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Human brain development exhibits several unique aspects,such as increased complexity and expansion of neuronal output,that have proven difficult to study in model organisms. As a result,in vitro approaches to model human brain development and disease are an intense area of research. Here we describe a recently established protocol for generating 3D brain tissue,so-called cerebral organoids,which closely mimics the endogenous developmental program. This method can easily be implemented in a standard tissue culture room and can give rise to developing cerebral cortex,ventral telencephalon,choroid plexus and retinal identities,among others,within 1-2 months. This straightforward protocol can be applied to developmental studies,as well as to the study of a variety of human brain diseases. Furthermore,as organoids can be maintained for more than 1 year in long-term culture,they also have the potential to model later events such as neuronal maturation and survival. View Publication

Human pluripotent stem cells (hPSCs) are sensitive to DNA damage and undergo rapid apoptosis compared to their differentiated progeny cells. Here,we explore the underlying mechanisms for the increased apoptotic sensitivity of hPSCs that helps to determine pluripotent stem cell fate. Apoptosis was induced by exposure to actinomycin D,etoposide,or tunicamycin,with each agent triggering a distinct apoptotic pathway. We show that hPSCs are more sensitive to all three types of apoptosis induction than are lineage-non-specific,retinoic-acid-differentiated hPSCs. Also,Bax activation and pro-apoptotic mitochondrial intermembrane space protein release,which are required to initiate the mitochondria-mediated apoptosis pathway,are more rapid in hPSCs than in retinoic-acid-differentiated hPSCs. Surprisingly,Bak and not Bax is essential for actinomycin-D-induced apoptosis in human embryonic stem cells. Finally,P53 is degraded rapidly in an ubiquitin-proteasome-dependent pathway in hPSCs at steady state but quickly accumulates and induces apoptosis when Mdm2 function is impaired. Rapid degradation of P53 ensures the survival of healthy hPSCs but avails these cells for immediate apoptosis upon cellular damage by P53 stabilization. Altogether,we provide an underlying,interconnected molecular mechanism that primes hPSCs for quick clearance by apoptosis to eliminate hPSCs with unrepaired genome alterations and preserves organismal genomic integrity during the early critical stages of human embryonic development. View Publication

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types,thus providing a platform for basic and clinical applications. However,pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here,we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival,self-renewal,and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored. View Publication

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g.,gene knock-in,knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs,microinjection of piPSCs into embryos,embryo transfer and production of chimeric animals based on successful protocols. View Publication

Diploidy is a fundamental genetic feature in mammals,in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species,but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes,leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics,such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover,we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts,they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation,alongside reduction in absolute gene expression levels and cell size. Surprisingly,we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state,but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo,despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development. View Publication

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus,hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks,however,conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology,retained a normal karyotype,and expressed characteristic hESC markers (OCT4,SSEA3,SSEA4 and TRA-1-60). Moreover,the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus,the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells. View Publication

Human embryonic stem cells (hESCs) are self-renewing,pluripotent cells derived from the inner cell mass of blastocysts,early-stage embryos,or blastomeres. hESCs can be propagated indefinitely in an undifferentiated state in vitro and have the ability to differentiate into all cell types of the body. Therefore,these cells can potentially provide an unlimited source of cells and hold promise for transplantation therapy,regenerative medicine,drug screening and discovery,and basic scientific research. Surplus human embryos donated for hESC derivation are extremely valuable,and inefficient derivation of hESCs would be a terrible waste of human embryos. Here,we describe a method for isolating hESC lines from human blastocysts with high efficiency. We also describe the methods for excising differentiated areas from partially differentiated hESC colonies and re-isolating undifferentiated hESCs from extremely differentiated hESC colonies. View Publication

Human somatic cells can be reprogrammed to the pluripotent state to become human-induced pluripotent stem cells (hiPSC). This reprogramming is achieved by activating signaling pathways that are expressed during early development. These pathways can be induced by ectopic expression of four transcription factors—Oct4,Sox2,Klf4,and c-Myc. Although there are many ways to deliver these transcription factors into the somatic cells,this chapter will provide protocols that can be used to generate hiPSC from lentiviruses. View Publication

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However,the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study,we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette,we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly,more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification,and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional,as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore,hESCs stably modified with the CD1d gene may serve as a convenient,unlimited,and competent DC source for iNKT cell-based cancer immunotherapy. View Publication

Human cord blood (CB)-derived CD133+ cells carry characteristics of primitive hematopoietic cells and proffer an alternative for CD34+ cells in hematopoietic stem cell (HSC) transplantation. To characterize the CD133+ cell population on a genetic level,a global expression analysis of CD133+ cells was performed using oligonucleotide microarrays. CD133+ cells were purified from four fresh CB units by immunomagnetic selection. All four CD133+ samples showed significant similarity in their gene expression pattern,whereas they differed clearly from the CD133- control samples. In all,690 transcripts were differentially expressed between CD133+ and CD133- cells. Of these,393 were increased and 297 were decreased in CD133+ cells. The highest overexpression was noted in genes associated with metabolism,cellular physiological processes,cell communication,and development. A set of 257 transcripts expressed solely in the CD133+ cell population was identified. Colony-forming unit (CFU) assay was used to detect the clonal progeny of precursors present in the studied cell populations. The results demonstrate that CD133+ cells express primitive markers and possess clonogenic progenitor capacity. This study provides a gene expression profile for human CD133+ cells. It presents a set of genes that may be used to unravel the properties of the CD133+ cell population,assumed to be highly enriched in HSCs. View Publication

Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently,murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4,Sox2,Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors,we have derived iPS cells from fetal,neonatal and adult human primary cells,including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency,and establish a method whereby patient-specific cells might be established in culture. View Publication