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TECHNOLOGY DEVELOPMENT

Markers and Methods for Cell Sorting of Human Embryonic Stem Cell-Derived Neural Cell Populations

Neuroregeneration Laboratories, Center for Neuroregeneration Research, Udall Parkinson's Disease Center of Excellence, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA

Key Words. Human embryonic stem cells • Neurons • Surface antigens • Fluorescence-activated cell sorting Immunomagnetic cell separation • Cell therapy • Parkinson disease • Neural lineage • Neural development

Correspondence: Ole Isacson, M.D., Center for Neuroregeneration Research, McLean Hospital/Harvard Medical School, 115 Mill Street, MRC130, Belmont, Massachusetts 02478, USA. Telephone: 617-855-3283; Fax: 617-855-3284; e-mail: isacson{at}hms.harvard.edu

Received November 15, 2006; accepted for publication May 17, 2007.
First published online in STEM CELLS EXPRESS   June 21, 2007.



Neural cells differentiated in vitro from human embryonic stem cells (hESC) exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. Here, we describe standard conditions for the use and discovery of markers for analysis and cell selection of hESC undergoing neuronal differentiation. To generate better-defined cell populations, we established a working protocol for sorting heterogeneous hESC-derived neural cell populations by fluorescence-activated cell sorting (FACS). Using genetically labeled synapsin-green fluorescent protein-positive hESC-derived neurons as a proof of principle, we enriched viable differentiated neurons by FACS. Cell sorting methodology using surface markers was developed, and a comprehensive profiling of surface antigens was obtained for immature embryonic stem cell types (such as stage-specific embryonic antigen [SSEA]-3, -4, TRA-1-81, TRA-1-60), neural stem and precursor cells (such as CD133, SSEA-1 [CD15], A2B5, forebrain surface embryonic antigen-1, CD29, CD146, p75 [CD271]), and differentiated neurons (such as CD24 or neural cell adhesion molecule [NCAM; CD56]). At later stages of neural differentiation, NCAM (CD56) was used to isolate hESC-derived neurons by FACS. Such FACS-sorted hESC-derived neurons survived in vivo after transplantation into rodent brain. These results and concepts provide (a) a feasible approach for experimental cell sorting of differentiated neurons, (b) an initial survey of surface antigens present during neural differentiation of hESC, and (c) a framework for developing cell selection strategies for neural cell-based therapies.

Disclosure of potential conflicts of interest is found at the end of this article.

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