Year 2 + NCE
The first aim of this project was to induce human embryonic stem cells (hESCs) into urothelium, which is the epithelial lining of the urinary tract. These cells are very specialized and unique. They are coated with a group of proteins, named uroplakins, which make the cells uniquely impermeable to many contaminants in the urine. Uroplakins are almost exclusively produced by urothelial cells, so they serve as a unique marker for these cells, and hence high expression of uroplakin is considered sine qua non for identifying urothelium. Thus, we are able to identify urothelial cells based on their expression of uroplakin proteins on the cell surface, as well as inside the cells, using various experimental techniques.
We evaluated numerous conditions to induce hESCs into urothelium. The first step in doing this was to induce hESCs into definitive endoderm (DE), which is a precursor stage of epithelial differentiation for the cells lining the intestines and urinary tract. We did this very efficiently and found that specialized urothelial cell culture medium with growth factor additives (uromedia) that we have optimized in our laboratory was able to induce about 20-30% of DE cells into urothelium, as determined by uroplakin expression. Since urothelium makes up less than 1% of the cells in the body, we believe we have developed a system that induces hESCs beyond their spontaneous preprogramming. We also tested the ability of growing DE with adult urothelium or adding growth factors produced by cultured adult urothelium or bladder smooth muscle to augment the differentiation, none of which succeeded in increasing urothelial cell differentiation in a statistically significant manner.
We were able to isolate the hESC-derived urothelium using a technique that sorts live cells based on protein expression (Fluorescence Activated Cell Sorting, or FACS). These cells, sorted based on uroplakin expression on the cell surface, are currently being cultured and tested to determine their protein expression and function.
The second aim of the project was to find transcription factors (TF) that are involved in directing the differentiation of hESCs into urothelium. Transcription factors affect the genetic mechanisms that lead to protein production. In our case, we were interested in what TFs were involved in the early expression of uroplakins as DE became urothelium. As there are hundreds of known TFs, we focused on those that have potential binding sites near the uroplakin genes. Using various techniques, we found that certain TFs were expressed in cells at the same time as uroplakins were first appearing. We were also able to see certain TFs disappear as hESCs became DE and then urothelium. Further work needs to be done to determine if these TFs are actually controlling the expression of the uroplakin genes or whether their co-expression is coincidental.
The third aim of the project was to determine if urothelium could be transplanted into a rodent bladder, survive and possibly incorporate into the organ. For injection, we used cultured urothelial cells that were labeled with a fluorescent genetic marker (green fluorescence protein – GFP) that allows them to be distinguished from the colorless host cells. GFP urothelial cells were obtained either from transgenic rats (all the cells in their body express GFP) or from urothelium infected with a specialized virus that transfers the GFP gene into the cells. GFP-urothelium was injected into the bladder walls of mice and rats. Ideally these cells should be injected under the bladder lining of these bladders. This potential space is microscopic and is difficult to infiltrate even with bladders outside the body on the laboratory bench. Therefore, getting into this space in a live animal under anesthesia was very difficult. Primarily, the cells were injected into the lamina propria, which is a space between the bladder muscle and epithelium. We were able to find viable transplanted cells within a few days of injection, but could not find cells at late time points after transplantation. This could be due to the cells not living, or it could be that very few cells did survive, but we were unable to find them.
Future work: We will continue to evaluate the urothelium produced from hESCs in this project. We hope that we can show that these cells appear and function just like normal urothelium, and thus be used for future tissue engineering applications.