Year 3

We have made significant progress in accomplishing the overall goals of our grant application. We have obtained skin
cell from 18 subjects with various inherited gastrointestinal disorders, and we are in the process of reprogramming
these cells to generate pluripotent stem cells. These cells have been used to generate intestinal epithelium that is
allowing us to model the disorders of patient with specific disorders of intestinal failure. We anticipate that this
approach will allow us to further develop tools that will let us understand the basis of chronic diarrhea and intestinal
failure in children.

We have also performed total genome (exome) sequencing of several subjects with unique forms of chronic diarrhea
and intestinal failure, and have identified several excellent candidate genes that likely account for these disorders. We
will continue to screen other subjects with similar disorders to determine the full range of genes that are likely
associated with these disorders.

We have also made considerable progress in our attempt to grow human small intestinal epithelium. In the prior year
we developed methods to grow intestinal epithelium obtained from human surgical samples by growing them in a
petri dish on a feeder layer of cells. We have now improved this method by eliminating the need of a feeder layer of
cells, and have developed ways of significantly expanding the gut stem cell in significant quantities to allow for
modeling. We have also adopted these methods to grow intestinal samples that are obtained from endoscopic
biopsies. We believe that this advancement will improve the usefulness of our culture system to samples obtained at
the time of biopsy.

Taken together, we have made considerable progress in developing the tools required to understand the genetic basis
of chronic diarrhea and intestinal failure in children. We are using a combination of genome sequencing, pluripotent
stem cells, and tissue (somatic) stem cells to decipher the biology of established disorders, and to discover new
disorders that have a significant impact on the growth and well being of children severe gastrointestinal disorders.