• Adipose-tissue derived stem cells
• chondrogenic differentiation
• epithelial differentiation
• neural differentiation
• PAD3
• pediatric stem cells

Background and aims: Stem cells derived from adipose tissue are a potential important source for autologous cell-based therapies, including treatment of birth-related defects. Critical for their use in therapeutic applications is to fully understand their differentiation potential and its consistency or variability among individuals, information still lacking, particularly in children. Therefore, the aim of this study was to better understand the differentiation potential of human adipose-derived stem cells (ADSCs) obtained from different pediatric patients with craniofacial defects, for future applications in autologous craniofacial reconstruction as a new approach to tissue repair in children.
To address these issues, I have investigated ADSCs phenotype and analysed expression levels of some differentiation markers and a molecule possibly involved in ADSCs differentiation, PAD3, an enzyme known to play a role in calcium-dependent processes. PAD enzymes citrullinate (change an arginine to citrulline) proteins and increased citrullination has been associated with several human diseases including neural or cartilage disorders, such as rheumatoid arthritis.
A crucial point of my research was to define the potentiality of the single cells, focusing my study on the clonal lines analysis, to assess whether cell lines derived from a single cell display a multilineage differentiation potential or whether each phenotype arises from a subset of committed progenitor cells that exist within a heterogeneous population.
Methods: I have cultured ADSCs, isolated from liposuction aspirates, both on plastic and on Matrigel or poly-L-lysin/laminin-coated plates to promote cell adhesion, migration, growth and differentiation. Cells differentiated in culture media containing lineage-specific induction factors were compared to cells grown in control medium, without differentiation factors. I have assessed the expression of some lineage-specific differentiation markers at the RNA and protein level using RT-qPCR and immunocytochemistry techniques after two, three and four weeks of differentiation. I have also analysed morphological differences between induced and non-induced cells using different microscopy techniques.
Results: ADSCs responded to chondrogenic, adipogenic, osteogenic, neural and epithelial induction by clearly changing their morphology in comparison with control cells which show a fibroblast-like cell shape and by up-regulating tissue-specific differentiation markers, independently from patient of origin.
Specifically, for chondrogenic-induced cells results of RT-qPCR analysis reveal an up-regulation of chondrogenic markers Aggrecan and Col II in 2 and 3 weeks induced ADSCs of patients H21 and H23.
Results of the analysis at clonal level of neurally-induced cells, show a consistent up-regulation of the Schwann cell marker P0, both at 2 and 4 weeks after neural induction, while mRNA levels of neuron-specific enoalse (NSE) remain unchanged between control and induced cells. Immunocytochemistry reveals a clearly different expression pattern of NF-200 in non-induced and neural-induced cells: in neural-induced cells, this protein is localized in filamentous structures both after 2 and 4 weeks after induction.
Following epithelial induction, mRNA up-regulation of the epithelial marker cytokeratin-18 (Ck-18) in epithelial-induced cells is observed at both 3 and 4 weeks after induction, whereas the mRNA levels of the tight junction protein, zonula occludens-1 (ZO-1) remain unchanged between non-induced and induced cells. However, translocation of the ZO-1 protein from the cytoplasm to the cell margin at points of cell-cell contact in the epithelial-induced cells, consistent with tight junction formation typical of epithelial cells, is observed. In addition, immunocytochemistry also demonstrates the induction of filamentous cytokeratin expression found in epithelial cells.
PAD3 mRNA is significantly up-regulated in neural-induced cells, showing results consistent among different clones. Also in chondrogenic-induced cells PAD3 expression levels are higher than in control cells.
Summary and conclusions: This study supports the hypothesis that ADSC cultures contain pluripotent adult stem cell and are not solely a mixed population of unipotent progenitor cells, extending this finding to non-mesenchymal lineages and pediatric ADSCs, that had not been previously studied at the clonal level. Together, the gene and protein expression results confirm that ADSCs can differentiate into cell lineages of mesodermal and non-mesodermal origin, demonstrating for the first time that ADSCs can undergo epithelial differentiation at clonal level. In addition, this study shows up-regulation of PAD3 upon differentiation suggesting a role for this citrullination in this process that will require further investigation.