Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul;3(7):801-8.
doi: 10.5966/sctm.2013-0211. Epub 2014 May 22.

A Defined, Controlled Culture System for Primary Bovine Chromaffin Progenitors Reveals Novel Biomarkers and Modulators

Affiliations
Free PMC article

A Defined, Controlled Culture System for Primary Bovine Chromaffin Progenitors Reveals Novel Biomarkers and Modulators

Jimmy Masjkur et al. Stem Cells Transl Med. .
Free PMC article

Abstract

We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.

Keywords: Cell culture; Differentiation; Self-renewal; Signal transduction.

Figures

Figure 1.
Figure 1.
Adult bovine adrenomedullary chromaffin progenitors can be efficiently cultured under defined conditions. (A): The diagram in the green box describes the culture techniques used. Cells were isolated from adult adrenals and plated directly in serum-free culture medium on precoated plates. bFGF was added to the medium daily to support the self-renewal state. Withdrawal of bFGF induced their differentiation. Differentiation was induced by replacing the expansion medium with bFGF-free NB/B27 medium. The diagram in the orange box shows the previously established culture method. Cells are dissociated from the adrenal medulla and selected through differential plating and culture in low attachment conditions in a serum-containing medium. Cells are then expanded and differentiated by subsequent plating in NB/B27 medium. (B): A time course of cell number in the expansion phase shows an initial decrease in cell number (likely, a selection event), followed by a gradual increase in cell number. (C): Immunocytochemical detection for Hes3 and EdU at 2 and 4 days following plating (magnification, ×10). (D, E): Graphs showing percentages of cells that express Hes3 (D) and percentages of Hes3+ cells that are labeled with EdU (E) at 2 and 4 days in culture (n = 1 in triplicate). (F): Expression of established markers of adrenal progenitor cells by reverse transcriptase polymerase chain reaction in isolated cells prior to plating, during the expansion phase (5 days), and after differentiation in NB medium (7 days). (G, H): During the expansion phase of cell culture (G), cells grow in monolayer form; several chromaffin progenitor markers are expressed, and the cells are small. Induction of differentiation (H) (these examples: N2 medium following bFGF withdrawal) maintains the cells in monolayer form and induces morphological changes (magnifications: TUJ1/DA, ×40; TH/DBH, ×10, CgA/PNMT, ×10). Abbreviations: bFGF, basic fibroblast growth factor; CgA, chromogranin A; d, day(s); DA, dopamine; DBH, dopamine-β-hydroxylase; Differ., differentiation; EdU, 5-ethynyl-2′-deoxyuridine; NB/27, Neurobasal/B27 medium; PNMT, phenylethanolamine N-methyltransferase; TH, tyrosine hydroxylase; TUJ1, βIII tubulin.
Figure 2.
Figure 2.
Adult bovine adrenomedullary cells cultured under defined conditions express Hes3. (A): The adult bovine adrenal medulla expresses Hes3 polymerase chain reaction (PCR) data of mRNA levels in samples from dissected adult bovine adrenal medulla as well as from isolated cells prior to plating; GAPDH is used as the housekeeping gene. (B): Fluorescence images of cells cultured for 5 days at the expansion phase and stained for Hes3 and other markers (magnification, ×40). (C): Hes3 subcellular localization varies; in this example, cells with nuclear and cells with cytoplasmic Hes3 are shown in the same field of view (magnification, ×20). (D): PCR detection of Hes3 in cell cultures demonstrates the loss of Hes3 mRNA following differentiation. (E): Cultured cells coexpress Hes3 in the expansion phase; most Hes3+ cells do not express chromogranin A or TH (magnification, ×20). (F): Split channel image showing two adjacent Hes3+ cells, and the cell with greater Hes3 immunoreactivity does not express CgA, whereas the cell exhibiting weaker immunoreactivity for Hes3 expresses CgA (magnification, ×40). (G): Example of Hes3+/TH+ cells in the expansion phase and loss of Hes3 expression following differentiation (magnification, ×20). (H): Fluorescence images demonstrating that following three passages, cell cultures maintain Hes3 expression; induction of differentiation results in the loss of Hes3 immunoreactivity. (I): After three passages, cells maintain neurogenic potential, generating TH+ and TUJ1+ cells after induction of differentiation (magnification, ×40). Abbreviations: d, day(s); DAPI, 4′,6-diamidino-2-phenylindole; Differ., differentiation; Exp., expansion; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TH, tyrosine hydroxylase; TUJ1, βIII tubulin.
Figure 3.
Figure 3.
Soluble factors regulate immature adult chromaffin progenitor cell growth and catecholamine content in culture. (A, B): Concomitant treatment with Delta4, Angiopoietin2, and JAK I during the expansion phase promotes cell growth in the expansion phase. In contrast, cholera toxin treatment has no effect on cell number (magnification, ×10). (C): The combined treatment during the expansion phase does not induce morphological changes to CgA+, TH+, or PNMT+ cells in the differentiation phase differentiation (2 days in basic fibroblast growth factor-free N2 medium and then 7 days in Neurobasal medium) (magnification, ×40). *, Statistical significance value of less than .05. Abbreviations: Ang2, Angiopoietin2; CgA, chromogranin A; Chol. T., cholera toxin; Cntrl, control; d, days; COMBO, combined treatment; DAPI, 4′,6-diamidino-2-phenylindole; JAK, Janus kinase; PNMT, phenylethanolamine N-methyltransferase; TH, tyrosine hydroxylase.
Figure 4.
Figure 4.
Cholera toxin regulates the morphology and catecholamine content of immature chromaffin cells in culture. (A, B): Treatment without (A) or with cholera toxin (B) during both the expansion and differentiation phases induces morphological changes to TH+ cells in the differentiation phase (2 days in basic fibroblast growth factor-free N2 medium and then 7 days in Neurobasal medium) (magnification, ×10; TH bottom, magnification, ×40). (C): Cholera toxin decreases the ratio of epinephrine to norepinephrine content. (D): Cholera toxin increases the content of dopamine relative to total catecholamines (epinephrine + norepinephrine + dopamine) in the expansion phase. (E–G): Graphs representing total cell number (E), total Hes3+ cell number (F), and percentage of total cells that express Hes3 in the expansion and differentiation stages (G), with and without cholera toxin treatment, demonstrate no significant effects. *, Statistical significance value of less than .05. Abbreviations: Chol. T., cholera toxin; Cntrl, control; %DA, percentage of DA ± SEM of total catecholamines; Differ., differentiation; EPI, epinephrine; Exp., expansion; NE, norepinephrine; TH, tyrosine hydroxylase.

Similar articles

See all similar articles

Cited by 2 articles

MeSH terms

LinkOut - more resources

Feedback