PITA Fiscal Year 2007 Projects

Biomedical and Health Engineering

Random Cell Fluorescent Tagging to Select New Stem Cell Behavior Marker Systems
In order to better understand the process of tissue formation and thus better equip ourselves to engineer replacement tissues, new technologies to measure, quantify and predict cell responses to multiple stimuli are required. This proposal describes the rationale and strategy to develop an enabling technology to quantify differentiative cell responses to multiple stimuli. Real-time of quantification of downstream signaling targets in living cells expressing reporter-tagged proteins will be our metric to detect cell differentiative response to multiple stimuli. Up to this point such cell lines have been sought by targeting genes, such as Osterix (Osx), that are known to be upregulated or downregulated at the mRNA level. This approach, however, has failed in many cases because the reporter-tagged protein is insufficiently abundant, short-lived, or because mRNA levels do not closely correlate with protein abundance. We propose here a new approach to develop new reporter cell-lines more efficiently. Our approach is to 1) randomly CD-tag cellular proteins; 2) use FACS (fluorescence activated cell sorting) to select for cells expressing morphogen (growth factor)-modulated proteins; and 3) use our 2D printing technology to create microenvironments of morphogens and to use the tagging system to observe a variety of cell behavioral responses (proliferation, migration, differentiation, and/or apoptosis) and to correlate the responses with tagged proteins levels and locations.

Successful completion of the proposed project will enable detection and quantification of cell response on engineered 2D and 3D biomimetic ECMs for applications in the regenerative medicine, tissue engineering, drug delivery and developmental biology fields. Our corporate partner SpectraGenetics here in Pittsburgh will benefit through the opportunity to commercialize the tagged genes and assay systems that will be generated and validated within the project.