Here, we describe the development of standard conditions that allow for the use and discovery of surface antigens as markers for analysis and cell selection of hESC undergoing neuronal differentiation. Although most opinions to date discuss the use of cell sorting to eliminate tumors or enrich neural precursors [6
], we believe that such methods are also critical with respect to developmental stage (time) and the heterogeneity of cell populations typical of hESC-derived cultures. An additional advantage illustrated by our studies is also the ability to isolate hESC-differentiated neurons for analysis in science and biomedicine. Using immunocytochemical and flow cytometric analysis of differentiating hESC, we characterized the temporal profile of a variety of surface antigens expressed throughout a neuronal cell differentiation protocol. In parallel, we optimized the conditions of FACS to enable growth and analysis of purified hESC-derived neural cells and mature neurons postsort. We found that identification of and selection according to cell surface antigens during neuronal differentiation of hESC (a) is feasible with the protocols described, (b) can be applied for developmental studies in neurobiology, and (c) might facilitate the development of cell therapeutic studies in human embryonic and neural stem cell research.
In the scientific and clinical contexts, the potential of embryonic stem cells for self-renewal and differentiation into any given tissue of the human body is both an opportunity for cell restoration and a concern, when noncontrolled growth occurs [5
]. Given the cellular heterogeneity observed during differentiation of hESC in vitro, eliminating the unwanted cell populations from further cultivation steps, prior to transplantation, would increase the purity of the graft and allow for a better defined cell composition to be transferred. The characterization of putatively therapeutic cell suspensions according to surface markers is likely a prerequisite to future applications in a clinical setting. Flow cytometry has previously been applied in the analysis of neural cells from fetal neural tissue [16
] and more recently in the prospective isolation of neural stem cells from the adult nervous system [54
]. Our studies show now that the principles of flow cytometric methodology can be applied in the analysis and purification of hESC-derived neural cells. By utilizing these advantages and by reducing physical stressors, we were thus able to purify viable neuronal cells for extended in vitro and in vivo characterization.
Analytically, FACS technology was successfully used here in a hESC differentiation paradigm to elucidate the characteristics of specific cellular subpopulations, thereby allowing further studies of critical steps in neural development. Additionally, the increasing necessity for cell selection is exemplified by the heterogeneity observed in this study () and others [2
]. Nonpurified embryonic stem cell populations have a broader developmental spectrum compared with fetal primary cultures. Although the surface antigens documented here represent an initial subset of immature, neural, and neuronal markers expressed during hESC differentiation, further characterization is needed.
The profiling of neural surface antigens initiated here forms the basis for exploring other markers and for investigating their mechanisms and function in the neural context. This profiling of surface marker antigens needs to be extended so that a more complete picture of neural cell-cell interaction can be achieved. Such studies are critical for further understanding of human neural development, the potential application of cell-based therapies of neurological diseases, and may also prove particularly useful in the diagnosis of neural tumors, where the same markers may have similar relevance.
In the studies described, we demonstrate that FACS can be used to monitor the presence of immature hESC during neuronal differentiation protocols (SSEA-3, -4, Tra-1-60, Tra-1-81) and, concluding from previous studies, may therefore be applied to avert tumor formation in neurotransplantation of ES-derived neural cells. By identifying a number of neural markers (SSEA-1, FORSE-1, CD29, CD146, A2B5, p75) present at neural stem and precursor stages of hESC differentiation, our methods allow for the separation of cell populations committed to specific lineages of neural differentiation. Such isolated cells could provide a major advantage both in experimental and potential cell therapy settings. Although the full development from a hESC to a differentiated postmitotic neuron in this protocol takes in the order of 30–40 days [2
], using an intermediate population, frozen or expanded, provides a shorter cell culture protocol [52
]. Moreover, differential expression of these antigens on neural precursor populations will allow for detailed studies, for example, of lineage restriction at this stage. For the later stages of hESC differentiation, an antibody targeting human specific NCAM (CD56) was used to monitor neural specification in vitro and in vivo. Such experiments indicated that hESC-derived neuronal cells can be FACS-purified, transplanted, and survive in the brain of a rodent model.
How can functional integration into synaptic circuitry, and ultimately the reconstitution of function in models of neurological disease, be demonstrated? For such situations, genetic approaches using transduction with fluorescence-labeled specific constructs such as synapsin-eGFP, or using fluorogenic substrates for enzymes such as aldehyde dehydrogenase, may be applied to isolate neural cell subpopulations. The successful cell selection of synapsin-eGFP+
cells demonstrates that even mature, neuronally differentiated cells can be FACS-purified. Synapsin expression correlates well with other synaptic markers such as Syntaxin (). An advantage of using gene-engineered cells is the avoidance of several washing and centrifugation steps during cell preparation prior to FACS, which can increase cell loss when using surface marker staining. When aiming for potential clinical applications though, the antibody-based fluorescent labeling will advance current approaches and make neural transplantation methods more feasible, as proven by clinical routines in hematological medicine [8
]. Although FACS for neuronal cells has been used in studies of animal models, so far it has not been widely applied in neurobiology, probably due to the specific inherent problems in using these fragile neuronal cell types. However, we demonstrate here that, with appropriate methodological adaptations, FACS can be used as a tool with great advantage in the studies of neuronal development and function. One advantage of using embryonic stem cell-derived neuronal cells is the large number of cells that can be generated in vitro. For example, in clinical trials of cellular transplantation therapy for Parkinson disease, approximately 1.1–1.3 × 106
cells are obtained per human fetal ventral midbrain dissected (sixth to ninth week of gestation). Using hESC derived neuronal cells at late stage of differentiation, in our hands more than 3 × 107
cells are obtained from one 10-cm dish, an amount of cells that allows the use of FACS to sort out rare subpopulations relevant to transplantation in neurological disease. The neuronal cell selection procedures described here enable researchers from different fields of neurobiology to use FACS as a method to isolate viable neuronal cell populations derived from hESC. Obviously this methodology can also be applied to mouse ES or primary neural cells. In the future, alternative cell selection procedures using microfluidics and optical switches [56
] may be used to facilitate the isolation and yield of hESC-derived cells for clinical applications.
Beyond the sorting aspect itself, flow cytometric methodology offers analytical options such as quantification according to intracellular stains of fixed cells [58
], viability assays using fluorescent DNA-binding dyes or caspase substrates (), cell cycle analysis, or 5-bromo-2′-deoxyuridine studies, which can be applied in neurobiological research. Multiparametric analysis using a combination of surface markers [11
], as well as the detection of phosphorylation states of intracellular proteins for the elucidation of cellular signaling networks [8
], provides further powerful analytical tools for studies of the nervous system. Testing drugs or factors on a defined population of primary or ESC-derived cells allows for a more precise analysis and understanding of their effects. The isolation of distinct neural cell subpopulations can thus promote our understanding of biology and cell-cell interactions, as in previous immunological and hematological research during the last decade [9
]. We believe that broader application of the methods presented here will enable scientific investigations of neural cell subsets derived from hESC to enhance our understanding of cellular developmental neurobiology and eventually lead to its translation to the treatment of neurological diseases.