Transplantation of adrenal cortical progenitor cells enriched by Nile red

Abstract

Background: The adrenal cortex may contain progenitor cells useful for tissue regeneration. Currently there are no established methods to isolate these cells.

Material and methods: Murine adrenal cells were sorted into a Nile-red-bright (NR(bright)) and a Nile-red-dim (NR(dim)) population of cells according to their degree of cholesterol content revealed by Nile red fluorescence. The cells were transplanted under the renal capsule to determine their ability for regeneration.

Results: The NR(bright) cells contained an abundance of lipid droplets, whereas the NR(dim) cells contained little. The NR(bright) cells expressed Sf1 and the more differentiated adrenal cortical genes, including Cyp11a1, Cyp11b1, and Cyp11b2, whereas the NR(dim) cells expressed Sf1 but not the more differentiated adrenal cortical genes. After 56 d of implantation in unilateral adrenalectomized mice, the NR(dim) cells expressed Sf1 and the more differentiated adrenal cortical genes, whereas the NR(bright) cells ceased to express Sf1 as well as the more differentiated adrenal cortical genes. NR(dim) cells also proliferated in the presence of basic fibroblast growth factor.

Conclusions: The population of NR(dim) cells contained adrenal cortical progenitor cells that can proliferate and give rise to differentiated daughter cells. These cells may be useful for adrenal cortical regeneration.

Figure 1

Nile Red and DAPI staining of adrenal gland, 200x. 7-μm adrenal gland sections were stained with Nile Red and DAPI to locate lipid droplets and nuclei. A. Nile Red staining of the adrenal gland showed the highest fluorescence within the zona fasciculata. B. DAPI staining of the adrenal gland showed a uniform distribution of nuclei. C. Merged picture of A and B. D and E, the fluorescent intensity of Nile Red and DAPI versus the radius of adrenal gland. m, medulla; f, zona fasciculata; g, zona glomerulosa; c, adrenal capsule.

Figure 2

The forward scattering, side scattering, and fluorescence characteristics of Nile Red stained adrenal cells. A. Forward and side scattering of all of the isolated adrenal cells. Staining of Nile Red does not change the forward and side scattering of the cells. B. Nile Red stained adrenal cells were discriminated by the green fluorescent (530/30) channel and the dark red channel (660/20). Two distinct populations, NR^bright and NR^dim, can be observed. C. Forward and side scattering of NR^bright cells. D. Forward and side scattering of NR^dim cells.

Figure 3

Nile Red staining of sorted adrenal cells, 200x. A-D; after sorting, sorted cells were fixed to slides and observed by a fluorescent microscope. A, D, and G,. All cells. B, E, and H, NR^bright cells. C, F, and I, NR^dim cells. A, B, and C were stained with Nile Red. Lipid droplets can be observed. D, E, and F, DAPI staining of the NR^bright cells to locate nuclei. G, H, and I, merged picture of Nile Red and Dapi staining.

Figure 4

Relative gene expression levels of Sf1, Cyp11b1, Cyp11b2, and Cyp11a1 of all adrenal cells, NR^bright cells, and NR^dim cells. The relative level of each adrenal-specific gene was normalized to that of pooled neonatal adrenal glands. Error bar represents standard deviation of measurements of retrieved implants from three animals. The levels of Sf1 were the same (p>0.05), where as the levels of Cyp11b1, Cyp11b2, and Cyp11a1 were significantly lower for the NR^dim cells.

Figure 5

Relative gene expression level of Sf1, Cyp11b1, Cyp11b2, and Cyp11a1 of NR^bright cells, and NR^dim cells that were implanted into the animal after 10, 28, and 56 days. The gene expression level of NR^bright cells, and NR^dim cells was normalized to that of all adrenal cells on the same day. A. Genes expressions levels of retrieved implants at day 0. B. Genes expressions levels of retrieved implants at day 10. C. Genes expressions levels of retrieved implants at day 28. D. Genes expressions levels of retrieved implants at day 56. The relative level of each adrenal-specific gene was normalized to that of all adrenal cells for implantation specimens. * indicates p<0.05 as compared to all adrenal cells, and # indicates p<0.05 as compared to NR^bright cells. Error bar represents standard deviation of measurements of retrieved implants from three animals.

Figure 6

The attachmentof NR^bright and NR^dim cells in KO medium (A) and growth with the aid of bFGF on day 7 (B) and day 14 (C) were investigated. D, Gene expression of NR^bright and NR^dim cells immediately after cell sorting. Both types of cells expressed similar levels of Sf1, but NR^dim cells expressed significantly lower levels of Cyp11b1 and Cyp11b2. Gene expression of NR^bright and NR^dim cells after 7 days (E) and 14 days (F) of culture in the KO medium. Error bar represents standard deviation of repeated measurements.