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

Abstract

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 21–79 days after benzalkonium chloride treatment. The presence of GFP+ cells, mature neurons, and mature glia was evaluated at post-transplant days 1, 6, and 9. Results Benzalkonium chloride-induced aganglionosis persisted for at least 85 days. 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.

Skin-derived precursors generate enteric-type neurons in aganglionic jejunum

“…SKPs were harvested following the protocol described by Biernaskie and colleagues. 25,27 For each SKP isolation, skin specimens were obtained from 2 pups for optimal yield. SKP isolations were performed among 3 litters, and cell yields were quantified with a hemacytometer.…”

“…27 The procedure resulted in a 1 cm-long isolated jejunal segment and a bypass jejunojejunostomy to restore intestinal continuity. Chemical denervation of the isolated jejunal segment was achieved by trans-serosal benzalkonium chloride (BAC, 0.2% w/v, Sigma-Aldrich) exposure for 20 minutes.…”

“…23–26 We recently discovered that SKPs are capable of gangliogenesis within aganglionic jejunum in vivo . 27 Nevertheless, the physiologic function of neurons in these SKP-derived ganglia is indistinct. Kwok and colleagues have shown that SKPs generate neurons capable of enteric neurotransmitter synthesis within intestinal explants.…”

Mouse model of endoscopically ablated enteric nervous system

“…In such a model, the ablated enteric nervous system is inevitably reconstituted through migration of enteric nervous elements from the healthy proximal and distal ends of the ablated segment [5]. An alternative configuration can be used where the intestinal segment of interest is taken out of continuity to prevent migration of enteric nervous cells [8]. However, in both techniques, multiple laparotomies would be needed to first induce ENS ablation and subsequently transplant neural cells, making this technique challenging in the murine model.…”

A novel in vivo model of permanent intestinal aganglionosis

“…Nevertheless, we have demonstrated the utility of this model system as a recipient for transplanted skin-derived precursor cells (SKPs) to regenerate enteric ganglia. 31 It remains unclear whether muscularis hypertrophy will influence our ability to detect motility changes induced by cellular regenerative therapies. We anticipate the behaviors of a variety of cellular therapeutic agents within this recipient model will require significant scientific scrutiny.…”