused by ventricular arrhythmia. The genetic type 3 of this syndrome (LQT3) is caused by gain-of-function mutations in the SCN5A cardiac sodium channel gene which mediates the fast Nav1.5 current during action potential initiation. Here, we report the analysis of LQT3 human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These were generated from a patient with a heterozygous p.R1644H mutation in SCN5A known to interfere with fast channel inactivation. LQT3 hiPSC-CMs recapitulated pathognomonic electrophysiological features of the disease, such as an accelerated recovery from inactivation of sodium currents as well as action potential prolongation, especially at low stimulation rates. In addition, unlike previously described LQT3 hiPSC models, we observed a high incidence of early after depolarizations (EADs) which is a trigger mechanism for arrhythmia in LQT3. Administration of specific sodium channel inhibitors was found to shorten action and field potential durations specifically in LQT3 hiPSC-CMs and antagonized EADs in a dose-dependent manner. These findings were in full agreement with the pharmacological response profile of the underlying patient and of other patients from the same family. Thus, our data demonstrate the utility of patient-specific LQT3 hiPSCs for assessing pharmacological responses to putative drugs and for improving treatment efficacies.
Subject(s)
Long QT Syndrome/metabolism , Myocytes, Cardiac/metabolism , NAV1.5 Voltage-Gated Sodium Channel/genetics , Cardiac Conduction System Disease , Cells, Cultured , Humans , Induced Pluripotent Stem Cells , Long QT Syndrome/genetics , Patch-Clamp Techniques , PhenotypeABSTRACT
Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large-scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high-efficiency procedure for generating CMs both in two-dimensional (2D) and three-dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost-efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin-free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time-course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture-induced maturation of the resulting CMs. This suggested that hPSC-CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC-CMs to functional readouts and thus define the cornerstones of culture-induced maturation.
Subject(s)
Cell Differentiation , Heart/physiology , Pluripotent Stem Cells/cytology , Humans , Mesoderm/cytology , Myocytes, Cardiac/cytologyABSTRACT
Conrad et al. have generated human adult germline stem cells (haGSCs) from human testicular tissue, which they claim have similar pluripotent properties to human embryonic stem cells (hESCs). Here we investigate the pluripotency of haGSCs by using global gene-expression analysis based on their gene array data and comparing the expression of pluripotency marker genes in haGSCs and hESCs, and in haGSCs and human fibroblast samples derived from different laboratories, including our own. We find that haGSCs and fibroblasts have a similar gene-expression profile, but that haGSCs and hESCs do not. The pluripotency of Conrad and colleagues' haGSCs is therefore called into question.
Subject(s)
Germ Cells/cytology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Adult , Animals , Biomarkers/analysis , Biopsy , Cells, Cultured , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling , Humans , Male , Mice , RNA, Messenger/analysis , RNA, Messenger/genetics , Reproducibility of Results , Testis/cytologyABSTRACT
Human embryonic stem cells (hESCs) can exit the self-renewal programme, through the action of signalling molecules, at any given time and differentiate along the three germ layer lineages. We have systematically investigated the specific roles of three signalling pathways, TGFß/SMAD2, BMP/SMAD1, and FGF/ERK, in promoting the transition of hESCs into the neuroectoderm lineage. In this context, inhibition of SMAD2 and ERK signalling served to cooperatively promote exit from hESC self-renewal through the rapid downregulation of NANOG and OCT4. In contrast, inhibition of SMAD1 signalling acted to maintain SOX2 expression and prevent non-neural differentiation via HAND1. Inhibition of FGF/ERK upregulated OTX2 that subsequently induced the neuroectodermal fate determinant PAX6, revealing a novel role for FGF2 in indirectly repressing PAX6 in hESCs. Combined inhibition of the three pathways hence resulted in highly efficient neuroectoderm formation within 4 days, and subsequently, FGF/ERK inhibition promoted rapid differentiation into peripheral neurons. Our study assigns a novel, biphasic role to FGF/ERK signalling in the neural induction of hESCs, which may also have utility for applications requiring the rapid and efficient generation of peripheral neurons.
Subject(s)
Embryonic Stem Cells/physiology , Fibroblast Growth Factors/metabolism , Neural Plate/cytology , Bone Morphogenetic Proteins/metabolism , Cell Line , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Eye Proteins/metabolism , Fibroblast Growth Factors/antagonists & inhibitors , Homeodomain Proteins/metabolism , Humans , MAP Kinase Signaling System , Nanog Homeobox Protein , Neural Plate/metabolism , Octamer Transcription Factor-3/metabolism , Otx Transcription Factors/metabolism , PAX6 Transcription Factor , Paired Box Transcription Factors/metabolism , Repressor Proteins/metabolism , Signal Transduction , Smad1 Protein/metabolism , Smad2 Protein/analysis , Smad2 Protein/metabolism , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/metabolismABSTRACT
Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.
Subject(s)
Cell Dedifferentiation , Cellular Reprogramming , Fetus/cytology , Neurons/cytology , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Animals , Biomarkers/analysis , Cell Differentiation , Cell Line , DNA Methylation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Gene Expression Profiling , Germ Layers/cytology , Germ Layers/metabolism , Humans , Kruppel-Like Factor 4 , Mice , Neurons/metabolism , Octamer Transcription Factor-3/geneticsABSTRACT
Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAHâ»/â» mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAHâ»/â»-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAHâ»/â» iPS cell lines, we aggregated FAHâ»/â»-iPS cells with tetraploid embryos and obtained entirely FAHâ»/â»-iPS cell-derived mice that were viable and exhibited the phenotype of the founding FAHâ»/â» mice. Then, we transduced FAH cDNA into the FAHâ»/â»-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell-derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.
Subject(s)
Fibroblasts/drug effects , Genetic Complementation Test/methods , Genetic Therapy/methods , Genetic Vectors/pharmacology , Hydrolases , Induced Pluripotent Stem Cells , Lentivirus , Tyrosinemias , Animals , Cell Survival , Cells, Cultured , Chromosomes/chemistry , Cyclohexanones/pharmacology , Disease Models, Animal , Female , Fetus , Fibroblasts/cytology , Humans , Hydrolases/deficiency , Hydrolases/genetics , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Liver/metabolism , Liver/pathology , Mice , Mice, Knockout , Nitrobenzoates/pharmacology , Pregnancy , Promoter Regions, Genetic , Spleen Focus-Forming Viruses/chemistry , Spleen Focus-Forming Viruses/genetics , Tetraploidy , Tyrosinemias/genetics , Tyrosinemias/metabolism , Tyrosinemias/pathology , Tyrosinemias/therapyABSTRACT
Cell therapeutic applications based on induced pluripotent stem cells (iPSCs) appear highly promising and challenging at the same time. Good manufacturing practice (GMP) regulations impose necessary yet demanding requirements for quality and consistency when manufacturing iPSCs and their differentiated progeny. Given the scarcity of accessible GMP iPSC lines, we have established a corresponding production workflow to generate the first set of compliant cell banks. Hence, these lines met a comprehensive set of release specifications and, for instance, displayed a low overall mutation load reflecting their neonatal origin, cord blood. Based on these iPSC lines, we have furthermore developed a set of GMP-compatible workflows enabling improved gene targeting at strongly enhanced efficiencies and directed differentiation into critical cell types: A new protocol for the generation of retinal pigment epithelium (RPE) features a high degree of simplicity and efficiency. Mesenchymal stromal cells (MSCs) derived from iPSCs displayed outstanding expansion capacity. A fully optimized cardiomyocyte differentiation protocol was characterized by a particularly high batch-to-batch consistency at purities above 95%. Finally, we introduce a universal immune cell induction platform that converts iPSCs into multipotent precursor cells. These hematopoietic precursors could selectively be stimulated to become macrophages, T cells, or natural killer (NK) cells. A switch in culture conditions upon NK-cell differentiation induced a several thousand-fold expansion, which opens up perspectives for upscaling this key cell type in a feeder cell-independent approach. Taken together, these cell lines and improved manipulation platforms will have broad utility in cell therapy as well as in basic research.
ABSTRACT
BACKGROUND: Transcription activator-like effector nucleases (TALENs) have emerged as a tool for enabling targeted gene editing and disruption in difficult systems, such as human pluripotent stem cells (hPSCs). The modular architecture of TAL effectors theoretically enables targeting of any genomic locus and several cloning systems for custom TALEN assembly have recently been established. However, there is a lack of versatile TALEN expression systems applicable to hPSCs. RESULTS: Here, we extend an existing TALE assembly system by a dual set of expression vectors for efficient application of TALEN technology in hPSCs. This is characterized by improved TALEN architecture as well as antibiotic resistance and fluorescent reporter cassettes, thus enabling enrichment for transfected cells. Improved functionality of the combined system was demonstrated by targeted disruption of the HPRT1 gene to create isogenic disease models of Lesch-Nyhan-Syndrome. Using female hPSCs, homozygous disruption of HPRT1 occurred at efficiencies of up to 15%. Differentiating isogenic knock-out cells both into central nervous system (CNS) as well as into sensory-like neurons recapitulated previously described phenotypes based on patient-specific induced PSCs and extended these findings to non-CNS neurons, respectively. CONCLUSION: The combined vector system allows for flexible and affordable generation of knock-out hPSCs lines, thus enabling investigation of developmental processes as well as the generation of isogenic disease models without the need for patient material.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/genetics , Endonucleases/genetics , Hypoxanthine Phosphoribosyltransferase/biosynthesis , Lesch-Nyhan Syndrome/genetics , Pluripotent Stem Cells/metabolism , Recombinant Fusion Proteins/genetics , Trans-Activators/genetics , Endonucleases/metabolism , Female , Gene Knockout Techniques , Gene Targeting , Genomics , Homozygote , Humans , Hypoxanthine Phosphoribosyltransferase/genetics , Lesch-Nyhan Syndrome/metabolism , Lesch-Nyhan Syndrome/pathology , Pluripotent Stem Cells/cytology , Sensory Receptor Cells/metabolismABSTRACT
Molecular mechanisms underlying the commitment of cells to the germ cell lineage during mammalian embryogenesis remain poorly understood due to the limited availability of cellular materials to conduct in vitro analyses. Although primordial germ cells (PGCs)--precursors to germ cells--have been generated from embryonic stem cells (ESCs)--pluripotent stem cells derived from the inner cell mass of the blastocyst of the early embryo in vitro-the simultaneous expression of cell surface receptors and transcription factors complicates the detection of PGCs. To date, only a few genes that mark the onset of germ cell commitment in the epiblast--the outer layer of cells of the embryo--including tissue non-specific alkaline phosphatase (TNAP), Blimp1, Stella and Fragilis--have been used with some success to detect PGC formation in in vitro model systems. Here, we identified 11 genes (three of which are novel) that are specifically expressed in male and female fetal germ cells, both in vivo and in vitro, but are not expressed in ESCs. Expression of these genes allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro.
Subject(s)
Gene Expression Profiling , Germ Cells/metabolism , Animals , Cells, Cultured , Embryonic Stem Cells/metabolism , Endodeoxyribonucleases/genetics , Female , Fetal Development/genetics , Genetic Markers , Humans , Male , Meiosis , Mice , Pluripotent Stem Cells/metabolism , Proto-Oncogene Proteins c-akt/geneticsABSTRACT
Viral myocarditis is pathologically associated with RNA viruses such as coxsackievirus B3 (CVB3), or more recently, with SARS-CoV-2, but despite intensive research, clinically proven treatment is limited. Here, by use of a transgenic mouse strain (TG) containing a CVB3ΔVP0 genome we unravel virus-mediated cardiac pathophysiological processes in vivo and in vitro. Cardiac function, pathologic ECG alterations, calcium homeostasis, intracellular organization and gene expression were significantly altered in transgenic mice. A marked alteration of mitochondrial structure and gene expression indicates mitochondrial impairment potentially contributing to cardiac contractile dysfunction. An extended picture on viral myocarditis emerges that may help to develop new treatment strategies and to counter cardiac failure.