Abstract
IntroductionIschemic stroke is a leading cause of death and disability, but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study, we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model.MethodsNeural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention.ResultsAfter 11 days of neural induction by using the small-molecule protocol, over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude, repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals.ConclusionsHuman embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo, enhance regenerative activities, and improve sensory recovery after ischemic stroke.
Highlights
Ischemic stroke is a leading cause of death and disability, but treatment options are severely limited
Various types of neural precursors, such as a conditionally immortalized cell line derived from human fetal tissue [7,8,9,10], lines derived from carcinomas [11,12], fetal neuronal stem cells [13,14], mouse neural precursors derived from the post-stroke cortex [15], region-specific murine embryonic precursors [16], and precursors derived from mouse [17,18,19] or human [20,21,22,23,24,25,26] embryonic stem cells have been used in experimental models
After 11 days of SMAD inhibition, cells had lost all detectable expression of pluripotency markers and had begun expressing neural precursor markers such as nestin, paired box gene 6 (PAX6), and sex-determining region Y-box 1 (SOX1)
Summary
Ischemic stroke is a leading cause of death and disability, but treatment options are severely limited. Approximately 795,000 people in the United States experience a stroke and it is the fourth leading cause of death when considered separately from other cardiovascular diseases. It is a leading cause of disability, and 26% of stroke survivors over 65 are still dependent on others for daily activities at 6 months after stroke [1]. Administration of tissue plasminogen activator in the acute phase of stroke is still the only US Food and Drug Administration-approved treatment for this prevalent cause of death and morbidity and its application is limited by a narrow therapeutic window and a number of complications [2]. Various types of neural precursors, such as a conditionally immortalized cell line derived from human fetal tissue ( in clinical trials) [7,8,9,10], lines derived from carcinomas [11,12], fetal neuronal stem cells [13,14], mouse neural precursors derived from the post-stroke cortex [15], region-specific murine embryonic precursors [16], and precursors derived from mouse [17,18,19] or human [20,21,22,23,24,25,26] embryonic stem cells have been used in experimental models
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