Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3ß and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.

The neural crest: a versatile organ system.

The neural crest is the name given to the strip of cells at the junction between neural and epidermal ectoderm in neurula-stage vertebrate embryos, which is later brought to the dorsal neural tube as the neural folds elevate. The neural crest is a heterogeneous and multipotent progenitor cell population whose cells undergo EMT then extensively and accurately migrate throughout the embryo. Neural crest cells contribute to nearly every organ system in the body, with derivatives of neuronal, glial, neuroendocrine, pigment, and also mesodermal lineages. This breadth of developmental capacity has led to the neural crest being termed the fourth germ layer. The neural crest has occupied a prominent place in developmental biology, due to its exaggerated migratory morphogenesis and its remarkably wide developmental potential. As such, neural crest cells have become an attractive model for developmental biologists for studying these processes. Problems in neural crest development cause a number of human syndromes and birth defects known collectively as neurocristopathies; these include Treacher Collins syndrome, Hirschsprung disease, and 22q11.2 deletion syndromes. Tumors in the neural crest lineage are also of clinical importance, including the aggressive melanoma and neuroblastoma types. These clinical aspects have drawn attention to the selection or creation of neural crest progenitor cells, particularly of human origin, for studying pathologies of the neural crest at the cellular level, and also for possible cell therapeutics. The versatility of the neural crest lends itself to interlinked research, spanning basic developmental biology, birth defect research, oncology, and stem/progenitor cell biology and therapy.

Parameters affecting efficiency of in ovo electroporation of the avian neural tube and crest.

BACKGROUND: Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quantitatively. RESULTS: Genome integrating pT2K-CAGGS-GFP and pCAGGS-T2TP transposase plasmids were co-electroporated into quail E2 embryo trunk neural tube and the proportion of GFP-expressing neural cells was counted 1 and 7 days later. Electroporation efficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 µg/µL and 3 pulses. Bilateral electroporation transfected more cells than unilateral but less than that anticipated by doubling the unilateral treatment. Holding the concentration of GFP plasmid constant and varying the transposase plasmid concentration revealed an optimum ratio of, in this case, 4:1 (1.2 µg/µL:0.3 µg/µL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivo with the transposase system, but declined with non-integrated plasmid. Transfection of neural crest cells was low if electroporated less than 6-8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this event be termed epithelio-mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigration from the neural epithelium. CONCLUSIONS: Co-electroporation in ovo must take into account plasmid(s) concentration and ratio, pulse number, pulse directionality, and timing.