Activin/Nodal Inhibition Alone Accelerates Highly Efficient Neural Conversion from Human Embryonic Stem Cells and Imposes a Caudal Positional Identity

Background Neural conversion from human embryonic stem cells (hESCs) has been demonstrated in a variety of systems including chemically defined suspension culture, not requiring extrinsic signals, as well as in an adherent culture method that involves dual SMAD inhibition using Noggin and SB431542 (an inhibitor of activin/nodal signaling). Previous studies have also determined a role for activin/nodal signaling in development of the neural plate and anterior fate specification. We therefore sought to investigate the independent influence of SB431542 both on neural commitment of hESCs and positional identity of derived neural progenitors in chemically defined substrate-free conditions. Methodology/Principal Findings We show that in non-adherent culture conditions, treatment with SB431542 alone for 8 days is sufficient for highly efficient and accelerated neural conversion from hESCs with negligible mesendodermal, epidermal or trophectodermal contamination. In addition the resulting neural progenitor population has a predominantly caudal identity compared to the more anterior positional fate of non-SB431542 treated cultures. Finally we demonstrate that resulting neurons are electro-physiologically competent. Conclusions This study provides a platform for the efficient generation of caudal neural progenitors under defined conditions for experimental study

Differentiation of neurons from neural precursors generated in floating spheres from embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Neural differentiation of embryonic stem (ES) cells is usually achieved by induction of ectoderm in embryoid bodies followed by the enrichment of neuronal progenitors using a variety of factors. Obtaining reproducible percentages of neural cells is difficult and the methods are time consuming.</p> <p>Results</p> <p>Neural progenitors were produced from murine ES cells by a combination of nonadherent conditions and serum starvation. Conversion to neural progenitors was accompanied by downregulation of <it>Oct4 </it>and <it>NANOG </it>and increased expression of <it>nestin</it>. ES cells containing a GFP gene under the control of the <it>Sox1 </it>regulatory regions became fluorescent upon differentiation to neural progenitors, and ES cells with a tau-GFP fusion protein became fluorescent upon further differentiation to neurons. Neurons produced from these cells upregulated mature neuronal markers, or differentiated to glial and oligodendrocyte fates. The neurons gave rise to action potentials that could be recorded after application of fixed currents.</p> <p>Conclusion</p> <p>Neural progenitors were produced from murine ES cells by a novel method that induced neuroectoderm cells by a combination of nonadherent conditions and serum starvation, in contrast to the embryoid body method in which neuroectoderm cells must be selected after formation of all three germ layers.</p

Neuroprotective Effect of Transplanted Human Embryonic Stem Cell-Derived Neural Precursors in an Animal Model of Multiple Sclerosis

BACKGROUND: Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination and suppress the inflammatory process. METHODS: We transplanted human embryonic stem cells (hESC)-derived early multipotent neural precursors (NPs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted NPs on the functional and pathological manifestations of the disease. RESULTS: Transplanted hESC-derived NPs significantly reduced the clinical signs of EAE. Histological examination showed migration of the transplanted NPs to the host white matter, however, differentiation to mature oligodendrocytes and remyelination were negligible. Time course analysis of the evolution and progression of CNS inflammation and tissue injury showed an attenuation of the inflammatory process in transplanted animals, which was correlated with the reduction of both axonal damage and demyelination. Co-culture experiments showed that hESC-derived NPs inhibited the activation and proliferation of lymph node-derived T cells in response to nonspecific polyclonal stimuli. CONCLUSIONS: The therapeutic effect of transplantation was not related to graft or host remyelination but was mediated by an immunosuppressive neuroprotective mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may serve as the first step towards further developments of hESC for cell therapy in MS

Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

BACKGROUND: The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. METHODS/FINDINGS: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. CONCLUSIONS/SIGNIFICANCE: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases

Human embryonic stem cell neural differentiation and enhanced cell survival promoted by hypoxic preconditioning

Transplantation of neural progenitors derived from human embryonic stem cells (hESCs) provides a potential therapy for ischemic stroke. However, poor graft survival within the host environment has hampered the benefits and applications of cell-based therapies. The present investigation tested a preconditioning strategy to enhance hESC tolerance, thereby improving graft survival and the therapeutic potential of hESC transplantation. UC06 hESCs underwent neural induction and terminal differentiation for up to 30 days, becoming neural lineage cells, exhibiting extensive neurites and axonal projections, generating synapses and action potentials. To induce a cytoprotective phenotype, hESC-derived neurospheres were cultured at 0.1% oxygen for 12 h, dissociated and plated for terminal differentiation under 21% oxygen. Immunocytochemistry and electrophysiology demonstrated the ‘hypoxic preconditioning' promoted neuronal differentiation. Western blotting revealed significantly upregulated oxygen-sensitive transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, while producing a biphasic response within HIF targets, including erythropoietin, vascular endothelial growth factor and Bcl-2 family members, during hypoxia and subsequent reoxygenation. This cytoprotective phenotype resulted in a 50% increase in both total and neural precursor cell survival after either hydrogen peroxide insult or oxygen–glucose deprivation. Cellular protection was maintained for at least 5 days and corresponded to upregulation of neuroprotective proteins. These results suggest that hypoxic preconditioning could be used to improve the effectiveness of human neural precursor transplantation therapies

Cell-Surface Marker Signatures for the Isolation of Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations

Synaptically-Competent Neurons Derived from Canine Embryonic Stem Cells by Lineage Selection with EGF and Noggin

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABAA- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model

Derivation of Xeno-Free and GMP-Grade Human Embryonic Stem Cells – Platforms for Future Clinical Applications

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs

Differentiation of Human Embryonic Stem Cells to Regional Specific Neural Precursors in Chemically Defined Medium Conditions

Background: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury. Methodology and Principal Findings: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis. Conclusions/Significance: These findings suggest that our differentiation protocol has the capacity to generate regionspecific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-cultur