Gabsang Lee

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest–related disorders.

Combined small molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors

Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types, including neurons. However, differentiation of hPSCs with extrinsic factors is a slow, step-wise process, mimicking the protracted timing of human development. Using a small-molecule screen, we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at >75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors, including tetrodotoxin (TTX)-resistant, SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development, suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain.

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

High-Throughput Screening Assay for the Identification of Compounds Regulating Self-Renewal and Differentiation in Human Embryonic Stem Cells

High-throughput screening (HTS) of chemical libraries has become a critical tool in basic biology and drug discovery. However, its implementation and the adaptation of high-content assays to human embryonic stem cells (hESCs) have been hampered by multiple technical challenges. Here we present a strategy to adapt hESCs to HTS conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice during differentiation. Global gene expression analysis upon drug treatment defines known and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the repertoire of chemical compounds for manipulating hESC fate. The availability of high-content assays should accelerate progress in basic and translational hESC biology.

Efficient Derivation of Functional Floor Plate Tissue from Human Embryonic Stem Cells

The floor plate (FP) is a critical signaling center during neural development located along the ventral midline of the embryo. Little is known about human FP development because of the lack of tissue accessibility. Here we report the efficient derivation of human embryonic stem cell (hESC)-derived FP tissue capable of secreting Netrin-1 and SHH and patterning primary and hESC derived tissues. FP induction in hESCs is dependent on early SHH exposure and occurs at the expense of anterior neurectoderm (AN). Global gene expression and functional studies identify SHH-mediated inhibition of Dkk-1 as key factor in FP versus AN specification. hESC-derived FP tissue is shown to be of anterior SIX6+ character but is responsive to caudalizing factors suppressing SIX6 expression and inducing a shift in usage of region-specific SHH enhancers. These data define the early signals that drive human FP versus AN specification and determine regional identity in hESC-derived FP.

Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression.

Patient-specific induced pluripotent stem cells (iPSCs) represent a novel system for modeling human genetic disease and could develop into a key drug discovery platform. We recently reported disease-specific phenotypes in iPSCs from familial dysautonomia (FD) patients. FD is a rare but fatal genetic disorder affecting neural crest lineages. Here we demonstrate the feasibility of performing a primary screen in FD-iPSC derived neural crest precursors. Out of 6,912 compounds tested we characterized 8 hits that rescue expression of IKBKAP, the gene responsible for FD. One of those hits, SKF-86466, is shown to induce IKBKAP transcription via modulation of intracellular cAMP levels and PKA dependent CREB phosphorylation. SKF-86466 also rescues IKAP protein expression and the disease-specific loss of autonomic neuron marker expression. Our data implicate alpha-2 adrenergic receptor activity in regulating IKBKAP expression and demonstrate that small molecule discovery in an iPSC-based disease model can identify candidate drugs for potential therapeutic intervention.Patient-specific induced pluripotent stem cells (iPSCs) represent a novel system for modeling human genetic disease and could provide a source of cells for large-scale drug-discovery screens. Here we demonstrate the feasibility of performing a primary screen in neural crest precursors derived from iPSCs that were generated from individuals with familial dysautonomia (FD), a rare, fatal genetic disorder affecting neural crest lineages. We tested 6,912 small-molecule compounds and characterized eight that rescued expression of IKBKAP, the gene responsible for FD. One of the hits, SKF-86466, was found to induce IKBKAP transcription through modulation of intracellular cAMP levels and PKA-dependent CREB phosphorylation. SKF-86466 also rescued IKAP protein expression and the disease-specific loss of autonomic neuronal marker expression. Our data implicate alpha-2 adrenergic receptor activity in regulating IKBKAP expression and demonstrate that small-molecule discovery using an iPSC-based disease model can identify candidate drugs for potential therapeutic intervention.

A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs.

The equivalence of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) remains controversial. Here we use genetically matched hESC and hiPSC lines to assess the contribution of cellular origin (hESC vs. hiPSC), the Sendai virus (SeV) reprogramming method and genetic background to transcriptional and DNA methylation patterns while controlling for cell line clonality and sex. We find that transcriptional and epigenetic variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detect between genetically matched hESCs and hiPSCs neither predict functional outcome nor distinguish an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional and epigenetic comparisons of pluripotent cell lines, explaining some of the previously observed differences between genetically unmatched hESCs and hiPSCs.

miR-371-3 Expression Predicts Neural Differentiation Propensity in Human Pluripotent Stem Cells

The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EPI-SCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR-371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior.

Modeling Neural Crest Induction, Melanocyte Specification, and Disease-Related Pigmentation Defects in hESCs and Patient-Specific iPSCs

Melanocytes are pigment-producing cells of neural crest (NC) origin that are responsible for protecting the skin against UV irradiation. Pluripotent stem cell (PSC) technology offers a promising approach for studying human melanocyte development and disease. Here, we report that timed exposure to activators of WNT, BMP, and EDN3 signaling triggers the sequential induction of NC and melanocyte precursor fates under dual-SMAD-inhibition conditions. Using a SOX10::GFP human embryonic stem cell (hESC) reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human NC induction. Subsequent maturation of hESC-derived melanocytes yields pure populations that match the molecular and functional properties of adult melanocytes. Melanocytes from Hermansky-Pudlak syndrome and Chediak-Higashi syndrome patient-specific induced PSCs (iPSCs) faithfully reproduce the ultrastructural features of disease-associated pigmentation defects. Our data define a highly specific requirement for WNT signaling during NC induction and enable the generation of pure populations of human iPSC-derived melanocytes for faithful modeling of pigmentation disorders.

Evaluation of Developmental Toxicants and Signaling Pathways in a Functional Test Based on the Migration of Human Neural Crest Cells

Background: Information on the potential developmental toxicity (DT) of the majority of chemicals is scarce, and test capacities for further animal-based testing are limited. Therefore, new approaches with higher throughput are required. A screening strategy based on the use of relevant human cell types has been proposed by the U.S. Environmental Protection Agency and others. Because impaired neural crest (NC) function is one of the known causes for teratologic effects, testing of toxicant effects on NC cells is desirable for a DT test battery. Objective: We developed a robust and widely applicable human-relevant NC function assay that would allow for sensitive screening of environmental toxicants and defining toxicity pathways. Methods: We generated NC cells from human embryonic stem cells, and after establishing a migration assay of NC cells (MINC assay), we tested environmental toxicants as well as inhibitors of physiological signal transduction pathways. Results: Methylmercury (50 nM), valproic acid (> 10 µM), and lead-acetate [Pb(CH3CO2)4] (1 µM) affected the migration of NC cells more potently than migration of other cell types. The MINC assay correctly identified the NC toxicants triadimefon and triadimenol. Additionally, it showed different sensitivities to various organic and inorganic mercury compounds. Using the MINC assay and applying classic pharmacologic inhibitors and large-scale microarray gene expression profiling, we found several signaling pathways that are relevant for the migration of NC cells. Conclusions: The MINC assay faithfully models human NC cell migration, and it reveals impairment of this function by developmental toxicants with good sensitivity and specificity.