Isolation and Culture of Skin-Derived Differentiated and Stem-Like Cells Obtained from the Arabian Camel (Camelus dromedarius)

Abstract

Elite camels often suffer from massive injuries. Thus, there is a pivotal need for a cheap and readily available regenerative medicine source. We isolated novel stem-like cells from camel skin and investigated their multipotency and resistance against various stresses. Skin samples were isolated from ears of five camels. Fibroblasts, keratinocytes, and spheroid progenitors were extracted. After separation of different cell lines by trypsinization, all cell lines were exposed to heat shock. Then, fibroblasts and dermal cyst-forming cells were examined under cryopreservation. Dermal cyst-forming cells were evaluated for resistance against osmotic pressure. The results revealed that resistance periods against trypsin were 1.5, 4, and 7 min for fibroblasts, keratinocytes, and spheroid progenitors, respectively. Furthermore, complete recovery of different cell lines after heat shock along with the differentiation of spheroid progenitors into neurons was observed. Fibroblasts and spheroid progenitors retained cell proliferation after cryopreservation. Dermal cyst-forming cells regained their normal structure after collapsing by osmotic pressure. The spheroid progenitors incubated in the adipogenic, osteogenic, and neurogenic media differentiated into adipocyte-, osteoblast-, and neuron-like cells, respectively. To the best of our knowledge, we isolated different unique cellular types and stem-like cells from the camel skin and examined their multipotency for the first time.

Keywords: camel; cell culture; differentiation; skin; stem cells.

Figure 1

Camel skin explants and outgrowths at P0 (passage zero). (A) Camel skin explant (arrowhead) with early outgrowth of monolayer of keratinocytes (black arrow) on day 10 of explant seeding. (B,C) Additional growth of keratinocytes (black arrow) and migration and appearance of cell clumps (arrowhead; later characterized as spheroid progenitors) fibroblast either scattered or in colony (thin arrows). Scale bars = 200 µm. (D) A magnified image of the fibroblast (thin arrows). Scale bar = 100 µm. All cultures were performed in 35 mm tissue culture dishes and maintained in the same conditions in humidified atmosphere at 38 °C under 5% CO₂.

Figure 2

Long-term culture of camel skin explants. Explants were cultured for 21 (A–C), and further, for 28 (D–F) days while changing the culture medium after every two days. (A) Distinguishing fibroblasts (black arrow) from keratinocytes (white arrow). (B) Appearance of small (arrowheads) and big (arrow) fluid-filled cysts. (C) Appearance of cuboidal cells (asterisk) with melanin crystals (arrows) (Scale bar = 100 µm). (D) Fibroblasts reached confluency on day 28. (E) The fluid-filled cysts increased in both number (arrowheads) and size (arrows). (F) Formation of epithelial-like sheet with uniform cuboidal cells. Scale bars for all, except (C) = 500 µm.

Figure 3

Sensitivity of different skin-derived cells to trypsinization. Incubation of primary cell culture with trypsin-EDTA for 5 min showed varied behavior. (A) Keratinocytes, the cell sheet, was distorted after 4.5 min (asterisk). (B) Fibroblasts, with round cells, were observed after 1.5 min of trypsinization (asterisk). (C) The fluid filled cyst layer showed resistance to trypsin for 5 min (arrow) indicating no change in cell morphology after trypsinization. (D) Cell clumps (spheroids) showed resistance to trypsin and the surrounding cells (arrows). Scale bar = 500 µm.

Figure 4

Sensitivity of different trypsin-sensitive skin-derived cells to heat shock. Dissociated fibroblasts (A), keratinocytes (B), and fluid filled cysts and clumps (C) were collected after 1.5, 4.5, and 7 min of trypsinization, respectively, washed in PBS, and cultured in new culture dishes. (A1) Fibroblast monolayer. (B1) Keratinocyte monolayer. (C1) Fluid-filled cysts and cell clumps (spheroid progenitors). Cells of (A1,B1,C1) were exposed to heat shock (45 °C for 2 h). (A2) Fibroblast architecture was disturbed. (B2) Architecture of keratinocytes was partially distorted. (C2) Spheroid progenitors and the surrounding cells were resistant to heat shock. (A3,B3,C3) show the corresponding cells after recovery at 38 °C for 4 days. (A3) Fibroblasts recovered and reached confluency. (B3) Keratinocytes recovered with increased nuclear size (arrows). (C3) Spheroid progenitors recovered with spontaneous differentiation into neuron-like axons (arrows) and cell bodies (arrowheads). Scale bar = 500 µm.

Figure 5

Fibroblast subculture and cryopreservation. Fibroblast were isolated morphologically and based in sensitivity to trypsin, were subcultured, and cryopreserved. (A) Fibroblasts at passage 1 (P1) at 70% confluency, and (B) magnified image to show spindle morphology (arrows). Cells were cultured to reach 90% confluency, harvested, and cryopreserved in liquid nitrogen. (C,D) Thawed and resurrected fibroblast at 90% confluency with clear spindle morphology (arrows). Scale bar of (A,C) = 500 µm. Scale bar of (B,D) = 100 µm.

Figure 6

Cystic cells subculture, cryopreservation, and characterization. (A) Cystic cell (arrows) monolayer was dissociated and cells were subcultured. (B) Groups of cells with cysts and individual cells with different fluid-filled compartments (arrows) at passage 1 (P1). (B1) Hoechst staining of nuclei of groups in (B). (C) Consistent formation of cystic cells (arrow) after cryopreservation of passage 3 (P3) with different numbers of fluid-filled compartments. (D) Single (arrowhead); double (arrow); and more than two compartments as distributed in the field. (D1) Hoechst staining of nuclei of cells shown in (D). (E) Exposure of the cystic cells (arrow) to osmotic stress (500 mOsm/L for 3 min) showed cyst collapse ((E1), arrow), which was reformed after recovery ((E2), arrow) at 300 mOsm/L for 15 min. Scale bar = 50 µm except for (A,C) are 500 µm.

Figure 7

Dermal spheroid characterization. Cell clumps or dermal spheroids were clearly observed either in primary culture (P0) and in P1 in form of solid clumps ((A), arrows) or fluid-filled clumps ((B), arrow) with resemblance to the embryoid bodies as observed in pluripotent stem cell culture. Scale bar = 500 µm. Individual spheroid ((C), arrow) were stained with Hoechst to reveal the uniform nuclear assembly of over than 100 cells to form the spheroids (C1). Scale bar = 100 µm.

Figure 8

Dermal spheroid differentiation. Individual dermal spheroids were collected and cultured in plain medium (A) showed formation of spindle-shaped cells. Scale bar = 100 µm. Spheroids were cultured in a medium containing 10 µg/mL of all-trans retinoic acid for neurogenic differentiation (B) with axon-like formation (arrows). Scale bar = 100 µm. Spheroids cultured in medium containing adipogenic-differentiation factors showed formation of lipid droplets uniformly distributed in polygonal cells after 10 days of culture (C) and were positive for oil-red stain after 18 days of culture ((D), red stain, arrow). Spheroids cultured in medium without differentiation factors (E) compared to spheroids cultured in a medium containing osteogenic-differentiation factors showed positively stained mineralization after Alizarin red staining ((F), red stain, arrow). Scale bar = 100 µm.