Neural progenitor cells from the proximal colons of patients with Hirschsprung disease divided and formed neurons and glia in their own distal aganglionic colon tissue, according to a study published in the January issue of Cellular and Molecular Gastroenterology and Hepatology.
The approach could lead to “a promising therapeutic strategy” for Hirschsprung disease, because autologous transplantation of nerve progenitor cells would prevent immune rejection, said Dr. Benjamin Rollo of Murdoch Children’s Research Institute in Victoria, Australia, and his associates. But they cautioned against reading too much into their findings, “as there are distributional hurdles to surmount” before cell therapy can be used alone or in combination with surgery.
Hirschsprung disease is a congenital disorder in which embryonic neural crest cells fail to form the distal part of the enteric nervous system. The resulting lack of bowel motility leads to severe constipation and potentially fatal megacolon. Treatment involves surgically resecting the aganglionic distal bowel and anastomosing the normal proximal bowel to the anorectum, but this approach itself can cause constipation as well as fecal soiling, the investigators noted (Cell Molec Gastroenterol Hepatol. 2015 Oct. 22 [doi: 10.1016/j.jcmgh.2015.09.007]).
For the study, they harvested tissue from the proximal (neuronal) and distal (aneuronal) margins of resection of the colons of 31 infants and children with Hirschsprung disease. They cultured cells from the myenteric plexus of the proximal colon, and separated out nerve cells by using flow cytometry for the p75 neural crest marker. To test whether these nerve cells could colonize aganglionic colon, they co-cultured them with aneural avian embryo gut and with patients’ own aneuronal colon muscle. In addition, they co-cultured embryonic mouse enteric nernous system cells with human aneuronal colon muscle. They used quantitative reverse transcriptase PCR and several other standard laboratory techniques to detect cellular markers and mitosis.
Ganglia from the patients’ proximal colons expressed the neural crest markers p75, SOX10, and HNK1, as well as several neuronal, neurite, and glial markers, the researchers reported. The proximal colon specimens also contained ENS progenitor cells, which were SOX10-positive but lacked neuronal or glial markers. The progenitor cells comprised less than 5% of all cells from the proximal colon, but proliferated fourfold more after supplementation with CHIR-99021, a selective inhibitor of glycogen synthase kinase 3. They successfully colonized avian aneural embryonic gut and autologous postnatal aneuronal colon tissue, and the latter also was colonized successfully by mouse enteric neural crest cells.
The study “fulfilled three key requirements” for transplantation – harvesting enteric nervous system cells from the proximal colons of patients with Hirschsprung disease; confirming that postnatal aneuronal colon tissue could support an enteric nervous system; and showing that enteric nervous system–derived cells could colonize both embryonic gut and autologous postnatal colon tissue, said the researchers. More work is needed to sufficiently expand embryonic neural crest stem or progenitor cells and transplant them over a large area of colon, although supplementation with growth factors and use of elastic polymer substrates might help, they added.
The National Health and Medical Research Council, Murdoch Children’s Research Institute, Graham Burke and Yvonne Spencer, the Federation of Chinese Associations, Fonds du Service de chirurgie pédiatrique et de Perfectionnement du CHUV, Fondation SICPA, and Société Académique Vaudoise funded the study. The researchers disclosed no conflicts of interest.