Neurons derived from human embryonic stem cells extend long-distance axonal projections through growth along host white matter tracts after intra-cerebral transplantation

Mark Denham,Bryan Leaw,Lachlan H Thompson,Clare L Parish,Christopher A Reid,Mirella Dottori,Steven Petrou,Jordan Wright

doi:10.3389/fncel.2012.00011

Mark Denham, Bryan Leaw + Show 6 more

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https://doi.org/10.3389/fncel.2012.00011

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Abstract

Human pluripotent stem cells have the capacity for directed differentiation into a wide variety of neuronal subtypes that may be useful for brain repair. While a substantial body of research has lead to a detailed understanding of the ability of neurons in fetal tissue grafts to structurally and functionally integrate after intra-cerebral transplantation, we are only just beginning to understand the in vivo properties of neurons derived from human pluripotent stem cells. Here we have utilized the human embryonic stem (ES) cell line Envy, which constitutively expresses green fluorescent protein (GFP), in order to study the in vivo properties of neurons derived from human ES cells. Rapid and efficient neural induction, followed by differentiation as neurospheres resulted in a GFP+ neural precursor population with traits of neuroepithelial and dorsal forebrain identity. Ten weeks after transplantation into neonatal rats, GFP+ fiber patterns revealed extensive axonal growth in the host brain, particularly along host white matter tracts, although innervation of adjacent nuclei was limited. The grafts were composed of a mix of neural cell types including differentiated neurons and glia, but also dividing neural progenitors and migrating neuroblasts, indicating an incomplete state of maturation at 10 weeks. This was reflected in patch-clamp recordings showing stereotypical properties appropriate for mature functional neurons, including the ability to generate action potentials, as well profiles consistent for more immature neurons. These findings illustrate the intrinsic capacity for neurons derived from human ES cells to integrate at a structural and functional level following transplantation.

Highlights

Substantial advances in pluripotent stem cell biology have fueled optimism for the development of stem cell-based procedures for brain repair
HESCs were grown on mitomycin-C treated mouse embryonic fibroblasts (MEFs) in hESC medium consisting of high-glucose Dulbecco’s modified Eagle’s medium (DMEM) without sodium pyruvate, supplemented with 1% insulin/transferrin/selenium, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids (NEAA), 2 mM glutamine, 25 U/ml penicillin, 25 μg/ml streptomycin, and 20% fetal calf serum (Hyclone) or on mitomycin-C treated human foreskin fibroblasts (HFF) in knockout serum replacement (KSR) media consisting of DMEM/nutrient mixture F-12, supplemented with 0.1 mM βmercaptoethanol, 1% NEAA, 2 mM glutamine, 25 U/ml penicillin, 25 μg/ml streptomycin, and 20% KSR
DISCUSSION there has been a significant period of rapid progress in the pluripotent stem cell field over the last decade, the success we have seen in the culture dish, allowing for the procurement of a diverse range of neuronal phenotypes from highly expandable stem cell populations, has been difficult to translate into an in vivo setting with effective and predictable outcomes in animal models of brain injury

Summary

Introduction

Substantial advances in pluripotent stem cell biology have fueled optimism for the development of stem cell-based procedures for brain repair. Clinical trials using fetal donor tissue in PD patients have provided proof-of-principle that new neurons, transplanted directly into the brain of the patient, can replace damaged circuitry with appropriate structural and functional features in order to significantly restore the disturbances in motor function associated with PD (Lindvall and Hagell, 2000; Lindvall and Bjorklund, 2004). In the context of brain repair, pluripotent stem cells possess attractive features including a capacity for large-scale expansion as a cell source for neural transplantation procedures and potential for differentiation into a range of potentially therapeutic cell types relevant for specific neurological conditions (Barberi et al, 2003). The rational development of stem cell-based transplantation procedures for brain repair requires a detailed understanding of the in vivo properties of stem cell-derived neurons, including their capacity for structural and functional incorporation into host circuitry

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