Systematic tracking of the biodistribution of enteric neural crest cells differentiated from human derived pluripotent stem cells in an in vivo mouse model
Introduction: Hirschsprung Disease (HD) is an enteric neuropathy that might be rescued with exogenous enteric neural crest cells (ENCC), but long-term models are fragile and don’t allow systematic injection assessment. Additionally, physical or transgenic creation of a niche for ENCC may impact biodistribution results. Therefore, we sought to systemically track human-derived ENCC in a mouse model with an intact enteric nervous system.
Methods: Induced pluripotent stem cells differentiated into ENCC were labeled with a fluorescent dye, Di-I. 100υl of media alone or with 1e7 cells were injected into the stomach subserosa of a NOD-SCID mouse (n= 2 control, 6 experimental). 16 biodistribution samples were taken from each mouse POD1 or POD3. ALU PCR was performed to screen samples for the presence of human cells. Immunofluorescent staining for LAMIN, a human cell marker, and TUJ1, a neuronal marker, were performed on ALU+ samples.
Results: ALU PCR had the highest signal in the stomach on POD 1 and POD 3 of experimental mice with no signal in our control mice. Di-I and LAMIN co-staining of ENCC neurospheres shows Di-I uptake on the neurosphere surface with poor central penetration while LAMIN stains throughout in vitro. Immunofluorescent staining of ALU+ samples revealed multiple Di-I/TUJ1+ cells within the injection site while LAMIN+ staining was not seen.
Conclusion: Di-I provides better identification of ENCC in vivo and, along with ALU PCR, enables systematic tracking at the cellular level in a mouse model. Biodistribution studies are critical for the application of cellular techniques for future human therapies.