Human cortical neurons rapidly generated by embryonic stem cell programming integrate into the stroke-injured rat cortex.

Abstract

Stem cell sources capable of producing appropriate cells for replacement will be necessary for functional repair of the injured brain. Here, we have determined whether transcription factor programming of human embryonic stem (hES) cells can be used to generate layer-specific cortical neurons capable of integrating into the stroke-injured rat cortex. Human embryonic stem cells were programmed via overexpression of neurogenin 2 (NGN2). After 7 days, hES-induced neurons (hES-iNs) were characterized in vitro using immunocytochemistry, RT-qPCR, and whole-cell patch-clamp. Cortical ischemic stroke was induced in rats via distal middle cerebral artery occlusion. Forty-eight hours later, hES-iNs were transplanted into the somatosensory cortex adjacent to the ischemic lesion. Three months thereafter, brains were analyzed for expression of neuronal markers, axonal myelination, and synapse formation using immunohistochemistry and immunoelectron microscopy (iEM). Overexpression of NGN2 in hES cells for 7 days generated excitatory neurons, expressing cortical markers at different stages of maturation. After transplantation, the hES-iNs expressed markers of both immature and mature neurons and of upper and deep cortical layers. The hES-iNs sent widespread projections to both hemispheres, and iEM revealed that they were myelinated by host oligodendrocytes and had formed efferent synaptic connections with host cortical neurons. The hES cells programmed via NGN2 overexpression gave rise to subtypes of cortical neurons, capable of integrating structurally into the injured brain, more rapidly than neurons produced by previous protocols. Functional characterization of the grafted hES-iNs and their impact on the balance between brain excitation and inhibition is now highly warranted. This new stem cell source should be considered when, in the future, the most suitable candidate will be selected for clinical translation.