Differentiation Of Human Pluripotent Stem Cells 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.

Sarcomeric tropomyosin expression during human iPSC differentiation into cardiomyocytes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1μ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and μ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1μ non-muscle isoform protein expression before and during cardiac differentiation.

Three-dimensional co-culturing of stem cell-derived cardiomyocytes and cardiac fibroblasts reveals a role for both cell types in Marfan-related cardiomyopathy

Pathogenic variants in the FBN1 gene, which encodes the extracellular matrix protein fibrillin-1, cause Marfan syndrome (MFS), which affects multiple organ systems, including the cardiovascular system. Myocardial dysfunction has been observed in a subset of patients with MFS and in several MFS mouse models. However, there is limited understanding of the intrinsic consequences of FBN1 variants on cardiomyocytes (CMs). To elucidate the CM-specific contribution in Marfan's cardiomyopathy, cardiosphere cultures of CMs and cardiac fibroblasts (CFs) are used. CMs and CFs were derived by human induced pluripotent stem cell (iPSC) differentiation from MFS iPSCs with a pathogenic variant in FBN1 (c.3725G>A; p.Cys1242Tyr) and the corresponding CRISPR-corrected iPSC line (Cor).Cardiospheres containing MFS CMs show decreased FBN1, COL1A2 and GJA1 expression. MFS CMs cultured in cardiospheres have fewer binucleated CMs in comparison with Cor CMs. 13% of MFS CMs in cardiospheres are binucleated and 15% and 16% in cardiospheres that contain co-cultures with respectively MFS CFs and Cor CFs, compared to Cor CMs, that revealed up to 23% binucleation when co-cultured with CFs. The sarcomere length of CMs, as a marker of development, is significantly increased in MFS CMs interacting with Cor CF or MFS CF, as compared to monocultured MFS CMs. Nuclear blebbing was significantly more frequent in MFS CFs, which correlated with increased stiffness of the nuclear area compared to Cor CFs.Our cardiosphere model for Marfan-related cardiomyopathy identified a contribution of CFs in Marfan-related cardiomyopathy and suggests that abnormal early development of CMs may play a role in the disease mechanism.

Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature invivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs

Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.

Characterization of human pluripotent stem cell differentiation by single-cell dual-omics analyses

Invivo differentiation of human pluripotent stem cells (hPSCs) has unique advantages, such as multilineage differentiation, angiogenesis, and close cell-cell interactions. To systematically investigate multilineage differentiation mechanisms of hPSCs, we constructed the invivo hPSC differentiation landscape containing 239,670 cells using teratoma models. We identified 43 cell types, inferred 18 cell differentiation trajectories, and characterized common and specific gene regulation patterns during hPSC differentiation at both transcriptional and epigenetic levels. Additionally, we developed the developmental single-cell Basic Local Alignment Search Tool (dscBLAST), an R-based cell identification tool, to simplify the identification processes of developmental cells. Using dscBLAST, we aligned cells in multiple differentiation models to normally developing cells to further understand their differentiation states. Overall, our study offers new insights into stem cell differentiation and human embryonic development; dscBLAST shows favorable cell identification performance, providing a powerful identification tool for developmental cells.

Tau tubulin kinase 1 and 2 regulate ciliogenesis and human pluripotent stem cells–derived neural rosettes

Primary cilia are key regulators of embryo development and tissue homeostasis. However, their mechanisms and functions, particularly in the context of human cells, are still unclear. Here, we analyzed the consequences of primary cilia modulation for human pluripotent stem cells (hPSCs) proliferation and differentiation. We report that neither activation of the cilia-associated Hedgehog signaling pathway nor ablation of primary cilia by CRISPR gene editing to knockout Tau Tubulin Kinase 2 (TTBK2), a crucial ciliogenesis regulator, affects the self-renewal of hPSCs. Further, we show that TTBK1, a related kinase without previous links to ciliogenesis, is upregulated during hPSCs-derived neural rosette differentiation. Importantly, we demonstrate that while TTBK1 fails to localize to the mother centriole, it regulates primary cilia formation in the differentiated, but not the undifferentiated hPSCs. Finally, we show that TTBK1/2 and primary cilia are implicated in the regulation of the size of hPSCs-derived neural rosettes.

Research Progress towards the Effects of Fatty Acids on the Differentiation and Maturation of Human Induced Pluripotent Stem Cells into Cardiomyocytes.

The incidence of cardiovascular disease has been continuously increasing. Because cardiomyocytes (CM) are non-renewable cells, it is difficult to find appropriate CM sources to repair injured hearts. Research of human induced pluripotent stem cell (hiPSC) differentiation and maturation into CM has been invaluable for the treatment of heart diseases. The use of hiPSCs as regenerative therapy allows for the treatment of many diseases that cannot be cured, including progressive heart failure. This review contributes to the study of cardiac repair and targeted treatment of cardiovascular diseases at the cytological level. Recent studies have shown that for differentiation and maturation of hiPSCs into CMs, fatty acids have a strong influence on cellular metabolism, organelle development, expression of specific genes, and functional performance. This review describes the recent research progress on how fatty acids affect the differentiation of hiPSCs into CMs and their maturation.

Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation

Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.

Designed peptide-grafted hydrogels for human pluripotent stem cell culture and differentiation.

Human pluripotent stem cells (hPSCs) have the ability to differentiate into cells derived from three germ layers and are an attractive cell source for cell therapy in regenerative medicine. However, hPSCs cannot be cultured on conventional tissue culture flasks but can be cultured on biomaterials with specific hPSC integrin interaction sites. We designed hydrogels conjugated with several designed peptides that had laminin-β4 active sites, optimal elasticities and different zeta potentials. A higher expansion fold of hPSCs cultured on the hydrogels was found with the increasing zeta potential of the hydrogels conjugated with designed peptides, where positive amino acid (lysine) insertion into the peptides promoted higher zeta potentials of the hydrogels and higher expansion folds of hPSCs when cultured on the hydrogels using xeno-free protocols. The hPSCs cultured on hydrogels conjugated with the optimal peptides showed a higher expansion fold than those on recombinant vitronectin-coated plates, which are the gold standard of hPSC cultivation dishes. The hPSCs could differentiate into specific cell lineages, such as mesenchymal stem cells (MSCs) and MSC-derived osteoblasts, even after being cultivated on hydrogels conjugated with optimal peptides for long periods of time, such as 10 passages.

Compact automated culture machine for human induced pluripotent stem cell maintenance and differentiation.

The technologies used to generate human induced pluripotent stem cell (iPSC) from somatic cells potentially enable the wide application of iPSC-derived differentiated cells in industrial research fields as a replacement for animals. However, as highly trained individuals are required to obtain reproducible results, this approach has limited social implementation. In the research field of iPSC, it is believed that documentable information is not enough for reproducing the quality of the differentiated cells. Therefore, automated culture machines for cell processing should make the starting of iPSC-using researches easier. We developed a programmable all-in-one automated culture machine, with dense and compact constitution that fits within a normal biosafety cabinet (200mm wide, 233mm height, and 110mm depth). This instrument was fabricated using novel x-y-z-axes-rail-system, such as an overhead traveling crane, in a factory, which served as the main handling machinery. This machine enabled stable and efficient expansion of human iPSC under the feeder-free condition, without karyotype alterations, and simultaneously differentiated the cells into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. Overall, this machine would facilitate the social implementation of human pluripotent stem cells and contribute to the accumulation of sharable knowledge for the standardization of the entire handling processes of iPSC in pharmaceutical, food, and cosmetic research.

MicroRNAs: Crucial Players in the Differentiation of Human Pluripotent and Multipotent Stem Cells into Functional Hepatocyte-Like Cells.

Hepatic disease negatively impacts liver function and metabolism. Primary human hepatocytes are the gold standard for the prediction and successful treatment of liver disease. However, the sources of hepatocytes for drug toxicity testing and disease modeling are limited. To overcome this issue, pluripotent stem cells (PSCs) have emerged as an alternative strategy for liver disease therapy. Human PSCs, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can self-renew and give rise to all cells of the body. Human PSCs are attractive cell sources for regenerative medicine, tissue engineering, drug discovery, and developmental studies. Several recent studies have shown that mesenchymal stem cells (MSCs) can also differentiate (or trans-differentiate) into hepatocytes. Differentiation of human PSCs and MSCs into functional hepatocytelike cells (HLCs) opens new strategies to study genetic diseases, hepatotoxicity, infection of hepatotropic viruses, and analyze hepatic biology. Numerous in vitro and in vivo differentiation protocols have been established to obtain human PSCs/MSCs-derived HLCs and mimic their characteristics. It was recently discovered that microRNAs (miRNAs) play a critical role in controlling the ectopic expression of transcription factors and governing the hepatocyte differentiation of human PSCs and MSCs. In this review, we focused on the role of miRNAs in the differentiation of human PSCs and MSCs into hepatocytes.

The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation toward the kidney lineage.

During embryonic development, the mesoderm undergoes patterning into diverse lineages including axial, paraxial, and lateral plate mesoderm (LPM). Within the LPM, the so-called intermediate mesoderm (IM) forms kidney and urogenital tract progenitor cells, while the remaining LPM forms cardiovascular, hematopoietic, mesothelial, and additional progenitor cells. The signals that regulate these early lineage decisions are incompletely understood. Here, we found that the centrosomal protein 83 (CEP83), a centriolar component necessary for primary cilia formation and mutated in pediatric kidney disease, influences the differentiation of human-induced pluripotent stem cells (hiPSCs) toward IM. We induced inactivating deletions of <i>CEP83</i> in hiPSCs and applied a 7-day in vitro protocol of IM kidney progenitor differentiation, based on timed application of WNT and FGF agonists. We characterized induced mesodermal cell populations using single-cell and bulk transcriptomics and tested their ability to form kidney structures in subsequent organoid culture. While hiPSCs with homozygous <i>CEP83</i> inactivation were normal regarding morphology and transcriptome, their induced differentiation into IM progenitor cells was perturbed. Mesodermal cells induced after 7 days of monolayer culture of <i>CEP83</i>-deficient hiPCS exhibited absent or elongated primary cilia, displayed decreased expression of critical IM genes (<i>PAX8</i>, <i>EYA1</i>, <i>HOXB7</i>), and an aberrant induction of LPM markers (e.g. <i>FOXF1</i>, <i>FOXF2</i>, <i>FENDRR</i>, <i>HAND1</i>, <i>HAND2</i>). Upon subsequent organoid culture, wildtype cells differentiated to form kidney tubules and glomerular-like structures, whereas <i>CEP83</i>-deficient cells failed to generate kidney cell types, instead upregulating cardiomyocyte, vascular, and more general LPM progenitor markers. Our data suggest that <i>CEP83</i> regulates the balance of IM and LPM formation from human pluripotent stem cells, identifying a potential link between centriolar or ciliary function and mesodermal lineage induction.

Key benefits of a microfluidic platform for cell culture at a clinically relevant scale

Watch the video or read the poster to learn:The capabilities of an automated cell therapy workstation for process development and manufacturing combining advanced end-to-end bioprocessing and analytical capabilities and seamless scale-up/downHighly efficient bioprocessing of primary T-cells, with 5-10x lower viral vector consumption, higher fold expansion, and tighter control of cell phenotype compared to conventional research cell culture vesselsBioprocessing of adherent mesenchymal stem cells in monolayer in the MicrofluidX platform, which has been shown to surpass the performance of conventional cell culture vessels with a 3x higher cell density while maintaining cell phenotypeLongitudinal studies over 6 passages of mesenchymal stem cells, which showed 50% higher average and 30% lower variability in cell densities compared to flask culture, while maintaining cell phenotype across all passagesJames Kusena&#160;is the VP Bioprocessing and Applications at MicrofluidX. James holds a PhD in human pluripotent stem cell culture and differentiation from Loughborough University, UK. He has 6 years of experience in cell therapy process development and manufacturing optimization. His background is in medicinal chemistry, international business and health economicsMohsen Shaeri is a Senior Scientist at the Cell and Gene Therapy Catapult, specializing in process development. He has 4 years of experience in microfluidic cell culture and over 12 years of experience in bioprocessing and automation, with a focus on cell therapies. He holds a PhD in Bioengineering from the University of Sheffield, UK.Pedro Costa is the Head of Operations at Stemmatters. Pedro holds a degree in Biochemistry from the University of Coimbra (Portugal) and a PhD in Biochemistry from the University of Coimbra in co-operation with Columbia University (NY, USA). He has over six years of experience in biomedical research, with a particular focus on nanocarrier development and functionalization, and in primary cell and stem cell isolation/culture, cell-based assays, and genome editing.

Heme oxygenase-1 induction promotes human pluripotent stem cell differentiation into endothelial cells and vascular repair

Background and Aims : Endothelial cells (ECs) have a crucial role in reendothelialization for repairing denuded arteries. Heme oxygenase-1 (HO-1) is a stress response protein that degrades prooxidant heme and generates molecules with antioxidative and anti-inflammatory properties. Given that redox status is critical in stem cell differentiation, our aims are to investigate the effects of HO-1 induction on human pluripotent stem cells (hPSCs) differentiation toward ECs and reendothelialization after arterial injury.

In vitro characterization of iridoid and phenylethanoid glycosides from Cistanche phelypaea for nutraceutical and pharmacological applications.

"Desert hyacinths" are a remarkable group of parasitic plants belonging to genus Cistanche, including more than 20 accepted species typically occurring in deserts or coastal dunes parasitizing roots of shrubs. Several Cistanche species have long been a source of traditional herbal medicine or food, being C.deserticola and C.tubulosa the most used in China. This manuscript reports the isolation and identification of some phenylethanoid and iridoid glycosides, obtained from the hydroalcoholic extract of C.phelypaea collected in Spain. The present study aims to characterize the antioxidant activity of C.phelypaea metabolites in the light of their application in nutraceutical and cosmeceutical industries and the effect of acetoside, the most abundant metabolite in C.phelypaea extract, on human keratinocyte and pluripotent stem cell proliferation and differentiation. Our study demonstrated that acetoside, besides its strong antioxidant potential, can preserve the proliferative potential of human basal keratinocytes and the stemness of mesenchymal progenitors necessary for tissue morphogenesis and renewal. Therefore, acetoside can be of practical relevance for the clinical application of human stem cell cultures in tissue engineering and regenerative medicine.

Integrative epigenomic and transcriptomic analysis reveals the requirement of JUNB for hematopoietic fate induction

Human pluripotent stem cell differentiation towards hematopoietic progenitor cell can serve as an in vitro model for human embryonic hematopoiesis, but the dynamic change of epigenome and transcriptome remains elusive. Here, we systematically profile the chromatin accessibility, H3K4me3 and H3K27me3 modifications, and the transcriptome of intermediate progenitors during hematopoietic progenitor cell differentiation in vitro. The integrative analyses reveal sequential opening-up of regions for the binding of hematopoietic transcription factors and stepwise epigenetic reprogramming of bivalent genes. Single-cell analysis of cells undergoing the endothelial-to-hematopoietic transition and comparison with in vivo hemogenic endothelial cells reveal important features of in vitro and in vivo hematopoiesis. We find that JUNB is an essential regulator for hemogenic endothelium specialization and endothelial-to-hematopoietic transition. These studies depict an epigenomic roadmap from human pluripotent stem cells to hematopoietic progenitor cells, which may pave the way to generate hematopoietic progenitor cells with improved developmental potentials.

Robust temporal map of human in vitro myelopoiesis using single-cell genomics

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

Temporal perturbation of histone deacetylase activity reveals a requirement for HDAC1-3 in mesendoderm cell differentiation.

Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation invitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation invitro and mouse gastrulation invivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1-3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations.

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR Tcells.

Unlimited generation of chimeric antigen receptor (CAR) Tcells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR Tcell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ Tcell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory Tcells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR Tcell characteristics. Expanded iPSC CD19-CAR Tcells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR Tcells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR Tcells also mediated potent antitumor activity invivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR Tcells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.