Developing a human pluripotent stem cell-based strategy for treating Hirschsprung disease

Hirschsprung disease is a life-threatening intestinal disorder caused by an absence of intrinsic nerve cells (aganglionosis) in the most distal gastrointestinal tract. It occurs in approximately 1 in 5000 live births, making it one of the most common congenital diseases affecting the gut. Given that intrinsic gut nerve cells (enteric neurons) mediate the contractions necessary for normal gut function, their absence in Hirschsprung patients causes severe constipation or intestinal obstruction. The only treatment available is surgical removal of the affected part of the bowel combined with a 'pull through' procedure, which entails connecting the healthy part of the gut to the anus. However, the surgery necessitates retention of part of the abnormal gut including the anal sphincter, which is likely to account in part for the long-term, often life-long, gastrointestinal problems and poor quality of life suffered by the majority of patients with Hirschsprung disease. Surgery, readmissions and outpatients hospital appointments required for management of this condition present a significant burden for the healthcare system. Recent advances in the understanding of development of the gut's intrinsic (or enteric) nervous system and pathogenesis of the disease, as well as considerable progress in regenerative medicine, have highlighted potential for alternative treatments, such as cell replacement therapy.

During normal embryonic development, the enteric nervous system is derived from a transient population of cells termed neural crest cells that migrate from the neural tube to innervate the gastrointestinal tract. In Hirschsprung disease these cells fail to colonize the distal gut leaving a variable segment of aganglionosis. Experiments in animal models have demonstrated that transplantation of enteric nervous system progenitor/stem cells (derivatives of the original neural crest cells and harvested from mouse gut explants or mouse embryonic stem cells) have the ability to differentiate into enteric neurons and colonize when transplanted into aganglionic gut explants. Similar neuronal progenitor cells obtained from human postnatal gut explants have been shown to effectively colonize and differentiate within aganglionic gut explants of Hirschsprung patients. However, the harvesting of such human nerve progenitor cells from gut is difficult and becomes more so with increasing post-natal age. Therefore, although such preclinical testing has demonstrated that cell therapy should be a viable option for curing Hirschsprung disease, the availability of human enteric neurons from post-natal gut remains a bottleneck for development of cell-based therapies.

An attractive alternative source is offered by pluripotent stem cells, where there is significant potential of generating appropriate cells for therapy both in terms of numbers and tailoring cell properties for the task. We have recently developed efficient protocols to generate human neural crest cells and enteric nervous system progenitors from human pluripotent stem cells. In this project, we will test the ability of these progenitors to correct for the lack of enteric neurons in animal and patient-derived models of Hirschsprung disease following transplantation. We will also optimise the current methods of generating and purifying human enteric nervous system progenitors from pluripotent stem cells
and will develop a new improved animal model of Hirschsprung disease. In this way, we will establish the pre-clinical basis for a regenerative medicine approach for treating Hirschsprung disease by transplantation of cells derived from human pluripotent stem cells to re-innervate the affected part of the gut. In the first instance our goal will be to improve the outcome of current treatments by innervation of the short section remaining after routine surgery.

Hirschsprung disease (HSCR) is the consequence of a failure of neural crest-derived enteric neural progenitor cells to colonise the distal gut during embryonic and fetal development. Nevertheless, such progenitors derived from mouse and human postnatal gut have been shown to effectively colonize, and differentiate within, aganglionic gut explants from animal models of HSCR and from affected patients, demonstrating that cell therapy should be a viable option for treatment. However, harvesting sufficient progenitor cells from human gut biopsies poses a significant hurdle which could be overcome by their production from differentiation of human pluripotent stem cells (hPSC). We have established an effective protocol to derive neural crest cells from hPSC in chemically defined, xeno-free conditions, and have shown that these can generate enteric neural progenitors which can colonise long term and efficiently the intestine of animals following transplantation and also produce enteric neurons in vitro. In this project we will establish the preclinical basis for a regenerative medicine approach to treating HSCR by transplantation of hPSC-derived enteric neural progenitors to re-innervate the affected part of the gut. To this end we will assess the ability of our hPSC-derived enteric neuron progenitors to correct for the lack of enteric neurons in both established and novel models of HSCR. We will also identify new specific cell surface markers to improve the isolation of enteric neuron progenitors suitable for transplantation and optimise the protocol for their generation. Finally, we will define the best-performing hPSC cell line for generating such progenitors in order to treat Hirschsprung disease.

The overall goal of this project is to develop a regenerative medicine approach for improving the treatment of Hirschsprung disease (HSCR). HSCR, affecting about 1 in 5000 live births, is one of the most common congenital diseases affecting the gut. It is a life-threatening intestinal disorder caused by the absence of enteric neurons in the most distal bowel, which loses its propulsive gut motility and ultimately results in severe constipation or intestinal obstruction. The only treatment involves surgical resection of the affected part of the bowel which is, in turn, associated with life-long gastrointestinal problems suffered by HSCR patients that contribute to poor life quality. Surgery, readmissions and outpatient hospital appointments required for management of HSCR present an ongoing significant burden for the healthcare system.

Recent advances in the understanding of development of the enteric nervous system (ENS, the intrinsic innervation of the gastrointestinal tract) and pathogenesis of HSCR have highlighted the potential for cell replacement therapy, but an adequate source of cells for transplantation remains problematic. However, our preliminary data indicate that generation of ENS progenitors from human pluripotent stem cells (hPSCs) in the petri dish could provide a viable route for effective treatments. The goal of the current project is to lay the foundations for clinical trials of the transplantation of hPSC-derived ENS progenitors to overcome the problems associated with current surgical treatments. The ultimate beneficiaries of this will be the children born with this condition; apart from greatly improving their quality of life, development of this approach would have an economic impact in reducing the costs of long term care. In the nearer term, the results will demonstrate to clinicians and industry the feasibility of using ENS progenitors derived from hPSCs in regenerative medicine for HSCR. To this end we have had discussions with the Cell Therapy Catapult and Takeda Pharmaceuticals, who have expressed an interest should we successfully demonstrate the potential of the approach. Another area of regenerative medicine for which the production of enteric neural cells from human pluripotent stem cells will be of value is in gut tissue engineering. For example, two of us, Nikhil Thapar and Paolo De Coppi, are part of a grouping, led by Paolo De Coppi, that has been awarded a Horizon 2020 grant for tissue engineering intestine for children with short bowel syndrome. Incorporating a neural element to the tissue engineered intestine will be essential and will be facilitated by the output from the current proposed project.

In addition to the anticipated impact for regenerative medicine, the tools developed in this project are likely to be of use to those studying both the development of other organs and tissues from derivatives of the neural crest, as well as development of the gut neuromusculature. In both cases the results may facilitate work to develop disease models for a wide array of conditions arising from defects in the underlying developmental mechanisms. Such models are likely to benefit, among others, pharmaceutical companies seeking relevant tools for drug development.