Isolation of neural crest derived chromaffin progenitors from adult adrenal medulla.

Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Unlike the closely-related sympathetic neurons, a subpopulation of proliferation-competent cells exists even in the adult. Here, we describe the isolation, expansion, and in vitro characterization of proliferation-competent progenitor cells from the bovine adrenal medulla. Similar to neurospheres, these cells, when prevented from adherence to the culture dish, grew in spheres, which we named chromospheres. These chromospheres were devoid of mRNA specific for smooth muscle cells (MYH11) or endothelial cells (PECAM1). During sphere formation, markers for differentiated chromaffin cells, such as phenylethanolamine-N-methyl transferase, were downregulated while neural progenitor markers nestin, vimentin, musashi 1, and nerve growth factor receptor, as well as markers of neural crest progenitor cells such as Sox1 and Sox9, were upregulated. Clonal analysis and bromo-2'-deoxyuridine-incorporation analysis demonstrated the self-renewing capacity of chromosphere cells. Differentiation protocols using NGF and BMP4 or dexamethasone induced neuronal or endocrine differentiation, respectively. Electrophysiological analyses of neural cells derived from chromospheres revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels and action potentials. Our study provides evidence that proliferation and differentiation competent chromaffin progenitor cells can be isolated from adult adrenal medulla and that these cells might harbor the potential for the treatment of neurodegenerative diseases, such as Parkinson's disease.

Chromaffin progenitor cells from the adrenal medulla.

Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Different lines of evidence suggest the existence of a subpopulation of proliferation-competent progenitor cells even in the adult state. The identification of sympathoadrenal progenitors in the adrenal would greatly enhance the understanding of adrenal physiology and their potential role in adrenal pathogenesis. Isolation and differentiation of these progenitor cells in culture would provide a tool to understand their development in vitro. Furthermore, due to the close relation to sympathetic neurons, these cells might provide an expandable source of cells for cell therapy in the treatment of neurodegenerative diseases. We therefore aim to establish protocols for the efficient isolation, enrichment and differentiation of chromaffin progenitor cells to dopaminergic neurons in culture.

Isolation, characterization, and differentiation of progenitor cells from human adult adrenal medulla.

Chromaffin cells, sympathetic neurons of the dorsal ganglia, and the intermediate small intensely fluorescent cells derive from a common neural crest progenitor cell. Contrary to the closely related sympathetic nervous system, within the adult adrenal medulla a subpopulation of undifferentiated progenitor cells persists, and recently, we established a method to isolate and differentiate these progenitor cells from adult bovine adrenals. However, no studies have elucidated the existence of adrenal progenitor cells within the human adrenal medulla. Here we describe the isolation, characterization, and differentiation of chromaffin progenitor cells obtained from adult human adrenals. Human chromaffin progenitor cells were cultured in low-attachment conditions for 10-12 days as free-floating spheres in the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor. These primary human chromosphere cultures were characterized by the expression of several progenitor markers, including nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox9, Sox10, Phox2b, and Ascl1 on the molecular level and of Sox9 on the immunohistochemical level. In opposition, phenylethanolamine N-methyltransferase (PNMT), a marker for differentiated chromaffin cells, significantly decreased after 12 days in culture. Moreover, when plated on poly-l-lysine/laminin-coated slides in the presence of FGF-2, human chromaffin progenitor cells were able to differentiate into two distinct neuron-like cell types, tyrosine hydroxylase (TH)(+)/ß-3-tubulin(+) cells and TH(-)/ß-3-tubulin(+) cells, and into chromaffin cells (TH(+)/PNMT(+)). This study demonstrates the presence of progenitor cells in the human adrenal medulla and reveals their potential use in regenerative medicine, especially in the treatment of neuroendocrine and neurodegenerative diseases.

Effects of dehydroepiandrosterone on proliferation and differentiation of chromaffin progenitor cells.

Dehydroepiandrosterone producing adrenocortical zona reticularis and the adrenal medulla are in direct contact and are highly intermingled in many species. This results in potentially strong paracrine influences of high local dehydroepiandrosterone concentrations on the adrenal medulla. Dehydroepiandrosterone has neuroprotective properties and increases neural stem cell proliferation and neurogenesis. Therefore, we aimed to establish its effects on chromaffin progenitor cell proliferation and differentiation. Previously, we successfully isolated chromaffin progenitors from bovine adrenal medulla in spherical cultures, so-called chromospheres. Seven days treatment of chromospheres with dehydroepiandrosterone at high concentrations (100 µM) hampered proliferation of chromaffin progenitors. Under differentiation conditions, dehydroepiandrosterone in the presence of retinoic acid, increased tyrosine hydroxylase and decreased dopamine-ß-hydroxylase mRNA expression. In addition, there was a tendency to increase dopamine contents. Dehydroepiandrosterone/retinoic acid is therefore suggested to induce dopaminergic differentiation from chromaffin progenitor cells. Furthermore, the high dehydroepiandrosterone concentrations present in the fetal and adult adrenal may play an important role in adrenomedullary cell proliferation and differentiation.

Osmoregulation and Na,K-ATPase expression in osmoregulatory organs of Scylla paramamosain.

Scylla paramamosain is a euryhaline marine crab that can actively regulate its osmotic pressure of the hemolymph. We investigated the expression of Na,K-ATPase in the osmoregulatory organs, including anterior gills, posterior gills, antennal glands, and midgut. Our cDNA sequencing of the Na,K-ATPase alpha-subunit revealed no isoforms in any of the four osmoregulatory organs studied. The full-length cDNA contains 3,866 bp that encode a complete open reading frame of 1,039 amino acids. The osmotic pressure of the hemolymph changed immediately after transference from 25 ppt to 5 and 45 ppt, and reached a new steady state within 3 days, showing the typical pattern of weak hyperosmoregulators. Expression of Na,K-ATPase alpha-subunit mRNA level was determined for individuals acclimated in 5, 25, and 45 parts per thousand (ppt) for 7 days. Compared to the mRNA level of those from 25 ppt, the level in the posterior gills from 5 ppt was significantly higher. Also, the mRNA levels in the anterior gills, posterior gills and antennal glands from 45 ppt were significantly higher than that of the respective organs in 25 ppt. This increase in the 5 ppt treatment may be responsible for the observed increase of Na,K-ATPase activity on day 14 when the posterior gills had a significantly higher activity than those in the other two salinity treatments. No difference was found in the protein level across salinities. In conclusion, the posterior gills play the most important role in osmoregulation in dilute seawater and the increase in Na,K-ATPase activity on the 7th day may be due to its gene transcription and/or mRNA translation.