Transplanted skin-derived precursor stem cells generate enteric ganglion-like structures in vivo

Introduction: Hirschsprung's disease is characterized by a developmental arrest of neural crest cell migration, causing distal aganglionosis. Transplanted cells derived from the neural crest may regenerate enteric ganglia in this condition. We investigated the potential of skin-derived precursor cells (SKPs) to engraft and to differentiate into enteric ganglia in aganglionic rat intestine in vivo.

Methods: Adult Lewis rat jejunal segments were separated from intestinal continuity and treated with benzalkonium chloride to induce aganglionosis. Ganglia were identified via immunohistochemical stains for S100 and β-III tubulin (TUJ1). SKPs were procured from neonatal Lewis rats expressing enhanced green fluorescent protein (GFP) and cultured in neuroglial-selective media. SKP cell line expansion was quantified, and immunophenotypes were assessed by immunocytochemistry. Aganglionic segments underwent SKP transplantation 21-79days after benzalkonium chloride treatment. The presence of GFP+cells, mature neurons, and mature glia was evaluated at posttransplant days 1, 6, and 9.

Results: Benzalkonium chloride-induced aganglionosis persisted for at least 85days. Prior to differentiation, SKPs expressed S100, denoting neural crest lineage, and nestin, a marker of neuronal precursors. Differentiated SKPs in vitro expressed GFAP, a marker of glial differentiation, as well as TUJ1 and several enteric neurotransmitters. After transplantation, GFP+structures resembling ganglia were identified between longitudinal and circular smooth muscle layers.

Conclusion: SKPs are capable of engraftment, migration, and differentiation within aganglionic rodent intestine in vivo. Differentiated SKPs generate structures that resemble enteric ganglia. Our observations suggest that SKPs represent a potential gangliogenic therapeutic agent for Hirschsprung's disease.