Regulation and patterning of cell differentiation and pluripotency (2011)

The development of a multicellular organism from an embryo is one of the nature's most remarkable phenomena. Deciphering how this transformation occurs is a fundamental challenge in biology with profound biomedical implications. Insights into the molecular signals guiding developmental patterning may provide design strategies to promote multicellular structure formation in applications such as tissue engineering and regenerative medicine. In this thesis, we explored the applications of controllable gene expression techniques in combination with engineering strategies in regulation and patterning of cell differentiation and pluripotency, by pursuing three related research projects: 1. Reversible immortalization of cardiomyocytes to enable their proliferation necessary for obtaining large cell numbers 2. Patterning of the delivery of Doxycycline (Dox), the expression modulator of inducible BMP-2 expression vector, to mesenchymal stem cells cultured in a microfluidic 3. Patterning of the Nanog gene expression in embryonic stem cells, using a microfluidic device, to establish differentiation - pluripotency boundaries that mimic the developmental processes in vivo. In the first project, we developed a novel strategy for controlled expansion of non-proliferating primary neonatal rat cardiomyocytes by lentivector-mediated cell immortalization, and then the reversal of the phenotype of expanded cells back to the cardiomyocytes state. Primary rat cardiomyocytes were transduced with simian virus 40 large T antigen (TAg), or with Bmi-1 followed by the human telomerase reverse transcriptase (hTERT) gene; the cells were expanded; and the transduced genes were removed by adenoviral vector expressing Cre recombinase. The TAg gene was more efficient in cell transduction than the Bmi-1/hTERT gene, based on the rate of cell proliferation. Immortalized cells exhibited the morphological features of dedifferentiation (increased vimentin expression, and reduced expression of troponin I and Nkx2.5) along with the continued expression of cardiac markers (α-actin, connexin-43, and calcium transients). After the immortalization was reversed, cells returned to their differentiated state, as evidenced by molecular and functional properties inherent to terminally differentiated cardiomyocytes. This strategy for controlled expansion of primary cardiomyocytes by reversible gene transfer could provide large amounts of a patient's own cardiomyocytes for cell therapy, and enable controlled in vitro study of cardiogenesis. In the second project, we developed a novel patterning strategy by using inducible gene expression systems in conjunction with simple multi-laminar fluidic techniques, which can directly pattern the expression of particular gene at transcriptional level. Using osteogenic differentiation of human mesenchymal stem cells as a model, we describe a novel approach to spatially regulate the expression and secretion of bone morphogenetic protein (BMP-2) in a two-dimensional field of cultured cells, by flow patterning the modulators of inducible BMP-2 gene expression. We first demonstrated a control of gene expression, and control of osteogenic differentiation of the cell line with inducible expression of BMP-2. Then we designed laminar flow systems, with patterned delivery of Doxycycline (Dox), the expression modulator of inducible BMP-2 expression vector. The patterned concentration profiles were verified by computational simulation and dye separation experiments. Experiments conducted in the flow systems for a period of three weeks showed the Dox concentration dependent osteogenic differentiation, as evidenced by mineral deposition. This strategy combining inducible gene expression with laminar flow technologies provides an innovative way to engineer tissue interfaces. In the third project, we further developed the patterning strategy for gene expression to form boundaries of different gene expression domains in cultures of mouse embryonic stem cells. Using Nanog safeguarded embryonic pluripotency as a model; we demonstrated controlled Nanog expression, which lead to controlled early differentiation under the exposure or withdrawal of varied small molecules, as evidenced by alkaline phosphatase (AP) staining, immunofluorescent staining, and gene expression analysis. By patterning Nanog gene expression, as well as soluble factors in the laminar fluidic system, we successfully developed varied differentiation - pluripotency boundaries between Nanog expressing pluripotency zones and Nanog suppressed early differentiation zones from the same population of cells, which mimic the development process in vivo. Mechanistic insights can be gained on dissecting the signaling pathways that drive multicellular patterning during the natural processes of embryonic and adult development. In summary, we demonstrated that controlled expansion of non-proliferating primary cells can achieved by reversible genetic manipulation, and that varied continuous, graded pluripotency - differentiation boundaries can established by patterning the expression of target genes via a simple laminar fluidic system. Taken together, these approaches provide innovative models to modulate cell function at the transcriptional level. Additional cooperative research was conducted during my graduate training. The manuscript of this study "Micropatterned Mammalian Cells Exhibit Phenotype-Specific Left-Right Asymmetry" was submitted to Proc Natl Acad Sci U S A., and it is currently under review. We attached this manuscript in appendix.