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Characterisation of Hep-G2 Conditioned Medium for Enhanced Mesoderm Differentiation of Es Cells: Application to Skeletal Tissue Engineering

Yunyi Kang1, Yu-shik Hwang2, and Athanasios Mantalaris1. (1) Department of Chemical Engineering and Chemical Technology, Imperial College London, South Kensington Campus, London, SW7 2AZ, United Kingdom, (2) Tissue Engineering & Regenerative Medicine Centre, Chelsea & Westminster Campus, Imperial College London, 369 Fulham Road, London, United Kingdom

As society is aging, the clinical need for bone and cartilage related treatment has also increased. There have been many approaches to replace impaired skeletal tissues. Among those approaches stem cell therapy has emerged as a promising way of achieving skeletal tissue regeneration overcoming limitations associated with autologous grafts (availability) and allogenic bone grafts (immune responses). Embryonic stem cells (ESCs) have recently become a potentially suitable cell source for skeletal tissue engineering applications since bone marrow derived mesenchymal stem cells present limitations such as difficulty of isolation from bone marrow and limited self-renewal capacity. Despite the considerable progress made in directing ESC differentiation to therapeutically useful lineages, several issues remain to be resolved before ESCs can be used for cell therapy: 1) increasing the efficiency of specific lineage generation, and 2) developing time- and cost-effective culture systems for controlling ESC differentiation. We have recently developed an efficient protocol to enhance mesodermal differentiation and thereby upregulate osteogenic differentiation of ESCs (Hwang et al., Tissue Engineering 2006). Specifically, murine ESCs (mESCs) were cultured in the presence of 50% conditioned medium (CM) from the human hepatocarcinoma cell line HepG2, which resulted enhanced mesoderm formation during embryoid body (EB) formation in the CM-treated mESCs. By varying the length of EB culture time, we achieved the selective control and stimulation of osteogenic differentiation and suppression of cardiogenic differentiation. Reducing the EB culture of the CM-treated mESCs to 1 day resulted in 5-10 fold enhancement of osteogenic differentiation, as determined by bone nodule formation, higher alkaline phosphatase activity, the presence of well-organised OB (osteoblast)-cadherin in the bone nodules, and increased cbfa-1/runx2 gene expression.

HepG2 cell line has a characteristic of visceral endoderm that was known as a source of signals during embryo development. This cell line secretes soluble signalling factors that are regarded as equivalent to the signals from visceral endoderm. The HepG2-cell-secreted signalling factors induce mesoderm effectively from embryonic stem cells preventing ectoderm development. Therefore elucidation of HepG2 cell-secreted factors will not only help us to understand complex signalling system of early embryo development in vivo but it will also give us a noble way of generating embryo stem cell derived final lineage cell source that can be used for skeletal tissue regeneration. To achieve identification of the active factors in the HepG2 CM, we adapted the HepG2 cell line in serum free culture and used proteomics method to identify key components in the HepG2 conditioned medium. Two-dimensional gel electrophoresis will separate proteins and the protein spots will be identified by peptide mass fingerprinting using MALDI-MS. However it is not easy to target all the proteins secreted by HepG2 cells since it will come up with numerous possibility of combination of factors. Therefore we compared the proteomes of two visceral endoderm-like cell line conditioned media, namely HepG2 and END-2. Then the common factors can be feasibly identified by DIGE. Once the list of candidate proteins was obtained, those factors are going to be validated using the loss and gain of function method for their mesoderm inducing activity and further osteogenic differentiation activity starting on mESCs. The final step will be the application of the identified molecules to the hESCs and introduce a noble process that can be practically exploited in the clinical skeletal tissue therapy.