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First published online June 21, 2007
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Submitted on November 15, 2006
Accepted on May 17, 2007

TECHNOLOGY DEVELOPMENT

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

Jan Pruszak 1, Kai Christian Sonntag 1, Moe Hein Aung 1, Rosario Sanchez-Pernaute 1, Ole Isacson 1*

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

* To whom correspondence should be addressed. E-mail: isacson{at}hms.harvard.edu.


  Abstract

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-GFP+ 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 ES cell types (such as SSEA-3, -4, TRA-1-81, TRA-1-60), neural stem and precursor cells (such as CD133, SSEA-1 [CD15], A2B5, FORSE-1, CD29, CD146, p75 [CD271]) and differentiated neurons (such as CD24 or NCAM [CD56]). At later stages of neural differentiation, the neural cell adhesion molecule 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 (1) a feasible approach for experimental cell sorting of differentiated neurons, (2) an initial survey of surface antigens present during neural differentiation of hESC, and (3) a framework for developing cell selection strategies for neural cell-based therapies.

Key Words. human embryonic stem cells, neurons, surface antigens, fluorescence-activated cell sorting, immunomagnetic cell separation, cell therapy, Parkinson disease, neural lineage, neural development




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