Human ESC-derived Neural Rosettes and Neural Stem Cell Progression

Developmental Biology Program, Division of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Cold Spring Harbor Symposia on Quantitative Biology 03/2009; 73:377-87. DOI: 10.1101/sqb.2008.73.052
Source: PubMed

ABSTRACT Neural stem cells (NSCs) are defined by their ability to self-renew while retaining differentiation potential toward the three main central nervous system (CNS) lineages: neurons, astrocytes, and oligodendrocytes. A less appreciated fact about isolated NSCs is their narrow repertoire for generating specific neuron types, which are generally limited to a few region-specific subtypes such as GABAergic and glutamatergic neurons. Recent studies in human embryonic stem cells have identified a novel neural stem cell stage at which cells exhibit plasticity toward generating a broad range of neuron types in response to appropriate developmental signals. Such rosette-stage NSCs (R-NSCs) are also distinct from other NSC populations by their specific cytoarchitecture, gene expression, and extrinsic growth requirements. Here, we discuss the properties of R-NSCs within the context of NSC biology and define some of the key questions for future investigation. R-NSCs may represent the first example of a NSC population capable of recreating the full cellular diversity of the developing CNS, with implications for both basic stem cell biology and translational applications in regenerative medicine and drug discovery.

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    ABSTRACT: The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.
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  • Frontiers in Bioscience 01/2012; 17(1):65. DOI:10.2741/3916 · 4.25 Impact Factor
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    ABSTRACT: Despite significant advances in commercially available media and kits and the differentiation approaches for human neural stem cell (NSC) generation, NSC production from the differentiation of human pluripotent stem cell (hPSC) is complicated by its time-consuming procedure, complex medium composition, and purification step. In this study, we developed a convenient and simplified NSC production protocol to meet the demand of NSC production. We demonstrated that NSCs can be generated efficiently without requirement of specific small molecules or embryoid body formation stage. Our experimental results suggest that a short suspension culture period may facilitate ectoderm lineage specification rather than endoderm or mesoderm lineage specification from hPSCs. The method developed in this study shortens the turnaround time of NSC production from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) differentiation. It provides a straightforward and useful strategy for generating NSCs that can benefit a wide range of research applications for human brain research.
    Journal of Biotechnology 08/2014; 188. DOI:10.1016/j.jbiotec.2014.07.453 · 2.88 Impact Factor


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