Lentiviral labeling of mouse and human enteric nervous system stem cells for regenerative medicine studies.

[hirschsprung disease]

Reliable methods of labeling human enteric nervous system (ENS ) stem cells for use in novel cell replacement therapies for enteric neuropathies are lacking . Here , we explore the possibility of using lentiviral vectors expressing fluorescent reporter genes to transduce , label , and trace mouse and human ENS stem cells following transplantation into mouse gut .Enteric nervous system precursors , including ENS stem cells , were isolated from enzymatically dissociated mouse and human gut tissues . Lentivirus containing eGFP or mCherry fluorescent reporter genes was added to gut cell cultures at a multiplicity of infection of 2 -5 . After fluorescence activated cell sorting for eGFP and subsequent analysis with markers of proliferation and cell phenotype , transduced mouse and human cells were transplanted into the gut of C 5 7 BL /6 and immune deficient Rag 2 -/gamma chain-/C 5 mice , respectively and analyzed up to 60 Â days later .Mouse and human transduced cells survived in vitro , maintained intense eGFP expression , proliferated as shown by BrdU incorporation , and formed characteristic neurospheres . When transplanted into mouse gut in vivo and analyzed up to 2 Â months later , transduced mouse and human cells survived , strongly expressed eGFP and integrated into endogenous ENS networks .Lentiviral vectors expressing fluorescent reporter genes enable efficient , stable , long -term labeling of ENS stem cells when transplanted into in vivo mouse gut . This lentiviral approach not only addresses the need for a reliable fluorescent marker of human ENS stem cells for preclinical studies , but also raises the possibility of using lentiviruses for other applications , such as gene therapy .

Diseases
Validation

Diseases presenting "integrated into endogenous ens networks" symptom

hirschsprung disease

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