INVITRO LABELING STRATEGIES FOR IDENTIFYING PRIMARY NEURAL TISSUE ANDA NEURONAL CELL-LINE AFTER TRANSPLANTATION IN THE CNS

Potential labels for identifying embryonic raphe neurons and a clonal, neuronally differentiating, raphe-derived cell line, RN33B, in CNS tr ansplantation studies were tested by first characterizing the labels i n vitro. The labels that were tested included 4,6-diamidino-2-phenylin dole hydrochloride, ,1'-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate, the Escherichia coli lacZ gene, Fast Blue, Fluoro-Gold, fluorescein-conjugated latex microspheres, fluorescein isothiocyanate- conjugated or nonconjugated Phaseolus vulgaris leucoagglutinin, methyl o-(6-amino-3'-imino-3H-xanthen-9-yl) benzoate monohydrochloride, or t etanus toxin C fragment. Subsequently, the optimal in vitro labels for embryonic raphe neurons and for RN33B cells were characterized in viv o after CNS transplantation. In vitro, 1,1'-dioctadecyl-3,3,3'-tetmmet hylindocarbocyanine perchlorate (DiI) optimally labeled embryonic neur ons. The Escherichia coli lacZ gene optimally labeled RN33B cells. Mos t labels were rapidly diluted in cultures of embryonic astrocytes and proliferating RN33B cells. Some labels were toxic and were often retai ned in cellular debris. In vivo, Dil was visualized in transplanted, D il-labeled raphe neurons, but not in astrocytes up to 1 mo posttranspl ant. DiI-labeled host cells were seen after transplantation of lysed, DiI-labeled cells. Beta-Galactosidase was visualized in transplanted, Escherichia coli lacZ gene-labeled RN33B cells after 15 days in vivo. No beta-galactosidase was seen in host cells after transplantation of lysed, lacZ-labeled RN33B cells. The results demonstrate that labels f or use in CNS transplantation studies should be optimized for the spec ific population of donor cells under study, with the initial step bein g characterization in vitro followed by in vivo analysis. Appropriate controls for false-positive labeling of host cells should always be as sessed.