Cell and Tissue Engineering

Liver-Specific Differentiation of Embryonic Stem Cells

Deriving new sources of hepatocytes is critical for further advancement of liver-directed cell therapies, bioartificial liver-assist devices and toxicology studies.  Embryonic stem cells (ESC) offer an ideal source of hepatocytes because these cells can proliferate indefinitely and can, hypothetically, be convinced to become any cell type.  However, convincing stem cells to become the desired adult cell type (differentiating stem cells) remains challenging as the signals required for this process remain to be poorly understood.  Hepatocytes are highly differentiated cells that carry on multiple functions in the body including serum protein synthesis, bile production and metabolism of xenobiotics.  Stem cell to hepatocyte transformation is therefore particularly challenging.  In order to expedite discovery of liver-specific stem cell niche our laboratory is developing novel cell culture tools including growth factor microarrays and micropatterned co-cultures.  These novel cell culture surfaces allow us to pinpoint the signaling molecules and heterotypic cellular interactions required for efficient differentiation of embryonic stem cells into hepatocytes.  Beyond in vitro discovery of differentiation inducers, we are also developing biomaterials scaffolds that will contain necessary signaling molecules and will be used as vehicles for stem cell transplantation to the liver.

Combining printed protein arrays and micropatterned co-cultures to guide stem cell differentiation . (A) Printed collagen spots (green fluorescence) adjacent to islands occupied by mouse ESCs. (B)  Seeding hepatocytes on the surface shown in (A) results in hepatocyte clusters being placed next to stem cell clusters.  (C) In order to analyze the extent of differentiation, stem cells were extracted from micropatterned co-cultures using laser microdissection and were analyzed by RT-PCR.  This analysis showed that presence of adult hepatocytes in micropatterned co-cultures induced stem cell to differentiate toward hepatic lineage.

People contributing to this research direction:  Dipali Patel, Elena Foster, Jungmok You, Amranul Haque, Nazgul Tuleova

Important recent papers on this topic: Lee, J. Y., Tuleuova, N., Jones, C.N., Ramanculov, E., Zern, M.A., Revzin, A., “Directing Hepatic Differentiation of Embryonic Stem Cells with Protein Microarray-Based Co-Cultures”, Integrative Biology, 2009, 1, 460-468 (journal cover)

Tuleuova, N., Lee, J.Y., Zern, M.A., Revzin, A. “Using growth factor arrays and micropatterned co-cultures to induce hepatic differentiation of embryonic stem cells” Biomaterials, 2010, 31, 9221-9231


Electroactive Surfaces for Micropatterning and Retrieval of Cells

The ability to exercise precise spatial and temporal control over cell-surface interactions is being leveraged in our laboratory to micropattern cells on surfaces or to retrieve cells from microfabricated surfaces.  We are employing conductive (gold or ITO) surfaces modified with cell adhesive or non-adhesive molecules that can be electrically stimulated, leading to selective adsorption or desorption of proteins and/or mammalian cells.  These “switchable” biointerfaces have been used to create micropatterned hepatocyte-fibroblast co-cultures (see Figure A).  Another application of this technology is in selective retrieval of cells from a micropatterned surface for downstream analysis.  This will allow to sort out a small number of cells from a desired location on the surface and will obviate the need to sacrifice a large number of cells to get functional information   Figure B shows selective retrieval of lymphocytes from antibody-modified gold electrodes.

Electroactive surfaces for cell culture and retrieval. (A) Groups of pepatocytes and fibroblasts micropatterned next to each other on individually addressable ITO electrodes.  (B) T-cells captured and then released from individually addressable gold electrodes.  Upper and lower electrodes were activated releasing cells, while the middle electrode was not electrically activated and still contains T-cells.

People contributing to this research direction:  Ying Liu, Jungmok You, Dong-Sik Shin, Yandong Gao, Ali Rahimian, Tam Vu, Kyungjin Son, Timothy Kwa, Qing Zhou

Important recent papers on this topic: Kim, M., Lee, J.Y., Shah, S.S., Tae, G., Revzin A. “On-cue Detachment of Hydrogels and Cells from Optically Transparent Electrodes”, Chemical Communications, 2009, 5865-5867

Shah, S., Howland, M., Chen, L., Silangcruz, J., Verkhoturov, S., Schweikert, E., Parikh, A., Revzin, A. “Micropatterning of Proteins and Mammalian Cells on Indium Tin Oxide.” ACS Applied Materials & Interfaces, 2009, 1, 2592-2601

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