In vivo transplantation of fetal human gut-derived enteric neural crest cells

Abstract

The prospect of using neural cell replacement for the treatment of severe enteric neuropathies has seen significant progress in the last decade. The ability to harvest and transplant enteric neural crest cells (ENCCs) that functionally integrate within recipient intestine has recently been confirmed by in vivo murine studies. Although similar cells can be harvested from human fetal and postnatal gut, no studies have as yet verified their functional viability upon in vivo transplantation. We sought to determine whether ENCCs harvested from human fetal bowel are capable of engraftment and functional integration within recipient intestine following in vivo transplantation into postnatal murine colon. Enteric neural crest cells selected and harvested from fetal human gut using the neurotrophin receptor p75^NTR were lentivirally labeled with either GFP or calcium-sensitive GCaMP and transplanted into the hindgut of Rag2⁻/γc⁻/C5⁻-immunodeficient mice at postnatal day 21. Transplanted intestines were assessed immunohistochemically for engraftment and differentiation of donor cells. Functional viability and integration with host neuromusculature was assessed using calcium imaging. Transplanted human fetal gut-derived ENCC showed engraftment within the recipient postnatal colon in 8/15 mice (53.3%). At 4 weeks posttransplantation, donor cells had spread from the site of transplantation and extended projections over distances of 1.2 ± 0.6 mm (n = 5), and differentiated into enteric nervous system (ENS) appropriate neurons and glia. These cells formed branching networks located with the myenteric plexus. Calcium transients (change in intensity F/F0 = 1.25 ± 0.03; 15 cells) were recorded in transplanted cells upon stimulation of the recipient endogenous ENS demonstrating their viability and establishment of functional connections.

This study demonstrates for the first time that human gut-derived ENCCs are capable of colonization of mouse colon, in vivo, forming both neurons and glia. Importantly, human gut-derived ENCCs make functional neuronal connections with the endogenous ENS. These findings increase the prospect of using human ENCC as a cell source for autologous cell replacement therapies to treat enteric neuropathies. ENCCs were isolated from fetal human gut by FACS for p75NTR before propagation in vitro and transplantation in vivo. Transplanted bowel was analyzed after 4 weeks by immunohistochemistry and calcium imaging. Transplanted cells displayed engraftment, spread, extension of projections, differentiation towards neurons and glia as well as functional connectivity with the endogenous ENS.

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