Abstract
Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson’s disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem cells constitute an alternative source of cells for transplantation in Parkinson’s disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content of β-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells (Caspase3) was reduced, whereas the expression of a cell proliferation marker (Ki67) was left unchanged. Increased expression of hypoxia inducible factor-1α and production of reactive oxygen species (ROS) in cultures exposed to CO may suggest a mechanism involving mitochondrial alterations and generation of ROS. In conclusion, the present procedure using controlled, short-term CO exposure allows efficient dopaminergic differentiation of human neural stem cells at low cost and may as such be useful for derivation of cells for experimental studies and future development of donor cells for transplantation in Parkinson’s disease.
Highlights
Parkinson’s disease is a neurodegenerative disorder affecting more than six million people worldwide [1]
To characterize and validate the reaction from the new carbon monoxide (CO)-releasing molecule (CORM) MePh2SiCO2H (Fig 1a), the CO concentration was measured in the gas chamber every minute throughout a 30 min exposure period (Fig 1b)
To investigate the effect of CO on the dopaminergic differentiation, hVMbclXL cells were differentiated for 6 days and received CO (12.5–100 ppm; 30 min) at days 0 and 4 (Fig 1c)
Summary
Parkinson’s disease is a neurodegenerative disorder affecting more than six million people worldwide [1]. The use of human fetal tissue is hampered by ethical concerns, suboptimal survival of grafted dopaminergic neurons, development of postgrafting dyskinesias in some patients, and the logistics related to collection and storage of the donor tissue [5,8,9,10,11,12,13]. NSCs are self-renewable multipotent cells that can be isolated from the developing and mature nervous system. Such cells may have significant advantages compared to human fetal tissue as they can be propagated to almost unlimited numbers of relatively homogenous cells in vitro and frozen without significant loss of viability [14,15]. Efficient, simple and costeffective protocols for controlled generation of functional dopaminergic neurons are still not available
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