Masanori Gomi

Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson's disease

For the safe clinical application of embryonic stem cells (ESCs) for neurological diseases, it is critical to evaluate the tumorigenicity and function of human ESC (hESC)‐derived neural cells in primates. We have herein, for the first time, compared the growth and function of hESC‐derived cells with different stages of neural differentiation implanted in the brains of primate models of Parkinsons disease. We herein show that residual undifferentiated cells expressing ESC markers present in the cell preparation can induce tumor formation in the monkey brain. In contrast, a cell preparation matured by 42‐day culture with brain‐derived neurotrophic factor/glial cell line‐derived neurotrophic factor (BDNF/GDNF) treatment did not form tumors and survived as primarily dopaminergic (DA) neurons. In addition, the monkeys with such grafts showed behavioral improvement for at least 12 months. These results support the idea that hESCs, if appropriately matured, can serve as a source for DA neurons without forming any tumors in a primate brain. STEM CELLS 2012;30:935–945

Tissue inhibitor of metalloproteinases protect blood-brain barrier disruption in focal cerebral ischemia

Enhanced matrix metalloproteinases (MMPs) can cause vasogenic edema and hemorrhagic transformation after cerebral ischemia, and affect the extent of ischemic injury. We hypothesized that the endogenous MMP inhibitors, tissue inhibitor of MMPs (TIMPs), were essential to protect against blood—brain barrier (BBB) disruption after ischemia by regulating the activities of MMPs. We confirmed the transition of MMP-2 and MMP-9, and the TIMPs family after 30 mins of middle cerebral artery occlusion, and elucidated the function of TIMP-1 and TIMP-2 in focal ischemia, using TIMP-1−/−and TIMP-2−/− mice. TIMP-1 mRNA expression was gradually increased until 24 h after reperfusion. In TIMP-1−/− mice, MMP-9 protein expression and gelatinolytic activity were significantly more augmented after cerebral ischemia than those in WT mice, and were accompanied by exacerbated BBB disruption, neuronal apoptosis, and ischemic injury. In contrast, TIMP-2 gene deletion mice exhibited no significant difference in MMP expressions and the degree of ischemic injury despite an increased Evans blue leakage. These results suggest that TIMP-1 inhibits MMP-9 activity and can play a neuroprotective role in cerebral ischemia.

Immune or inflammatory response by the host brain suppresses neuronal differentiation of transplanted ES cell–derived neural precursor cells

Embryonic stem (ES) cells are a promising donor source for transplantation therapy, but several problems must be solved before they can be clinically useful. One of these is the host immune reaction to allogeneic grafts. In this article, we examine the effect of the host immune reaction on survival and differentiation of grafted ES cell–derived neural precursor cells (NPCs). We induced NPCs from mouse ES cells by stromal cell–derived inducing activity and then transplanted them into mouse brains with or without administering the immunosuppressant cyclosporine A (CsA). Two and 8 weeks following transplantation, the accumulation of host‐derived microglia/macrophages and lymphocytes was observed around the graft. This effect was reduced by CsA treatment, although no significant difference in graft volume was observed. These data suggest that an immune response occurs in allografts of ES cell–derived NPCs. Intriguingly, however, the ratio of neurons to astrocytes in the graft was higher in immunosuppressed mice. Because inflammatory or immune cells produce various cytokines, we examined the effect of IL‐1β, IL‐6, IFN‐γ, and TNF‐α on the differentiation of NPCs in vitro. Only IL‐6 promoted glial cell fate, and this effect could be reversed by the addition of an IL‐6 neutralizing antibody. These results suggest that allogeneic ES cell–derived NPCs can cause an immune response by the host brain, but it is not strong enough to reject the graft. More important, activated microglia and lymphocytes can suppress neuronal differentiation of grafted NPCs in vivo by producing cytokines such as IL‐6.

Attenuation of neuronal degeneration in thioredoxin-1 overexpressing mice after mild focal ischemia

Thioredoxin (Trx) is a 12-kDa protein ubiquitously expressed in all living cells that fulfills a variety of biological functions related to cell proliferation and apoptosis. It is characterized by the highly conserved reduction/oxidation (redox)-active site sequence Trp-Cys-Gly-Pro-Cys-Lys. Trx acts as a powerful antioxidant and plays an important role in maintaining critical protein thiols in the reduced state. Moreover, it has been shown to scavenge reactive oxygen species (ROS) and to protect against oxidative stress. We have reported that Trx-1 protects against neuronal damage during focal ischemia. However, the mechanisms underlying this protective effect and the effect of Trx-1 on neuronal apoptosis during ischemia have not been fully clarified. In this study, we analyzed the effect of Trx-1 overexpression against neuronal degeneration after a short duration of transient brain ischemia. Mild focal ischemia was reported to induce neuronal death through apoptosis. We employed Fluorojade-B staining to detect neuronal degeneration. In Trx transgenic mice, a smaller number of Fluorojade-B-positive neurons were detected after ischemia-reperfusion than in wild-type mice. In addition, we detected cleaved caspase-3- and TUNEL-positive cells, which indicated caspase-dependent apoptosis. Fewer caspase-3- and TUNEL-positive neurons were detected after ischemia-reperfusion in Trx transgenic mice than in wild-type mice. Furthermore, Akt signaling was reported to play a role in neuronal survival in Trx-1 overexpressing mice. After ischemia-reperfusion, Western blot and immunohistochemical analysis indicated that phosphorylation of Akt was enhanced in Trx transgenic mice after ischemia-reperfusion. Intraventricular injection of LY294002,which is a phosphoinositide 3-kinase (PI3K), vanished the neuroprotective effect in Trx-1 transgenic mice. These results indicate that Trx-1 overexpression protects neurons from apoptosis after ischemia-reperfusion.

Functional recovery of the murine brain ischemia model using human induced pluripotent stem cell-derived telencephalic progenitors

Induced pluripotent stem (iPS) cells possess the properties of self-renewal and pluripotency, similar to embryonic stem cells. They are a good candidate as a source of suitable cells for cell replacement therapy. In this study, we transplanted human iPS cell-derived neural progenitors into an ischemic mouse brain. Human iPS cells were differentiated into neuronal progenitors by serum-free culture of embryoid body-like aggregates (SFEBs). Focal cerebral ischemia was induced by occluding the middle cerebral artery using the intraluminal filament technique. Donor cells were transplanted into the ischemic lateral striatum 1 week after ischemia induction. Cells survived at the transplantation site, with migration of a proportion of cells along the external capsule and corpus callosum. Cells that were positive for the basal telencephalon marker, Nkx2.1, migrated into the basal part of the telencephalon. The pallial telencephalon marker, Emx1, was detected in cells that had migrated into the pallial part of the telencephalon. SFEBs differentiated into various types of neurons, and a retrograde tracer labeling study showed that differentiated cells integrated into host neural circuitry. Behavioral recovery was significantly enhanced in the transplanted group. Our results suggest that human iPS cell-derived neuronal progenitors survive and migrate in the ischemic brain, and contribute toward functional recovery via neural circuit reconstitution.

Single and local blockade of interleukin-6 signaling promotes neuronal differentiation from transplanted embryonic stem cell-derived neural precursor cells.

Safe and efficient transplantation of embryonic stem (ES) cells to the brain requires that local inflammatory and immune responses to allogeneic grafts are inhibited. To investigate cytokines that affect graft cell survival and differentiation, we used stromal cell‐derived inducing activity to induce the differentiation of neural progenitor cells (NPCs) from mouse ES cells and transplanted the NPCs into mouse brain. Examination of surrounding brain tissue revealed elevated expression levels of interleukin (IL)‐1β, IL‐4, and IL‐6 in response to NPC transplantation. Among these, only IL‐6 reduced neuronal differentiation and promoted glial differentiation in vitro. When we added anti‐IL‐6 receptor antibodies to NPCs during transplantation, this single and local blockade of IL‐6 signaling reduced the accumulation of host‐derived leukocytes, including microglia. Furthermore, it also promoted neuronal differentiation and reduced glial differentiation from the grafted NPCs to an extent similar to that with systemic and continuous administration of cyclosporine A. These results suggest that local administration of anti‐IL‐6 receptor antibodies with NPCs may promote neuronal differentiation during the treatment of neurological diseases with cell replacement therapy.

Chronic venous congestion following embolization of spinal dural arteriovenous fistula

The authors present a case of spinal dural arteriovenous fistula with fluctuations in symptoms following embolization. Superselective injection of 33% N-butyl cyanoacrylate into the feeding vessel resulted in the complete occlusion of the fistula with traversal of the nidus. The subsequent venous congestion was progressive and treatable with anti-thrombin therapy. Extended medication with dual antiplatelet therapy was required because dose reduction to aspirin monotherapy worsened symptoms. In this case, it took > 2 months for the patients symptoms to stabilize. The duration of progressive venous thrombosis after embolization of a spinal dural arteriovenous fistula is not well known, nor is the most adequate treatment. Although it is presumed that prevention of venous thrombosis is best achieved with anticoagulation, dual antiplatelet therapy can be a substitute for patients with poor compliance.

Transplantation of telencephalic neural progenitors induced from embryonic stem cells into subacute phase of focal cerebral ischemia

Cerebral ischemia causes neuronal death and disruption of neural circuits in the central nervous system. Various neurological disorders caused by cerebral infarction can severely impair quality of life and are potentially fatal. Functional recovery in the chronic stage mainly depends on physical treatment and rehabilitation. We aim to establish cell therapy for cerebral ischemia using embryonic stem (ES) cells, which have self-renewing and pluripotent capacities. We previously reported that the transplanted monkey and mouse ES cell-derived neural progenitors, by stromal cell-derived inducing activity method, could survive and differentiate into various types of neurons and glial cells, and form the neuronal network in basal ganglia. In this report, we induced the differentiation of the neural progenitors from mouse ES cells using the serum-free suspension culture method and confirmed the expression of various basal ganglial neuronal markers and neurotransmitter-related markers both in vitro and in vivo, which was thought to be suitable for replacing damaged striatum after middle cerebral artery occlusion. This is the first report that used selectively induced telencephalic neural progenitors into ischemia model. Furthermore, we purified the progenitors expressing the neural progenitor marker Sox1 by fluorescence-activated cell sorting and Sox1-positive neural progenitors prevented tumor formation in ischemic brain for 2 months. We also analyzed survival and differentiation of transplanted cells and functional recovery from ischemic damage.