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      Derivation of High Purity Neuronal Progenitors from Human Embryonic Stem Cells

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          Abstract

          The availability of human neuronal progenitors (hNPs) in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs) in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use.

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          Feeder-free growth of undifferentiated human embryonic stem cells.

          C. Xu, M Inokuma, J. Denham … (2001)
          Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.
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            Directed differentiation of telencephalic precursors from embryonic stem cells.

            Kiichi Watanabe, Daisuke Kamiya, Ayaka Nishiyama … (2005)
            We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.
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              Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice.

              Brian Cummings, Nobuko Uchida, Stanley Tamaki … (2005)
              We report that prospectively isolated, human CNS stem cells grown as neurospheres (hCNS-SCns) survive, migrate, and express differentiation markers for neurons and oligodendrocytes after long-term engraftment in spinal cord-injured NOD-scid mice. hCNS-SCns engraftment was associated with locomotor recovery, an observation that was abolished by selective ablation of engrafted cells by diphtheria toxin. Remyelination by hCNS-SCns was found in both the spinal cord injury NOD-scid model and myelin-deficient shiverer mice. Moreover, electron microscopic evidence consistent with synapse formation between hCNS-SCns and mouse host neurons was observed. Glial fibrillary acidic protein-positive astrocytic differentiation was rare, and hCNS-SCns did not appear to contribute to the scar. These data suggest that hCNS-SCns may possess therapeutic potential for CNS injury and disease.
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                Author and article information

                Contributors
                : Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2011
                Publication date (Electronic): 6 June 2011
                Volume: 6
                Issue: 6
                Electronic Location Identifier: e20692
                Affiliations
                [1 ]Reeve-Irvine Research Center, Sue and Bill Gross Stem Cell Research Center, Department of Anatomy and Neurobiology, School of Medicine, Sue & Bill Gross Hall, a CIRM Institute, University of California Irvine, Irvine, California, United States of America
                [2 ]California Stem Cell, Inc., Irvine, California, United States of America
                [3 ]Pfizer Regenerative Medicine, Cambridge, Massachusetts, United States of America
                Baylor College of Medicine, United States of America
                Author notes

                Conceived and designed the experiments: GN MMS SNP SR AJP MEC JDM CNA HSK. Performed the experiments: GN MMS SNP SR. Analyzed the data: GN MMS SNP SR AJP HSK. Contributed reagents/materials/analysis tools: JDM CNA HSK. Wrote the paper: GN MMS SNP SR HSK.

                Article
                Publisher ID: PONE-D-11-00126
                DOI: 10.1371/journal.pone.0020692
                PMC ID: 3108963
                PubMed ID: 21673956
                SO-VID: 629b1881-782c-439f-ac66-86b6c165c622
                Copyright © Nistor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                Date received : 10 December 2010
                Date accepted : 10 May 2011
                Page count
                Pages: 11
                Categories
                Subject: Research Article
                Subject: Biology
                Subject: Developmental Biology
                Subject: Stem Cells
                Subject: Embryonic Stem Cells
                Subject: Neural Stem Cells
                Subject: Stem Cell Lines
                Subject: Cell Differentiation
                Subject: Cell Fate Determination
                Subject: Molecular Cell Biology
                Subject: Cellular Types
                Subject: Stem Cells
                Subject: Embryonic Stem Cells
                Subject: Neural Stem Cells
                Subject: Stem Cell Lines
                Subject: Neurons
                Subject: Neuroscience
                Subject: Developmental Neuroscience
                Subject: Neural Stem Cells
                Subject: Cellular Neuroscience

                ScienceOpen disciplines: Uncategorized
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                ScienceOpen disciplines: Uncategorized

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