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Parthenogenetic embryonic stem (ES) cells with two oocyte-derived genomes (uniparental) have been proposed as a source of autologous tissue for transplantation. The therapeutic applicability of any uniparental cell type is uncertain due to the consequences of genomic imprinting that in mammalian uniparental tissues causes unbalanced expression of imprinted genes. We transplanted uniparental fetal liver cells into lethally irradiated adult mice to test their capacity to replace adult hematopoietic tissue. Both maternal (gynogenetic) and paternal (androgenetic) derived cells conveyed long-term,multilineage reconstitution of hematopoiesis in recipients,with no associated pathologies. We also establish that uniparental ES cells can differentiate into transplantable hematopoietic progenitors in vitro that contribute to long-term hematopoiesis in recipients. Hematopoietic tissue in recipients maintained fidelity of parent-of-origin methylation marks at the Igf2/H19 locus; however,variability occurred in the maintenance of parental-specific methylation marks at other loci. In summary,despite genomic imprinting and its consequences on development that are particularly evident in the androgenetic phenotype,uniparental cells of both parental origins can form adult-transplantable stem cells and can repopulate an adult organ. View Publication

AbstractBackgroundCancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating cancers such as hematological malignancies and breast cancers. However,approaches to obtain cancer‐reactive TCR‐T cells have been unsuccessful.MethodsHere,we developed a novel strategy to screen for cancer‐targeted TCR‐T cells using a special humanized mouse model with person‐specific immune fingerprints. Rare steady‐state circulating hematopoietic stem and progenitor cells were expanded via three‐dimensional culture of steady‐state peripheral blood mononuclear cells,and then the expanded cells were applied to establish humanized mice. The human immune system was evaluated according to the kinetics of dendritic cells,monocytes,T‐cell subsets,and cytokines. To fully stimulate the immune response and to obtain B‐cell precursor NAML‐6‐ and triple‐negative breast cancer MDA‐MB‐231‐targeted TCR‐T cells,we used the inactivated cells above to treat humanized mice twice a day every 7 days. Then,human T cells were processed for TCR β‐chain (TRB) sequencing analysis. After the repertoires had been constructed,features such as the fraction,diversity,and immune signature were investigated.ResultsThe results demonstrated an increase in diversity and clonality of T cells after treatment. The preferential usage and features of TRBV,TRBJ,and the V–J combination were also changed. The stress also induced highly clonal expansion. Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice.ConclusionsWe constructed a personalized humanized mouse model to screen cancer‐targeted TCR‐T pools. Our platform provides an effective source of cancer‐targeted TCR‐T cells and allows for the design of patient‐specific engineered T cells. It therefore has the potential to greatly benefit cancer treatment. Cancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating malignancies but with limited source. We applied steady‐state peripheral blood mononuclear cells via three‐dimensional culture to construct humanized mouse model for cancer‐targeted TCR‐T repertoire screening. View Publication

INTRODUCTION: The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells,thereby providing a novel cell-therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming. METHODS: We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones. RESULTS: Ectopic expression of OCT4,SOX2,KLF4,and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers,including SSEA-4,TRA-1-60,and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes,such as LIN28,TERT,DPPA4,and PODXL,in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation,HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers,including insulin-producing cells through endodermal lineage,verifying the pluripotency of the blood-derived iPSC clones. CONCLUSIONS: Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming,mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation,especially from diabetes patients complicated by slow-healing wounds. View Publication

BACKGROUND Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage,differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally,current differentiation strategies are hampered by inefficiency,lack of reproducibility,and use of animal-derived products. METHODS To direct the differentiation of hESCs to endothelial subtypes,H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3),basic fibroblast growth factor (bFGF),bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation,the endothelial progenitor cells (CD34(+)CD31(+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore,the safety and functionality of these cells upon in vivo transplantation were characterized. RESULTS Sequential modulation of hESCs with GSK-3 inhibitor,bFGF,BMP4 and VEGF resulted in stages reminiscent of primitive streak,early mesoderm/lateral plate mesoderm,and endothelial progenitors under feeder- and serum-free conditions. Furthermore,these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically,the endothelial progenitors differentiated to venous ECs in the absence of VEGF,and to arterial phenotype under low concentrations of VEGF. Additionally,these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore,these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse. CONCLUSIONS We report a simple,rapid,and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery,disease modeling and regenerative medicine in the future. View Publication

Human embryonic stem cells (hESCs) are a promising cell source for tissue engineering and regenerative medicine,but before they can be used in therapies,we must be able to accurately identify the state and progeny of hESCs. One of the most commonly used methods for identification is flow cytometry. Many flow cytometry applications use antibodies to detect the amount of antigen present on/in a cell. This allows for the identification of unique cell populations or the tracking of expression changes within a population during differentiation. The results are typically presented as a percentage of positively expressing cells (%Pos) for a marker of choice,relative to a negative control. However,this reporting term is vulnerable to distortion from outliers and inaccuracy from loss of information about the population's fluorescence intensity. In this article,we describe an alternate strategy that uses the normalized median fluorescence intensity (nMFI),in which the MFI of the stained sample is normalized to the MFI of the negative control,as the reporting term to more accurately describe a population of cells in culture. We observed that nMFI provides a more accurate representation for the quality of a starting population and comparing data of different experimental runs. In addition,we demonstrated that the nMFI is a more sensitive measure of pluripotent and differentiation markers expression changes during hESC differentiation into three germ layer lineages. View Publication

BACKGROUND Efficient slow freezing protocols within serum-free and feeder-free culture systems are crucial for the clinical application of human embryonic stem (hES) cells. Frequently,however,hES cells must be cryopreserved as clumps when using conventional slow freezing protocols,leading to lower survival rates during freeze-thaw and limiting their recovery and growth efficiency after thawing,as well as limiting downstream applications that require single cell suspensions. We describe a novel method to increase freeze-thaw survival and proliferation rate of single hES cells in serum-free and feeder-free culture conditions. METHODS hES cells maintained on Matrigel-coated dishes were dissociated into single cells with Accutase and slow freezing. After thawing at 37 degrees C,cells were cultured in mTeSR medium supplemented with 10 microM of Rho-associated kinase inhibitor Y-27632 for 1 day. RESULTS The use of Y-27632 and Accutase significantly increases the survival of single hES cells after thawing compared with a control group (P textless 0.01). Furthermore,by treatment of hES cell aggregates with EGTA to disrupt cell-cell interaction,we show that Y-27632 treatment does not directly affect hES cell apoptosis. Even in the presence of Y-27632,hES cells deficient in cell-cell interaction undergo apoptosis. Y-27632-treated freeze-thawed hES cells retain typical morphology,stable karyotype,expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. CONCLUSIONS The method described here allows for cryopreservation of single hES cells in serum-free and feeder-free conditions and therefore we believe this method will be ideal for current and future hES cell applications that are targeted towards a therapeutic end-point. View Publication

Many human embryonic stem (hES) and induced pluripotent stem (hiPS) cell differentiation protocols begin with the formation of three-dimensional aggregates of cells called embryoid bodies (EBs). Traditional EB formation methods result in a heterogeneous population of EB sizes and shapes,which then undergo heterogeneous differentiation efficiencies. AggreWell(TM)400 and AggreWell(TM)800 use the spin-EB method to force the aggregation of a defined number of cells,thereby controlling EB size and generating a population of uniform EBs. Moreover,the dense array of microwells on the bottom surface of AggreWell(TM)400 provide for the rapid and simple production of thousands of EBs at a time. View Publication

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks,we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells,we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes,which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions,which can then be employed to direct cellular differentiation down desired pathways. View Publication

The development of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) facilitates in vitro studies of human disease mechanisms,speeds up the process of drug screening,and raises the feasibility of using cell replacement therapy in clinics. However,the study of genotype-phenotype relationships in ESCs or iPSCs is hampered by the low efficiency of site-specific gene editing. Transcription activator-like effector nucleases (TALENs) spurred interest due to the ease of assembly,high efficiency and faithful gene targeting. In this study,we optimized the TALEN design to maximize its genomic cutting efficiency. We showed that using optimized TALENs in conjunction with single-strand oligodeoxynucleotide (ssODN) allowed efficient gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb can be accomplished at high efficiencies. We established human tumor cell lines and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21) gene and miR-9-2 gene. These cell lines provide a robust platform to dissect the roles these genes play during cell differentiation and tumorigenesis. We also observed that the endogenous homologous chromosome can serve as a donor template for gene editing. Overall,our studies demonstrate the versatility of using ssODN and TALEN to establish genetically modified cells for research and therapeutic application. View Publication

Our study highlights the role of epigenetic machinery in transformation of normal pluripotent stem cells to variant pluripotent state. We demonstrate the importance of non-histone protein methylation,which underlie the EMT and abnormal differentiation behaviour of variant hESCs. View Publication