A Novel Xenogeneic Co-Culture System to Examine Neuronal Differentiation Capability of Various Adult Human Stem Cells

Background: Targeted differentiation of stem cells is mainly achieved by the sequential administration of defined growth factors and cytokines, although these approaches are quite artificial, cost-intensive and time-consuming. We now present a simple xenogeneic rat brain co-culture system which supports neuronal differentiation of adult human stem cells under more in vivo-like conditions. Methods and Findings: This system was applied to well-characterized stem cell populations isolated from human skin, parotid gland and pancreas. In addition to general multi-lineage differentiation potential, these cells tend to differentiate spontaneously into neuronal cell types in vitro and are thus ideal candidates for the introduced co-culture system. Consequently, after two days of co-culture up to 12% of the cells showed neuronal morphology and expressed corresponding markers on the mRNA and protein level. Additionally, growth factors with the ability to induce neuronal different iation in stem cells could be found in the media supernatants of the co-cultures. Conclusions: The co-culture system described here is suitable for testing neuronal differentiation capability of numerous types of stem cells. Especially in the case of human cells, it may be of clinical relevance for future cell-based therapeutic applications

The effect of brain co-culture on the protein expression of skin-derived and glandular stem cells.

<p>Co-cultivation with rat brain biopsies led to comparable changes in the expression of some structure proteins in all three human stem cell populations. In the case of Nestin-expressing cells a distinctive decrease by up to one third could be detected, with the slight exception of the SDSC (<b>A–F</b>). The percentage of NF-positive cells increased in all populations up to 4-fold, accompanied by the general exhibition of an elongated morphology resembling uni- and bipolar neurons (<b>G–L</b>; arrows). α-SMA-positive cells decreased in number and/or showed a visible degradation of the actin filaments (<b>M–R</b>). Nuclei were counterstained with DAPI (blue). Blue bars represent the total cell count (via DAPI) in each image, red and green bars the relative amount of positively stained cells.</p

Neuron-like development in skin- and gland-derived stem cells during co-culture.

<p>An immunocytochemical staining revealed the co-localization of the neuroprogenitor marker Nestin and the neuronal cell marker Neurofilament in co-culture stimulated SDSC, PDSC and PSC (<b>A</b>). Furthermore, the establishment of axon-like cell processes with lengths up to 450 µm could be detected after co-cultivation (<b>B</b>).</p

Immunocytochemical characterization of skin- and gland-derived stem cells concerning their stemness.

<p>SDSC, PDSC and PSC generally express the stem cell markers Nestin, Sox 2 and Oct 4 as well as marker proteins for protein synthesis (Vigilin) and proliferation (Ki67). Nuclei were counterstained with DAPI (blue).</p

Immunocytochemical characterization of skin- and gland-derived stem cells concerning their differentiation capability.

<p>The used stem cell lines (SDSC, PDSC, PSC) spontaneously differentiate <i>in vitro</i> and, in the course of this differentiation, express marker proteins for the ectodermal (NF, GFAP, CK18, panCK) and mesodermal (α-SMA) germ layer. Nuclei were counterstained with DAPI (blue).</p

Detection of secreted growth factors via microarray.

<p>Supernatants of co-cultures and controls were tested for the presence of growth factors via a microarray-based technique (<b>A</b>). The processed arrays were semi-quantitatively analyzed by fluorescence, whereby representative images for every approach are shown (<b>B</b>). Growth factors that were found in at least four of five experiments were comparatively described in their expression based on the fluorescence signals (blue = weak/+; green-yellow = moderate/++; red-white = strong/+++) (<b>C</b>).</p