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 Differentiation Toward Regional Specific Neural Precursors

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that have the capacity to differentiate into a wide variety of cell types. This potentiality represents a promising source to overcome many human diseases by providing an unlimited supply of all cell types, including cells with neural characteristics. Therefore, this review summarizes early neural development and the potential of hESCs to differentiate under in vitro conditions, examining at the same time the potential use of differentiated hESCs for therapeutic applications for neural tissue and cell regeneration

Dual embryonic origin of the mammalian enteric nervous system.

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut

Oct4 Is Required ∼E7.5 for Proliferation in the Primitive Streak

<div><p>Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, <i>in vivo</i>, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ∼E6.0–E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ∼E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ∼E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.</p></div

Tetraploid chimeras indicate that Oct4 is not required extraembryonically ∼E7.5, while diploid chimeras indicate that Oct4^+/+ can compensate for Oct4^−/− cells embryonically.

<p>All embryos transferred to a surrogate and depicted or described in panels A–E were induced with tamoxifen ∼E6.0 and ∼E6.5 to compensate for the variability in developmental timing associated with transfer (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1Q</a>,AB). Scale bars in panels A–D are 200 um. <b>A</b> A representative embryo from aggregation of Oct4^+/+ RFP ES cells with a tetraploid Oct4^f/f embryo. Oct4^f/f extraembryonic tissue yielded E9.5 chimeric embryos with no phenotype. <b>B</b> An Oct4^f/f;CreER^T2+/− E9.5 embryo with the Oct4^{COND MUT} phenotype. <b>C</b> Oct4 depletion in extraembryonic tissue is compatible with WT development. A representative chimera consisting of RFP+ Oct4^+/+ ES cell derived embryo and tetraploid Oct4^f/f;CreER^T2+/− extraembryonic tissue. The embryo has turned (compare panel ‘B’ where the tail is behind to panel ‘C’ where it is in front), undergone NTC and posterior extension (compare the lack of somites and short tail in panel ‘B’ to the somites and full-length tail in ‘C’). <b>D</b> The most severe embryonic defect observed in a diploid chimeras consisting of Oct4^+/+ and Oct4^f/f;CreER^T2+/− cells. The neural tube is open between closure points 1 and 2, indicated here with a black bracket. All Oct4^{COND MUT} features aside from cranial NTC defect, which is still present in 5/16 mosaic embryos, are rescued by Oct4^+/+ cells in these diploid chimeras (16/16). For example, this embryo has ‘turned’ such that it faces its tail and the posterior has extended normally. <b>E</b> Quantification of the genotypes and phenotypes of recovered chimeric embryos.</p

Pathway enrichment and confirmation of a subset of differentially expressed genes following Oct4 depletion.

<p>Litters depicted in ‘A,B’ were induced with tamoxifen ∼E7.0 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>E). The FDR for reported enrichments in ‘A,B’ is <0.001, based on 1000 random permutations of annotated genes. <b>A</b> Unsupervised clustering of relative (Oct4^f/f;CreER^T2+/−/Oct4^f/f littermates) gene expression sub-setted for Oct4 binding targets following Oct4 depletion. Enrichment for the same pathways in the global differential expression set and subset directly targeted by Oct4 support the utility of sub-setting for Oct4 binding targets in identifying primary effects of Oct4 depletion and the relevance of these primary effects to the Oct4^{COND MUT} phenotype in that they appear amplified into effects on the overall gene expression profile (see blue script in panel ‘A’ and ‘B’ for these). The most enriched pathway is provided for each cluster, and an additional pathway provided (in black text) for the cluster where the most enriched pathway in the Oct4 target set did not translate to a global change. <b>B</b> Unsupervised clustering of global differential expression (same dataset as panel ‘A’): Oct4^f/f;CreER^T2+/−/Oct4^f/f. The most enriched pathway and binding factor are provided for each cluster (black text), while primary effects that translated to enriched effects in the global set are in blue text. <b>C</b> Confirmation of expression change for select genes by quantitative PCR in independent litters (Oct4^f/f;CreER^T2+/−/Oct4^f/f ±s.e.m.). Litters were induced with tamoxifen ∼E7.0 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>G).</p

Decreased proliferation in the primitive streak occurs coincident with Oct4 depletion.

<p>The embryos depicted in panels ‘A–H’ were administered tamoxifen ∼E7.0 and ∼E7.5 and dissected 24 hrs ATA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>C). Scale bars in ‘A–H’ are 50 µm. <b>A, B</b> Specification of Chordin in the node still occurs after Oct4 depletion <b>A</b> Oct4^f/f 24 hrs ATA. <b>B</b> Oct4^f/f;CreER^T2+/− 24 hrs ATA. <b>C, D</b> The expression domain of p-Smad1 is altered after Oct4 depletion. <b>C</b> Oct4^f/f 24 hrs ATA <b>D</b> Oct4^f/f;CreER^T2+/− 24 hrs ATA. <b>E,F</b> Distribution of apoptotic Caspase-3+ cells 24 hrs ATA. <b>E</b> Oct4^f/f<b>F</b> Oct4^f/f;CreER^T2+/−. <b>G,H</b> Distribution of Phospho-histone-3 (Ph3), which marks proliferating cells, 24 hrs ATA. Proliferation is significantly reduced in the primitive streak (bracketed by a white line) of Oct4^f/f;CreER^T2+/− embryos. (G) Oct4^f/f (H) Oct4^f/f;CreER^T2+/−. <b>I</b> Quantification of apoptotic frequency ±s.e.m. in Oct4^f/f and Oct4^f/f;CreER^T2+/− embryos (F_1,44 = 13.16, p<0.05 2-way ANOVA, *p<0.05, **p<0.01 Bonferroni posttest). <b>J</b> Quantification of proliferation frequency ±s.e.m. in Oct4^f/f and Oct4^f/f;CreER^T2+/− embryos 24 hrs ATA (F_1,68 = 3.28, p<0.05 2-way ANOVA, **p<0.01 Bonferroni posttest). Oct4 removal only effects proliferation significantly in the primitive streak. <b>K</b> Distribution of gene expression changes in Oct4^f/f;CreER^T2+/− embryos. Relative (Oct4^f/f;CreER^T2+/−/Oct4^f/f) transcript abundance ±s.e.m. was quantified 24 hrs ATA by QPCR (F_2,8 = 12.14, p<0.05 2-way ANOVA, ***p<0.001 Bonferroni posttest). Embryos were administered tamoxifen ∼E7.0 and ∼E7.5 and dissected 24 hrs ATA for the experiment in panel ‘K’ (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>H).</p