br Introduction The in vitro

Introduction The in vitro differentiation of progenitor vip receptor allows researchers to manipulate cellular environment, signaling and interactions without the interference of other cell/tissue types. These differentiation models are not only useful for increasing the understanding of stem cell kinetics but are also applicable to tissue engineering as a system to investigate tissue formation in vitro. It has also been previously documented that the treatment of progenitor cells with factors which enhance differentiation in vitro can have a positive effect on tissue formation in vivo (Siddappa et al., 2008; Song et al., 2009). Thus, to allow the use of stem cells as a cell source for clinical applications, establishment of defined differentiation conditions for specific stem cell populations is of utmost importance. When considering osteogenic differentiation most work has been carried out on bone marrow mesenchymal stem cells (BM-MSCs). The current gold standard for osteogenic differentiation of this cell type is regarded as basal medium containing 10% fetal bovine serum (FBS), dexamethasone (Dex), β-glycerophosphate (β-GP) and l-ascorbic acid-2-phosphate (AsAP) (Jaiswal et al., 1997). This formulation has been shown to exhibit a robust differentiation for BM-MSCs, however some caveats exist for alternative cell populations. For example, Dex causes a dual differentiation of adipose derived cells to both adipocytes and osteoblasts under osteogenic conditions (Arutyunyan et al., 2009). This has been attributed to Dex potentially augmenting differentiation of MSCs and not specifically directing differentiation (Oshina et al., 2007). This effect is unsurprising considering most progenitor cells are developmentally in various stages of differentiation and residing within different niches. Alternative osteogenic stimulators have been described including 1,25-dihydroxyvitamin D3 (VitD3) (Liu et al., 1999), all trans-retinoic acid (atRA) (Wan et al., 2006), cyclic AMP (cAMP) (Siddappa et al., 2008) and bone morphogenic protein 2 (BMP2) (Rickard et al., 1994), however confounding results on osteogenic differentiation have also been documented with many of these. For example Dex has been shown to block osteogenic differentiation of murine osteoprogenitor cells with VitD3 shown to down regulate osteocalcin (OCN) within the same cell type (Lian et al., 1997). Additionally, FBS within cell culture media can also have a negative effect on differentiation due to endogenous growth factors interfering with cellular processes such as BMP signaling (Zilberberg et al., 2007). A major limitation in studies aiming to elucidate differentiation conditions is the absence of investigating interactions between inductive agents. This limitation is perhaps due to the absence of quantitative experimental designs for the assessment of multiple factors in a multiplex assay, which can be used to define factors and interactions to promote cellular differentiation. We herein provide a novel methodology to elucidate effects of known osteogenic factors on the differentiation of human periosteal derived cells (hPDCs). These are progenitor cells which are known to be involved in bone development and postnatal repair processes and are therefore a promising starting population for clinically relevant skeletal tissue engineering (Roberts et al., 2011). The analysis of differentiation achieved through a factorial design strategy which allows the direct contribution of each factor to measurable outcomes to be evaluated along with potential interactions thereof. This work represents a first step to elucidate specific factors which can enhance the osteogenic differentiation of hPDCs. Additionally, it provides a novel strategy for the investigation of stem cell differentiation for the development of cell-customized in vitro conditions as part of a tissue engineering approach.
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