Abstract
Hypoxic-ischemic encephalopathy (HIE) at birth could cause cerebral palsy (CP), mental retardation, and epilepsy, which last throughout the individual's lifetime. However, few restorative treatments for ischemic tissue are currently available. Cell replacement therapy offers the potential to rescue brain damage caused by HI and to restore motor function. In the present study, we evaluated the ability of embryonic stem cell-derived neural progenitor cells (ES-NPCs) to become cortical deep layer neurons, to restore the neural network, and to repair brain damage in an HIE mouse model. ES cells stably expressing the reporter gene GFP are induced to a neural precursor state by stromal cell co-culture. Forty-hours after the induction of HIE, animals were grafted with ES-NPCs targeting the deep layer of the motor cortex in the ischemic brain. Motor function was evaluated 3 weeks after transplantation. Immunohistochemistry and neuroanatomical tracing with GFP were used to analyze neuronal differentiation and axonal sprouting. ES-NPCs could differentiate to cortical neurons with pyramidal morphology and expressed the deep layer-specific marker, Ctip2. The graft showed good survival and an appropriate innervation pattern via axonal sprouting from engrafted cells in the ischemic brain. The motor functions of the transplanted HIE mice also improved significantly compared to the sham-transplanted group. These findings suggest that cortical region specific engraftment of preconditioned cortical precursor cells could support motor functional recovery in the HIE model. It is not clear whether this is a direct effect of the engrafted cells or due to neurotrophic factors produced by these cells. These results suggest that cortical region-specific NPC engraftment is a promising therapeutic approach for brain repair.
Highlights
Hypoxic-ischemic encephalopathy (HIE) represents a major cause of brain damage in the fetus and newborn infants, occurring in about 20 per 1000 term live birth, and in nearly 60% of very low birth weight newborns (MacDonald et al, 1980; Gunn, 2000)
Cells were plated at low density, 106 per 10 cm dish coated with gelatin (Millipore Bioscience Research Reagents; Embryonic stem (ES)-006-B), for 10 days in “Maintenance medium” consisting of Glasgow modified Eagle’s medium (GMEM; Sigma) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 2 mM L-glutamine (Gibco-Invitrogen), 0.1 mM non-essential amino acid (NEAA;Gibco-Invitrogen), 1 mM sodium pyruvate (Sigma), 0.1 mM 2-mercaptoethanol (Sigma), and 2000 units/ml leukemia inhibitory factor (LIF; Esgro, Chemicon) under Geneticin selection (1.5 μg/ml; Invitrogen)
Our goal was to establish a therapeutic strategy for hypoxia-ischemia encephalopathy (HIE) using NPCs grafted into the brains of injured mice
Summary
Hypoxic-ischemic encephalopathy (HIE) represents a major cause of brain damage in the fetus and newborn infants, occurring in about 20 per 1000 term live birth, and in nearly 60% of very low birth weight newborns (MacDonald et al, 1980; Gunn, 2000). Hypoxic ischemia (HI) stress triggers neuronal and glial injury leading to necrosis secondary to cellular edema, lysis, and the resulting apoptosis in delayed cellular injury (DelivoriaPapadopoulos and Mishra, 1998; Nakajima et al, 2000; Northington et al, 2001). These cellular injuries tended to occur in the subplate neurons and resulted in periventricular leukomalacia (PVL) due to immaturity of the cerebral vasculature or its vulnerability (Volpe, 2001; McQuillen et al, 2003). Each of these areas is composed of six layers of glutamatergic pyramidal neurons with distinct firing patterns, specific axonal and dendritic projection patterns (Connors and Gutnick, 1990; Tseng and Prince, 1993), and unique gene expression profiles including bHLH transcriptional factors, such as neuroD, neuroD2, and Math, which
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