The generation of induced pluripotent stem cells (iPSCs) presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. While several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPSCs. The rapid expansion of a minor population of reprogrammed cells can also obscure investigation of relevant processes. Understanding of the biological mechanisms essential for successful iPSC generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that reprogramming follows an orderly sequence of stage transitions marked by changes in cell surface markers CD44 and ICAM1, and a Nanog-GFP reporter. RNA-sequencing (RNA-seq) analysis of these populations demonstrates two waves of pluripotency gene up-regulation, and unexpectedly, transient up-regulation of multiple epidermis-related genes, demonstrating that reprogramming is not simply the reversal of normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and this improved understanding of the reprogramming process will lead to novel reprogramming strategies.

Pancreatic Ductal Adenocarcinoma (PDAC) continues to have a dismally low survival rate due to late diagnosis and poor treatment options. Therefore, there is a need to understand the early stages and progression of the disease. PDAC is known to have two types of cells of origin: ductal cells or acinar cells. Since acinar-derived PDAC is thought to be the more malignant of the two, it was chosen as the focus of this work. Most studies of acinar cells as they relate to PDAC are accomplished by using animal models such as genetically engineered mouse models. While this method yields a large amount of insight into the progression of the disease and the role of specific genes, it has the drawbacks of being very time and resource intensive. The quicker and less costly alternative is <i>in vitro </i>culture. Specifically, here we have developed a microfluidic model which can incorporate a key aspect of the extracellular matrix (ECM), type I collagen, and mimics the 3D geometry of an <i>in vivo </i>acinus. Most attempts at <i>in vitro </i>culture have been limited by the fact that isolated acinar cells show a decrease in the amount of digestive enzymes they secrete as culture continues. For this reason, we are using a reprogrammed cancer cell line. These cells can be induced with doxycycline to express PTF1a, which allows the cells to adapt acinar characteristics, such as the production of digestive enzymes. We were able to successfully culture and induce PTF1a in these cells within our chip. We showed that the cells exhibit no invasion into the collagen matrix once PTF1a is expressed, thus eliminating a key aspect of cancer cell culture. The cells grown in the chip are confirmed to be producing PRSS2, the digestive enzyme trypsinogen. Collectively, this suggests that we have produced healthy acinar cells growing in the same configuration that they would <i>in vivo. </i>This has many applications in the study of pancreatic ductal adenocarcinoma, as we have developed way to culture reprogramed cancer cells as their benign precursors and maintain acinar characteristics <i>in vitro.</i> It will also have applications in the study of many other pancreatic diseases by providing an <i>in vitro</i> model of a healthy, functional acinus.