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Isolation of Definitive Zone and Chromaffin Cells Based upon Expression of CD56 (Neural Cell Adhesion Molecule) in the Human Fetal Adrenal Gland

Department of Laboratory Medicine (M.O.M.) and Center for Reproductive Sciences (J.V.R., M.N., H.I., R.B.J.), Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143

Address all correspondence and requests for reprints to: Marcus O. Muench, Ph.D., Department of Laboratory Medicine, University of California, San Francisco, 533 Parnassus Avenue, Room U-440, San Francisco, California 94143-0793. E-mail: muench{at}itsa.ucsf.edu.

The cortex of the human midgestation adrenals comprises a thin layer of cells, the definitive zone (DZ) that surrounds the larger, inner fetal zone (FZ). CD56 expression was observed by immunohistochemistry on DZ cells and isolated groups of cells within the FZ. CD56 mRNA expression also was detected among DZ cells but not selected sections of FZ cells isolated by laser capture microdissection. Flow cytometric analysis of dispersed adrenal cells indicated CD56 expression on a subset of adrenal cells lacking expression of hematopoietic (CD45⁺ and CD235a⁺) and endothelial (CD31⁺) cell markers. The CD56⁺CD31^-CD45^-CD235a^- cells were isolated by discontinuous-gradient centrifugation and fluorescence-activated cell sorting. The purified cells were enriched for DZ cells based on expression of mRNA for metallopanstimulin-1, nephroblastoma overexpressed gene, and 3-ß-hydroxysteroid dehydrogenase. P450c17 mRNA expression also was detected among a subset of CD56⁺ cells consistent with expression of low levels of this protein in some DZ cells. The presence of a subpopulation of chromaffin cells among the CD56⁺ population also was shown by the expression of chromogranin A mRNA. These findings indicate that CD56 expression can be used to isolate DZ and chromaffin cells to further study their functional and developmental properties.

This work was supported in part by NIH Grants DK59301 (to M.O.M.) and HD08478 (to R.B.J.). J.R. is the recipient of a fellowship from the Endocrine Fellows Foundation.

M.O.M. and J.V.R. contributed equally to this work.

Abbreviations: Chr-A, Chromogranin A; DZ, definitive zone; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FZ, fetal zone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 3-ßHSD, 3-ß-hydroxysteroid dehydrogenase; LDLR low-density lipoprotein receptor; lin^-, lineage^-; mAb, monoclonal antibody; Mps-1, metallopanstimulin-1; NovH, nephroblastoma overexpressed gene; PE, phycoerythrin; PI, propidium iodide; P450scc, P450 side-chain cleavage enzyme; TZ, transitional zone.

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