MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons.

Mutations in the methyl-DNA-binding protein MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). How MECP2 contributes to transcriptional regulation in normal and disease states is unresolved; it has been reported to be an activator and a repressor. We describe here the first integrated CUT&Tag, transcriptome, and proteome analyses using human neurons with wild-type (WT) and mutant MECP2 molecules.

Functional analysis of CX3CR1 in human induced pluripotent stem (iPS) cell-derived microglia-like cells.

Microglia are the primary immune cells of the central nervous system and crucial to proper development and maintenance of the brain. Microglia have been recognized to be associated with neurodegenerative diseases and neuroinflammatory disorders. CX3C chemokine receptor 1 (CX3CR1), which is specifically expressed in microglia, regulates microglia homeostatic functions such as microglial activation and is downregulated in aged brain and disease-associated microglia in rodents, yet its role in human microglia is not fully understood.

Efficient derivation of microglia-like cells from human pluripotent stem cells.

Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions.

Genetic and chemical correction of cholesterol accumulation and impaired autophagy in hepatic and neural cells derived from Niemann-Pick Type C patient-specific iPS cells.

Niemann-Pick type C (NPC) disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs) using transcription activator-like effector nucleases (TALENs).

Identification and rescue of α-synuclein toxicity in Parkinson patient-derived neurons.

The induced pluripotent stem (iPS) cell field holds promise for in vitro disease modeling. However, identifying innate cellular pathologies, particularly for age-related neurodegenerative diseases, has been challenging. Here, we exploited mutation correction of iPS cells and conserved proteotoxic mechanisms from yeast to humans to discover and reverse phenotypic responses to α-synuclein (αsyn), a key protein involved in Parkinson's disease (PD).

Global transcriptional and translational repression in human-embryonic-stem-cell-derived Rett syndrome neurons.

Rett syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors.

Molecular signatures of human induced pluripotent stem cells highlight sex differences and cancer genes.

Although human induced pluripotent stem cells (hiPSCs) have enormous potential in regenerative medicine, their epigenetic variability suggests that some lines may not be suitable for human therapy. There are currently few benchmarks for assessing quality. Here we show that X-inactivation markers can be used to separate hiPSC lines into distinct epigenetic classes and that the classes are phenotypically distinct.

Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells.

Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however, present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure-function relationships between material properties and biological performance.

Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations.

The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations.

Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing.

Metastable pluripotent states in NOD-mouse-derived ESCs.

Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming.

Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons.

Reprogramming of murine and human somatic cells using a single polycistronic vector.

Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, which support efficient polycistronic expression from a single promoter.

A drug-inducible system for direct reprogramming of human somatic cells to pluripotency.

Current approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous "secondary" somatic cells, which carry the reprogramming factors as defined doxycycline (DOX)-inducible transgenes.

Nanoparticles for gene transfer to human embryonic stem cell colonies.

We develop biodegradable polymeric nanoparticles to facilitate nonviral gene transfer to human embryonic stem cells (hESCs). Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self assembly of cationic, hydrolytically degradable poly(beta-amino esters) and plasmid DNA. By varying the end group of the polymer, we can tune the biophysical properties of the resulting nanoparticles and their gene-delivery efficacy.

Isolation and characterization of human embryonic stem cells.

The derivation of human embryonic stem (hES) cells is a challenging procedure. The isolation and maintenance of hES is visually and manually complicated, involving mechanical or enzymatic passaging using either collagenase or trypsin. This chapter describes detailed protocols that have been used for the derivation, maintenance, and characterization of hES cells in vitro along with protocols to test their differentiation potential in vivo.

Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions.

Research on the cell fate determination of embryonic stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human embryonic stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine factors affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs.

Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces.

Background: Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins.

Directed neuronal differentiation of human embryonic stem cells.

Background: We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII).

Human embryonic stem cell lines derived from discarded embryos.

Human pluripotent embryonic stem (ES) cells have important potential in regenerative medicine and as models for human preimplantation development; however, debate continues over whether embryos should be destroyed to produce human ES cells. We have derived four ES cell lines on mouse embryonic fibroblast cells in medium supplemented with basic fibroblast growth factor, human recombinant leukemia inhibitory factor, and fetal bovine serum. The source of these cell lines was poor-quality embryos that in the course of routine clinical practice would have been discarded.