Fig 1.
Monolayer fibroblasts cell cultures express markers associated with SKPs.
DF cells isolated from skin biopsies from giant panda (a), red panda (e) and Asiatic lion (i) could be cultured as monolayers. Giant panda (b-d), red panda (f-h) and Asiatic lion (j-l) monolayer DF cultures all expressed vimentin, fibronectin and versican. Nuclei were stained with DAPI. Scale Bar = 100μm.
Fig 2.
Giant panda fibroblasts, but not red panda or Asiatic lion fibroblasts, are able to generate m-SKPs after passage and cryopreservation.
Fibroblast cultures from red panda (a) and Asiatic lion (b) were unable to generate m-SKPs. Female giant panda fibroblasts cultures were able to generate m-SKPs (c). Buccal mucosa cell cultures from the male giant panda could also generate m-SKPs (d) after cryopreservation of cells, m-SKPs generated from the male giant panda could be passaged to p1 (e) and p2 (f). Scale Bar = 100μm.
Fig 3.
Giant panda buccal mucosa m-SKPs are able to differentiate along various cell lineages.
Giant panda cells from passage 3 m-SKPs cultured in adipogenic and osteogenic differentiation media exhibited adipogenic differentiation (a) and osteogenic differentiation (c) as shown by oil Red-O and Von Kossa staining, respectively. Control cultures (b) and (d) did not stain positively for adipogenic or osteogenic differentiation, respectively. Giant panda m-SKPs cultured in neuronal differentiation media differentiated along neuronal lineages as shown by βIII tubulin immunostaining (e). βIII tubulin staining was negative in SKP cells cultured in control media (f). The Schwann cell marker S100β was up-regulated in cells cultured in Schwann cell differentiation media and cell morphology was characteristic of Schwann cells (g) when compared to cells cultured in control media (h). Nuclei were stained with DAPI in (e-h). Scale Bar = 100μm.
Fig 4.
Expression of SKP protein markers is maintained and ABCG2 expression is up-regulated in giant panda m-SKPs.
Expression of SKP associated markers was analysed in m-SKPs generated and passaged from giant panda male buccal mucosa cell cultures. Fibronectin (a), nestin (b), vimentin (c), and versican (d) are expressed in m-SKPs. P75 similarly is expressed both in monolayer (e) and m-SKP (f) cells. ABCG2 expression is not expressed in giant panda monolayer cultures (g) but is induced in m-SKPs (h). Nuclei were stained with DAPI. Scale bar = 100μm.
Fig 5.
m-SKP yield does not correlate with α-SMA expression in red panda, Asiatic lion or giant panda buccal mucosa fibroblasts.
Monolayer cultures of red panda (a) and Asiatic lion (b) dermal fibroblasts strongly expressed α-SMA, compared to monolayer cultures of giant panda buccal mucosa fibroblasts (c) where α-SMA expression was greatly reduced. Some, but not all giant panda SKP spheroids displayed α-SMA expression (d). Nuclei were stained with DAPI. Scale Bar = 100μm.