Urs Vossmerbaeumer1, 4, Sandra Kuehl¹, Stefanie Ohnesorge², Minna Haapalahti³, Harald Klueter³, Jost B. Jonas¹, Hermann-Josef Thierse² and Karen Bieback³
Retinal pigment epithelial phenotype induced in human adipose tissue-derived mesenchymal stromal cells
The non-exudative form of age-related macular degeneration (ARMD) is characterized by a progressive decay of retinal pigment epithelium cells at the posterior pole of the eye. As mesenchymal stromal cells (MSC) have been shown to differentiate into various cell types beyond their natural mesodermal lineage to ectodermal lineages, we investigated whether we can induce a phenotype displaying retinal pigment epithelium (RPE) characteristics. This transdifferentiation process, as typed by specific protein markers also involves functional features. We investigate the responsiveness to α-Melanocyte Stimulating Hormone ( α-MSH). Also the current study analyses to what extent up- or downregulation of specific proteins can be traced over the period of differentiation.
Human MSC were isolated from liposuction material and characterized for lineage specific undifferentiation properties. Differentiation toward the RPE lineage was triggered by exposure to conditioned medium from either human or porcine RPE cells with selective vasoactive intestinal peptide (VIP) addition. Resulting cell populations were assessed for expression of RPE-specific markers by immunofluorescence, quantitative real time (RT)-polymerase chain reaction (PCR) and Western blotting. The potential for pigment synthesis was assessed by the response to a 3 day treatment with 10-5 mM melanocyte-stimulating hormone (α-MSH). Immunochemical -tyrosinase staining served to prove melanin synthesis and was quantified by counting 5 randomly selected, discontiguous fields of vision. For proteomic analysis, cells were lysed using a standard lysis protocol Proteomic analysis of total protein was performed by classical and differential 2D gel electrophoresis (2DE, DIGE). Focusing was achieved with an IPGphor system and for 2-D separation the Ettan II Dalt system was used (GE Healthcare). Parallel assessment of the material was done with Flamingo staining and with standard DIGE analysis. The gels were analyzed in a Fuji FLA 5100 fluorescence scanner (Fuji) and data decoded by Delta2D software (Decodon). Subsequently, differential regulated proteins were subjected to mass spectrometric analysis.
Induction of differentiation into RPE–like cells by conditioned medium and/or VIP led to expression of RPE markers Bestrophin (77.6–88.9%), Cytokeratine 8/18 (52.6–57.6%) and RPE 65 (57.44–61.26%) irrespective of the culture condition used. 34.9 ± 8.7% of undifferentiated MSC contained pigmented granula after exposure to α-MSH. Following RPE directed stimulation, α-MSH treatment increased pigmentation from baseline to 2.4 ± 2.3% to 75.7 ± 2.3% (CM) and 80.3 ± 0.4% (CM + VIP), respectively. 2DE comparison of protein expression patterns of native vs. induced MSC showed induction and inhibition for single proteins before and after the differentiation stimulus as visualized by distinct spots in the gels. Results from experiments with Flamingo stains are in line with DIGE analysis, thus providing complementary information. Proteomic pattern analysis revealed signals that were either detectable pre- or post induction.
MSC are shown to express RPE markers upon induction with either RPE-conditioned medium and/or VIP. The gain of basic functional features of RPE cells was indicated by melanin synthesis. This alludes to a differentiation potential of MSC into the neuroectodermal lineage, yielding cells with phenotypic characteristics of RPE cells.
Induction of RPE directed differentiation enhances MSH susceptibility significantly with melanin (as assessed by Tyrosinase) being found in up to 80% of the cultured and stimulated cells. The experiments demonstrated that human MSC display a differential proteomic expression pattern with significant alteration after induction of RPE-directed differentiation. It remains subject to further research to determine whether these in vitro data correspond to in vivo conditions. Reconstructive therapies of degenerative retinal disease could stem from such implicit differentiational plasticity crossing the natural destination limits of a mesenchymal cell type. Current research includes identification of regulated proteins as found in DIGE-experiments using mass spectrometry. Besides established differentiation markers novel proteins apt to indicate transition in differential status might be identified.