Giant panda fibroblasts, but not red panda or Asiatic lion fibroblasts, are able to generate m-SKPs after passage and cryopreservation.

<p>Fibroblast cultures from red panda (<b>a</b>) and Asiatic lion (<b>b</b>) were unable to generate m-SKPs. Female giant panda fibroblasts cultures were able to generate m-SKPs (<b>c</b>). Buccal mucosa cell cultures from the male giant panda could also generate m-SKPs (<b>d</b>) after cryopreservation of cells, m-SKPs generated from the male giant panda could be passaged to p1 (<b>e</b>) and p2 (<b>f</b>). Scale Bar = 100μm.</p

m-SKP yield does not correlate with α-SMA expression in red panda, Asiatic lion or giant panda buccal mucosa fibroblasts.

<p>Monolayer cultures of red panda (<b>a</b>) and Asiatic lion (<b>b</b>) dermal fibroblasts strongly expressed α-SMA, compared to monolayer cultures of giant panda buccal mucosa fibroblasts (<b>c</b>) where α-SMA expression was greatly reduced. Some, but not all giant panda SKP spheroids displayed α-SMA expression (<b>d</b>). Nuclei were stained with DAPI. Scale Bar = 100μm.</p

Expression of SKP protein markers is maintained and ABCG2 expression is up-regulated in giant panda m-SKPs.

<p>Expression of SKP associated markers was analysed in m-SKPs generated and passaged from giant panda male buccal mucosa cell cultures. Fibronectin (<b>a</b>), nestin (<b>b</b>), vimentin (<b>c</b>), and versican (<b>d</b>) are expressed in m-SKPs. P75 similarly is expressed both in monolayer (<b>e</b>) and m-SKP (<b>f</b>) cells. ABCG2 expression is not expressed in giant panda monolayer cultures (<b>g</b>) but is induced in m-SKPs (h). Nuclei were stained with DAPI. Scale bar = 100μm.</p

Giant panda buccal mucosa m-SKPs are able to differentiate along various cell lineages.

<p>Giant panda cells from passage 3 m-SKPs cultured in adipogenic and osteogenic differentiation media exhibited adipogenic differentiation (<b>a</b>) and osteogenic differentiation (<b>c</b>) as shown by oil Red-O and Von Kossa staining, respectively. Control cultures (<b>b</b>) and (<b>d</b>) 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 (<b>e</b>). βIII tubulin staining was negative in SKP cells cultured in control media (<b>f</b>). The Schwann cell marker S100β was up-regulated in cells cultured in Schwann cell differentiation media and cell morphology was characteristic of Schwann cells (<b>g</b>) when compared to cells cultured in control media (<b>h</b>). Nuclei were stained with DAPI in (<b>e-h</b>). Scale Bar = 100μm.</p

Monolayer fibroblasts cell cultures express markers associated with SKPs.

<p>DF cells isolated from skin biopsies from giant panda (<b>a</b>), red panda (<b>e</b>) and Asiatic lion (<b>i</b>) could be cultured as monolayers. Giant panda (<b>b-d</b>), red panda (<b>f-h</b>) and Asiatic lion (<b>j-l</b>) monolayer DF cultures all expressed vimentin, fibronectin and versican. Nuclei were stained with DAPI. Scale Bar = 100μm.</p

Monolayer cultured dermal fibroblasts yield m-SKPs after passage and cryopreservation.

<p><b>a </b><b><i>i</i></b><b>)</b> Skin biopsies are dissociated <i>1)</i> and the resulting DF suspension is cultured in SKP proliferation media <i>2)</i>. SKPs are formed after approximately 7 days <i>3)</i>, at which point they can be assessed for marker expression <i>4)</i>, differentiated down neural and mesenchymal lineages <i>5)</i> or maintained <i>via</i> passage <i>6)</i>. <b>a </b><b><i>ii</i></b><b>)</b> DF isolated from skin biopsies are cultured as monolayers <i>7)</i> which can be passaged <i>8)</i> and cryopreserved <i>9</i>). These DF cultures form m-SKPs upon incubation with SKP proliferation media. <b>b)</b> Phase contrast images of <b>b </b><b><i>i</i></b><b>)</b> original monolayer normal adult human DF, <b>b </b><b><i>ii</i></b><b>)</b> m-SKPs derived from the DF culture in <b>b </b><b><i>i</i></b><b>), b </b><b><i>iii</i></b><b>)</b> collagenase XI treated m-SKPs (from <b>b </b><b><i>ii</i></b><b>)</b> in single cell suspension and <b>b </b><b><i>iv</i></b><b>)</b> passaged m-SKPs (derived from single cell suspension in <b>b </b><b><i>iii</i></b><b>)</b>. c) There was no difference in m-SKP yield from matched DF cultures expanded to p3, or those expanded to p3 after cryopreservation at p1. (DF – dermal fibroblasts, (m)-SKP – (monolayer) skin derived precursor).</p

m-SKPs express markers associated with traditionally isolated SKPs.

<p><b>a)</b> In response to m-SKP formation cryopreserved normal adult human DF from both low (p3 hair dense and hair sparse) and high (p12 hair dense) passage and cryopreserved diseased (acute progeria) human DF from intermediate passage (p8) up-regulate expression of nestin and versican protein. Moreover, nestin and versican expression are maintained in passaged m-SKPs (p2) derived from cryopreserved low passage normal adult human DF. DF express fibronectin which is maintained after m-SKP formation. <b>b)</b> Nestin mRNA expression is induced in p2 normal adult human DF upon m-SKP formation and maintained after passaging. <b>c)</b> m-SKPs formed from both low (p3) and high (p12) passage cryopreserved normal adult human DF express similar levels of <i>Dermo-1</i>, <i>Slug</i>, <i>Pax-3</i>, <i>Nestin</i>, <i>Nanog</i>, <i>Oct-4</i>, <i>Sox-2</i> and <i>Sox-9</i>. Levels of <i>p75NTR</i> and <i>Snail</i>, however, are reduced in m-SKPs formed from high (p12) passage cryopreserved normal adult human DF. (m-SKP – monolayer skin derived precursor, DF – dermal fibroblasts).</p

Mouse vibrissae, but not human follicle dermal papillae form m-SKPs.

<p>Normal adult human DP at p4 did not form m-SKPs, with cells remaining attached to the culture vessel surface. However, mouse DP at p3 formed small m-SKP cultures expressing versican, fibronectin and nestin. Mouse DP m-SKPs took longer to form and were smaller than human DF m-SKPs. (m-SKP – monolayer skin derived precursor, DP – dermal papilla).</p

m-SKP Yields from adult hair dense and hair sparse and neonatal monolayer cultures.

<p>Counts of total m-SKPs formed from dermal fibroblast cells previously cultured as monolayers (*denotes m-SKPs formed from one specimen that contained both hair sparse and hair dense sites and ^†denotes donors that formed fibroblast m-SKPs but did not form papilla m-SKPs. Counting was performed “blind”.</p

Densitometric analysis of percent decrease in <i>p75NTR</i> and <i>Snail</i> expression (p12 versus p3) in four populations of fibroblast m-SKPs.

<p>Densitometric analysis of percent decrease in <i>p75NTR</i> and <i>Snail</i> expression (p12 versus p3) in four populations of fibroblast m-SKPs.</p