Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use.

Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson's disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson's disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge.

HSP90-incorporating chaperome networks as biosensor for disease-related pathways in patient-specific midbrain dopamine neurons.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors.

CryoPause: A New Method to Immediately Initiate Experiments after Cryopreservation of Pluripotent Stem Cells.

Human pluripotent stem cells (PSCs) provide an unlimited cell source for cell therapies and disease modeling. Despite their enormous power, technical aspects have hampered reproducibility. Here, we describe a modification of PSC workflows that eliminates a major variable for nearly all PSC experiments: the quality and quantity of the PSC starting material.

Bringing Neural Cell Therapies to the Clinic: Past and Future Strategies.

Cell replacement therapy in the nervous system has a rich history, with ∼40 years of research and ∼30 years of clinical experience. There is compelling evidence that appropriate cells can integrate and function in the dysfunctioning human nervous system, but the clinical results are mixed in practice. A number of factors conspire to vary patient outcome: the indication, cell source, patient selection, and team performing transplantation are all variables that can affect efficacy.

Derivation of Diverse Hormone-Releasing Pituitary Cells from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) provide an unlimited cell source for regenerative medicine. Hormone-producing cells are particularly suitable for cell therapy, and hypopituitarism, a defect in pituitary gland function, represents a promising therapeutic target. Previous studies have derived pituitary lineages from mouse and human ESCs using 3D organoid cultures that mimic the complex events underlying pituitary gland development in vivo.

Neural Crest Cells from Dual SMAD Inhibition.

Neural crest (NC) cells are migratory multipotent progenitors that delaminate from the neural tube during embryonic development and give rise to various cell types in different organs. These cells are a transient embryonic cell population and therefore difficult to obtain from primary sources. Deriving NC from human pluripotent stem cells offers an alternative way to provide large-scale human NC cells for developmental and disease-related studies.

Interrogation of a context-specific transcription factor network identifies novel regulators of pluripotency.

The predominant view of pluripotency regulation proposes a stable ground state with coordinated expression of key transcription factors (TFs) that prohibit differentiation. Another perspective suggests a more complexly regulated state involving competition between multiple lineage-specifying TFs that define pluripotency. These contrasting views were developed from extensive analyses of TFs in pluripotent cells in vitro.

miR-18b and miR-518b Target FOXN1 during epithelial lineage differentiation in pluripotent cells.

MicroRNAs (miRNAs) regulate myriad biological processes; however, their role in cell fate choice is relatively unexplored. Pluripotent NT2/D1 embryonal carcinoma cells differentiate into an epithelial/smooth muscle phenotype when treated with bone morphogenetic protein-2 (BMP-2). To identify miRNAs involved in epithelial cell development, we performed miRNA profiling of NT2/D1 cells treated with BMP-2 at 6, 12, and 24 h, and on days 6 and 10.

Human iPSC-based modeling of late-onset disease via progerin-induced aging.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging.

The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells.

Fragile X syndrome (FXS) is caused by a CGG repeat expansion in the FMR1 gene that appears to occur during oogenesis and during early embryogenesis. One model proposes that repeat instability depends on the replication fork direction through the repeats such that (CNG)n hairpin-like structures form, causing DNA polymerase to stall and slip. Examining DNA replication fork progression on single DNA molecules at the endogenous FMR1 locus revealed that replication forks stall at CGG repeats in human cells.

Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment.

Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy.

The endothelial cell line bEnd.3 maintains human pluripotent stem cells.

Endothelial cells line blood vessels and coordinate many aspects of vascular biology. More recent work has shown that endothelial cells provide a key niche in vivo for neural stem cells. In vitro, endothelial cells secrete a factor that expands neural stem cells while inhibiting their differentiation.

Converting human pluripotent stem cells to neural tissue and neurons to model neurodegeneration.

Human embryonic stem cells (hESCs) and the related induced pluripotent stem cells (hiPSCs) have attracted considerable attention since they can provide an unlimited source of many different tissue types. One challenge of using pluripotent cells is directing their broad differentiation potential into one specific tissue or cell fate. The cell fate choices of extraembryonic, endoderm, mesoderm, and ectoderm (including neural) lineages represent the earliest decisions.

Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation.

Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, and we demonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction.

BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.

Human embryonic stem cells (hESCs) have enormous potential for applications in basic biology and regenerative medicine. However, harnessing the potential of hESCs toward generating homogeneous populations of specialized cells remains challenging. Here we describe a novel technology for the genetic identification of defined hESC-derived neural cell types using bacterial artificial chromosome (BAC) transgenesis.

Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library.

Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated.

In vivo egress of an alphaherpesvirus from axons.

Many alphaherpesviruses establish a latent infection in the peripheral nervous systems of their hosts. This life cycle requires the virus to move long distances in axons toward the neuron's cell body during infection and away from the cell body during reactivation. While the events underlying entry of the virion into neurons during infection are understood in principle, no such consensus exists regarding viral egress from neurons after reactivation.