Masaomi Koyanagi

Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model

Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell-derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell-derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.

Fluorescence‐Activated Cell Sorting–Based Purification of Embryonic Stem Cell–Derived Neural Precursors Averts Tumor Formation after Transplantation

The differentiation of dopaminergic (DA) neurons from mouse embryonic stem cells (ESCs) can be efficiently induced, making these neurons a potential source for transplantation as a treatment for Parkinsons disease, a condition characterized by the gradual loss of midbrain DA neurons. One of the major persistent obstacles to the successful implementation of therapeutic ESC transplantation is the propensity of ESC‐derived grafts to form tumors in vivo. To address this problem, we used fluorescence‐activated cell sorting to purify mouse ESC‐derived neural precursors expressing the neural precursor marker Sox1. ESC‐derived, Sox1+ cells began to express neuronal cell markers and differentiated into DA neurons upon transplantation into mouse brains but did not generate tumors in this site. In contrast, Sox1− cells that expressed ESC markers frequently formed tumors in vivo. These results indicate that Sox1‐based cell sorting of neural precursors prevents graft‐derived tumor formation after transplantation, providing a promising strategy for cell transplantation therapy of neurodegenerative disorders.

Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.

Rho‐GTPase has been implicated in the apoptosis of many cell types, including neurons, but the mechanism by which it acts is not fully understood. Here, we investigate the roles of Rho and ROCK in apoptosis during transplantation of embryonic stem cell‐derived neural precursor cells. We find that dissociation of neural precursors activates Rho and induces apoptosis. Treatment with the Rho inhibitor C3 exoenzyme and/or the ROCK inhibitor Y‐27632 decreases the amount of dissociation‐induced apoptosis (anoikis) by 20–30%. Membrane blebbing, which is an early morphological sign of apoptosis; cleavage of caspase‐3; and release of cytochrome c from the mitochondria are also reduced by ROCK inhibition. These results suggest that dissociation of neural precursor cells elicits an intrinsic pathway of cell death that is at least partially mediated through the Rho/ROCK pathway. Moreover, in an animal transplantation model, inhibition of Rho and/or ROCK suppresses acute apoptosis of grafted cells. After transplantation, tumor necrosis factor‐α and pro‐nerve growth factor are strongly expressed around the graft. ROCK inhibition also suppresses apoptosis enhanced by these inflammatory cytokines. Taken together, these results indicate that inhibition of Rho/ROCK signaling may improve survival of grafted cells in cell replacement therapy.

Neural precursor cells derived from human embryonic brain retain regional specificity.

Recent studies have revealed that neural precursor cells can be expanded not only from the subventricular zone and hippocampus but also from other regions of the human embryonic brain. To determine the regional differences of these precursor cells, we divided the brain of a 9‐week‐old human embryo into four parts, i.e., telencephalon, diencephalon, mesencephalon, and rhombencephalon. All cultures of the tissues yielded neurospheres, and these spheres gave rise to neurons, astrocytes, and oligodendrocytes. An analysis of clonal populations revealed that these precursor cells were multipotent, and two region‐specific differences in neural precursor cells were revealed: 1) The precursor cells from the rostral part of the brain tended to proliferate faster than those from the caudal part, and 2) the precursor cells from the diencephalon and mesencephalon gave rise to more tyrosine hydoxylase (TH)‐positive neurons than those from the telencephalon and rhombencephalon. When 50‐day‐cultured spheres were caused to differentiate, the percentage of TH‐positive cells per total cell population was 1.2% for diencephalic and mesencephalic precursors, whereas it was 0.4% for telencephalic and rhombencephalic ones. Furthermore, the TH‐positive cells from diencephalic and mesencephalic precursors were large, multipolar, and γ‐aminobutyric acid (GABA)‐negative, which suggested that these cells were midbrain dopaminergic neurons. In contrast, TH‐positive cells from telencephalic and rhombencephalic precursors were small, bipolar, and GABA‐positive. These results suggest that human neural precursor cells might have the potential to differentiate into a variety of cells but retain regional specificity.

Generation of graftable dopaminergic neuron progenitors from mouse ES cells by a combination of coculture and neurosphere methods

Parkinsons disease is characterized by a loss of midbrain dopamine (DA) neurons and is generally viewed as a potential target for stem cell therapy. Although several studies have reported the generation of postmitotic DA neurons from embryonic stem (ES) cells, it is unknown whether the proliferative progenitors of DA neurons can be isolated in vitro. To investigate this possibility, we have developed a combined approach in which ES cells are cocultured with PA6 stromal cells to expose them to stromal cell‐derived inducing activity (SDIA) and are then cultured as neurospheres. Mouse ES cell colonies were detached from PA6 feeder cells after 8 days of SDIA treatment and then expanded as spheres for another 4 days in serum‐free medium supplemented with fibroblast growth factor‐2. The spheres exhibited neural stem cell characteristics and contained few DA neurons at this stage of culture. After being induced to differentiate on polyornithine/laminin‐coated dishes for 7 days, these spheres generated DA neurons in vitro at a relatively low frequency. Intriguingly, addition of PA6 cell conditioned medium to the sphere culture medium significantly increased the percentage of DA neurons to 25–30% of the total number of neurons. Transplantation of conditioned medium‐treated day 4 spheres, which contained DA neuron progenitors, into the mouse striatum resulted in the generation of a significant number of graft‐derived DA neurons. These findings suggest that progenitors of DA neurons are generated and can proliferate in ES cell‐derived neurospheres induced by serial SDIA and PA6 conditioned medium treatment.

Estrogen promotes differentiation and survival of dopaminergic neurons derived from human neural stem cells

To investigate the effect of estrogen on neuronal differentiation, especially on dopaminergic (DA) neurons, human neural stem cells (NSCs) were differentiated in the presence of 17β‐estradiol. NSCs gave rise to tyrosine hydroxylase (TH)‐positive neurons in vitro, the proportion of which was increased by 17β‐estradiol. Increase in TH‐positive neurons was abrogated by an estrogen receptor (ER) antagonist, ICI182780, suggesting ERs play a role in differentiation of DA neurons. The observation that ERs were expressed in both proliferating NSCs and postmitotic DA neurons suggested that increase in TH‐positive neurons was due to induction and support of DA neurons. 17β‐Estradiol also increased the number of DA neurons derived from human NSCs in vivo when the cells were grafted into mouse brains. These results support a possible role for estrogen in the transplantation of NSCs for Parkinsons disease.

Meningeal cells induce dopaminergic neurons from embryonic stem cells.

Neural induction of midbrain dopaminergic (DA) neurons from embryonic stem (ES) cells can be achieved by culturing them on a bone marrow‐derived stromal cell line, PA6, which possesses stromal cell‐derived inducing activity (SDIA). The mechanism of SDIA is unknown, but clinical application of ES cell transplantation requires the use of defined factors for DA neuron induction. Here, we demonstrate that meningeal cells harvested from the developing dura can induce DA neuron differentiation from mouse and human ES cells, as assessed by midbrain DA marker expression and secretion of DA in response to potassium stimuli. Intriguingly, the inductive strength of meningeal cells depends on their developmental stage, with those harvested from embryonic day 18 embryos showing the highest activity. Among six soluble factors known to be involved in DA neuron differentiation, only Wnt‐5a and transforming growth factor‐β3 were expressed by both meningeal and PA6 cells, and the expression of Wnt‐5a correlated with the DA neuron induction activity of these cells. Furthermore, the induction of DA neuron differentiation by PA6 cell‐conditioned medium was reversed by addition of a Wnt‐5a neutralizing antibody, whereas recombinant Wnt‐5a promoted DA neuron induction when cells were cultured on Matrigel. These results indicate that meningeal cells can be used as feeders to induce DA neurons from ES cells, and that Wnt‐5a plays an important role in DA neuron induction by SDIA.

Reduced cerebrovascular reserve is associated with an increased risk of postoperative ischemic lesions during carotid artery stenting

Background Reduced cerebrovascular reserve (CVR) is associated with increased risk of ischemic events in carotid steno-occlusive diseases. Objective To determine whether pretreatment CVR can predict postoperative ischemic lesions after carotid artery stenting (CAS) by retrospective analysis. Methods We retrospectively reviewed the medical records of 46 patients (42 men; mean age 74.2±8.3 years) who underwent CAS and preprocedural cerebral blood flow measurement by quantitative single-photon emission CT. Ischemic lesions were evaluated by diffusion-weighted image (DWI) within 72 h after the intervention. We also evaluated plaque characteristics using black-blood MR plaque imaging. Results New ipsilateral DWI-positive lesions were found in 11 cases (23.9%). Patients were classified into two groups based on the presence or absence of new DWI-positive lesions, and no significant differences in characteristics were found between the DWI-positive and DWI-negative groups, except for age and CVR of the ipsilateral middle cerebral artery (MCA) territory. The DWI-positive group was significantly older than the DWI-negative group (79.7±4.1 vs 72.5±8.6 years; p=0.0085) and had lower average regional CVR (1.4±18.2% vs 22.4±25.8%; p=0.016). MR plaque imaging showed no significant difference in relative overall plaque MR signal intensity between the two groups (1.53±0.37 vs 1.34±0.26; p=0.113). In multivariate logistic regression analysis, lower CVR of the ipsilateral MCA territory (<11%) was the only independent risk factor for new ischemic lesions following CAS (OR=6.99; 95% CI 1.17 to 41.80; p=0.033). Conclusions Impaired pretreatment CVR was associated with increased incidence of new infarction after CAS.

Effect of intrathecal milrinone injection via lumbar catheter on delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage

OBJECTIVE Delayed cerebral ischemia (DCI) is an important complication after aneurysmal subarachnoid hemorrhage (aSAH). Although intrathecal milrinone injection via lumbar catheter to prevent DCI has been previously reported to be safe and feasible, its effectiveness remains unknown. The goal of this study was to evaluate whether intrathecal milrinone injection treatment after aSAH significantly reduced the incidence of DCI. METHODS The prospectively maintained aSAH database was used to identify patients treated between January 2010 and December 2015. The cohort included 274 patients, with group assignment based on treatment with intrathecal milrinone injection or not. A propensity score model was generated for each patient group, incorporating relevant patient variables. RESULTS After propensity score matching, 99 patients treated with intrathecal milrinone injection and 99 without treatment were matched on the basis of similarities in their demographic and clinical characteristics. There were significantly fewer DCI events (4% vs 14%, p = 0.024) in patients treated with intrathecal milrinone injection compared with those treated without it. However, there were no significant differences between the 2 groups with respect to their 90-day functional outcomes (46% vs 36%, p = 0.31). The likelihood of chronic secondary hydrocephalus, meningitis, and congestive heart failure as complications of intrathecal milrinone injection therapy was also similar between the groups. CONCLUSIONS In propensity score-matched groups, the intrathecal administration of milrinone via lumbar catheter showed significant reduction of DCI following aSAH, without an associated increase in complications.

Endovascular Therapy for Ruptured Cerebral Aneurysms in the Elderly: Poor Accessibility of the Guiding Catheter and Use of Local Anesthesia as the Predictors of Procedure-Related Rupture.

BACKGROUND Endovascular therapy is favored for ruptured intracranial aneurysms in the elderly. However, poor accessibility to the aneurysm through the parent artery and use of local anesthesia in this age group may predispose to intraprocedural complications. OBJECTIVE To evaluate whether age-related poor access to the ruptured target aneurysm and use of local anesthesia are associated with increased incidence of procedure-related rupture during endovascular embolization. METHODS A total of 117 patients with 117 ruptured aneurysms underwent endovascular embolization at a single institution. Correlation of increasing age with poor accessibility of the guiding catheter was analyzed. In addition, the distance from the aneurysm to the guiding catheter was investigated to identify an association with incidence of procedure-related rupture. Correlation of local anesthesia with procedure-related rupture was also evaluated in the multivariable analysis. RESULTS Increasing age was significantly associated with poor accessibility of the guiding catheter (P = .001, Mann-Whitney U test). Procedure-related rupture occurred in 9 of 117 aneurysms (7.7%). Longer distance between distal aneurysms and low-positioned guiding catheters carried a higher risk of procedure-related rupture than a shorter distance between proximal aneurysms and high-positioned guiding catheters (odds ratio, 19.3; 95% confidence interval, 1.84-201; P = .01, multivariable analysis). Use of local anesthesia was also a significant risk factor of procedure-related rupture by multivariable analysis. CONCLUSION Increasing age was correlated with poor accessibility of the guiding catheter in endovascular embolization of ruptured intracranial aneurysms. Distally located aneurysms treated through a low-positioned guiding catheter and use of local anesthesia increased the risk of procedure-related rupture.