Monday, October 9, 2006 - 2:31 PM
11473

Plastic Surgery Research Council 2005 Clifford C. Snyder, MD Award: Nuclear Cloning by Fusion of Somatic Cells

Jason Pomerantz, MD and Helen Blau, PhD.

Introduction: Fusion of different somatic cell types results in nuclear reprogramming, the adoption of new gene expression profiles in response to cytoplasmic factors. We investigated techniques to reprogram nuclei from fibroblasts into mononucleate muscle cells capable of proliferation and differentiation. This approach, if successful, would allow production of cell types of interest from individual patients without the requirement for oocytes or ES cells. Methods: Polyethylene glycol was used to artificially fuse myoblasts with fibroblasts that stably expressed GFP and a neomycin resistance gene. The resulting fused cells consisted of a higher proportion of muscle to non-muscle nuclei and were allowed to differentiate for two days in conditions favoring muscle differentiation. Cultures were treated with cytosine arabinoside to kill unfused cells that continued to synthesize DNA and then with the purine analog, myoseverin, that causes cellularization of myotubes by interfering with microtubule structure. Neomycin and serum were used to select cells and re-stimulate proliferation respectively. Surviving cells were analyzed clonally for myogenic differentiation, by morphology and muscle marker expression. Results: RT-PCR analysis of muscle gene activation in non-muscle nuclei confirmed that non-muscle nuclei recapitulate the muscle differentiation pattern of gene expression after cell fusion. Non-muscle nuclei remained distinct, intact and did not synthesize DNA while existing in syncytia with muscle nuclei. Myoseverin treatment resulted in cellularization of cell fusion products resulting in a significant proportion of mononuclear cells. Re-stimulation with growth medium in the presence of selection agents resulted in the proliferation of neomycin resistant, GFP positive clones. A small percentage of these clones exhibited myogenic differentiation characteristics including fusion to form myotubes and expression of myosin heavy chain. Conclusion: Our results suggest that it is possible to recover and grow somatic cells whose nuclei have been reprogrammed by cell fusion and exposure to factors present in the cytoplasm of a different cell type. Fusion of somatic cells in vitro and subsequent reprogramming of gene expression may represent one viable method of directed, tissue specific cloning that bypasses the use of embryonic stem cells.