The proposed work represents a significant step forward from current methodologies that may enable a new level of understanding of the genetic networks that may be important in stem cell differentiation or self-renewal. Although this methodology will be initially applied to a subpopulation of bone marrow derived mesenchymal stem cells (MSC), the broader applicability of this approach will likely impact other fields of biotechnology, bioengineering and medicine.
Broader impacts
A key outcome of the proposed work will be the development of a novel high-throughput technology that can be used to study diverse biological systems. Specifically, the proposed project will lead to the development of a genome-wide lentivirus library and lentivirus microarrays that will be made available to the scientific and engineering communities and that will be applicable to a wide range of cell types and bioengineering problems. The same concept can be expanded to libraries of antisense-RNA that can be used in loss-of-function studies, and these will be similarly disseminated. It will also produce quantitative kinetic data sets that will be available to the scientific community via an internet database. It will allow engineers to take a leading role in the growing field of functional genomics and encourage the application of engineering approaches to understanding stem cell differentiation in a quantitative manner. The PIs will make a concerted effort to recruit underrepresented minority and women graduate students for the Department of Chemical and Biological Engineering and this project, and has supervised a large fraction of female and minority students. The PI's former doctoral students are employed by diverse pharmaceutical, biotechnology, and engineering companies.
The major goal of this project was to develop an array of viruses (LentiViral Array, LVA) to transfer reporter genes into adult stem cells as a way of monitoring which genes and pathways are activated during differentiation of these cells into the three well-known lineages i.e. fat, bone or cartilage. Intellectual Merit During the award period our laboratory has made significant progress in this direction and developed the technologies and know-how necessary to realize the proposed work. To achieve these goals we had to develop several technologies. First, we developed a novel viral vector that enables quantitative measurement of gene or pathway activity in real time. This was a big step forward as it allowed us to obtain quantitative measurements over time, thereby providing temporal information as cells were differentiating. Next we developed a novel method to immobilize viral particles on hydrogels, enabling the generation of microarrayed devices with immobilized viral particles. In addition, we developed our second-generation vector that enables dynamic monitoring of gene and pathway activity while at the same time knocking down expression of some genes, in order to study whether they are necessary in the differentiation process. We demonstrated the LVA concept by generating arrays that were used to measure the activity of several genes and pathways as cells responded to inflammatory stimuli. Finally, we generated a library of >45 viruses to monitor stem cell differentiation and used it to identify novel pathways that maybe involved in this process. Our results revealed that the LVA could capture the dynamics of gene and pathway activation in real time, as stem cells differentiate. In addition, when combined with large libraries of signaling pathway inhibitors (chemical or siRNA libraries), the LVA can be used to uncover novel genes and signaling pathways affecting complex biological processes such as stem cell differentiation or reprogramming. Broader Impact This work developed a novel technology that can be used by many laboratories to monitor stem cells as they respond to different signals from their microenvironment ranging from soluble signals to mechanical cues transmitted from their 2D or 3D substrate. In addition to stem cells, this technology can be adopted to study other biological processes such as inflammation, or cancer progression and metastatic potential. Therefore, we expect that it may enable other investigators to address many different questions in a high-throughput manner (many genes or pathways can be studied at the same time; we employed 45), thereby saving time in identifying the most important ones for more in depth study. In addition, several graduate and undergraduate students have been trained at the interface of engineering and molecular/cellular biology in the duration of this project. In total seven graduate students (2 M.S. and 5 Ph.D. students) as well as four undergraduate students have worked on various aspects of this project. Three of these PhD and both M.S. students have since graduated and are employed in major companies such as Bristol-Myers Squibb, MedImmune, Shire Human Genetic Therapies and Meso Scale Discovery. The other two students are still in my laboratory finishing their PhD work. In addition, two of the undergraduate students have since joined graduate programs to pursue their PhD. Therefore, this work has had a significant impact on the research careers of many graduate and undergraduate students. Finally, the work has been disseminated in seven publications, sixteen meeting abstracts and more than ten invited talks in academic departments and hospitals around the country.
