[show abstract][hide abstract] ABSTRACT: In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.
[show abstract][hide abstract] ABSTRACT: We examined the gene expression and DNA methylation of 49 human induced pluripotent stem cells (hiPSCs) and 10 human embryonic stem cells and found overlapped variations in gene expression and DNA methylation in the two types of human pluripotent stem cell lines. Comparisons of the in vitro neural differentiation of 40 hiPSCs and 10 human embryonic stem cells showed that seven hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. These data demonstrated a subset of hiPSC lines that have aberrant gene expression and defective potential in neural differentiation, which need to be identified and eliminated before applications in regenerative medicine.
Proceedings of the National Academy of Sciences 11/2013; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Surgical intervention is expected to improve the quality of life in patients with intractable epilepsy by providing adequate seizure control. Although many previous studies showed various rates of seizure freedom, definite conclusions have not yet been made regarding outcomes. In order to clarify the long-term postoperative outcome for a period up to 10 years, a retrospective review of our patients was performed longitudinally by using the survival analysis method. The postoperative state of epilepsy in 76 patients who underwent resection surgery was assessed based on Engel's criteria. In addition, Kaplan-Meier survival analysis was used to calculate the probability of seizure freedom. In this patient group, abnormal lesion were detected by MRI in 70 out of 76 cases, and the ictal onset zone was finally identified within temporal lobe in 51 cases. The most favorable outcome, defined as Engel Class Ia, was observed in 26 (37%), 24 (40%), and 18 (41%) cases at 2, 5, and 10 years after surgery, respectively. The Kaplan-Meier survival curve in the overall group estimated the probability of seizure freedom as 75% (95% confidence interval [CI] 70-80%), 67% (62-72%), and 51% (45-57%) at 2, 5, and 10 years follow up, respectively. Half of all seizure recurrences occurred within the first 2 postoperative years. In this study, we showed that long-term favorable outcome of seizure control following resection surgery can be achieved in more than half of the patients.
[show abstract][hide abstract] ABSTRACT: Our work and the study of Bilican et al. highlight the need for complementary assays to detect subtle phenotypic differences between control and mutant induced pluripotent stem cell lines.
Science translational medicine 06/2013; 5(188):188lr2. · 10.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Animals that possess regenerative abilities are widespread in the animal kingdom. Hydra,
planarian, zebrafish, newt and axolotl are known prominent species, and the cellular aspects
of the stem cell system for regeneration are well elucidated. However, few animals can be
used to investigate the molecular basis of neuronal regeneration, in spite of the presence of
prominent regenerative animals, as mentioned above. Planarians, for instance, can regenerate
a functional brain after amputation in a few days, even from non-brain tissue. Newts can
regenerate several tissues and organs (i.e., lens, limbs, jaws, hearts and tails) with recovery of
function and physiology after injury or tissue removal. These animals achieve regeneration
of missing nervous system utilizing stem cells. However, it is difficult to regenerate
nervous system in mammalians, including human beings, although these animals possess
neural stem cells. Therefore, regenerative animals provide unique opportunities to investigate
the generation and utilization of stem cells to repair lost or injured tissue in non-regenerative
animals. On the other hand, the successful derivation of neural cells from human embryonic
stem cells (ESCs) and induced pluripotent stem cells (iPSCs) under in vitro conditions
provides a new experimental strategy for clinical translation. In other words, although human
beings lack regenerative abilities, the new clinical strategy of “regenerative medicine,”
including cell-transplantation therapy, has been developed to recover lost neural functions by
using stem cells. This research field has become a greatly advancing scientific field worldwide. In this chapter, we focus on the molecular systems of generation of functional dopaminergic
(DA) neurons in vivo and/or in vitro in regenerative and non-regenerative animals. The first
topic investigates how regenerative animals recruit new DA neurons from stem cells after
injury. The second topic explores how to generate DA neurons from mammalian ESCs and
iPSCs under in vitro conditions. The third topic evaluates clinical applications for human neural
disease, especially Parkinson’s disease.
[show abstract][hide abstract] ABSTRACT: Pluripotent stem cells are promising potential sources for cell replacement therapy and are useful research tools for exploring disease mechanisms. Neural cells are one of the cell types that have been most efficiently differentiated through several established protocols. This chapter describes the feeder-free floating aggregation culture system for the induction of dopaminergic neurons. This method is simple and highly efficient for the production of dopaminergic neurons. It has several advantages for application in clinical usage in comparison to the other protocols using either feeder cells or Matrigel.
Methods in molecular biology (Clifton, N.J.) 01/2013; 1018:11-9.
[show abstract][hide abstract] ABSTRACT: Induced pluripotent stem cells (iPSCs) provide the potential for autologous transplantation using cells derived from a patient's own cells. However, the immunogenicity of iPSCs or their derivatives has been a matter of controversy, and up to now there has been no direct comparison of autologous and allogeneic transplantation in the brains of humans or nonhuman primates. Here, using nonhuman primates, we found that the autologous transplantation of iPSC-derived neurons elicited only a minimal immune response in the brain. In contrast, the allografts caused an acquired immune response with the activation of microglia (IBA-1(+)/MHC class II(+)) and the infiltration of leukocytes (CD45(+)/CD3(+)). Consequently, a higher number of dopaminergic neurons survived in the autografts. Our results suggest that the autologous transplantation of iPSC-derived neural cells is advantageous for minimizing the immune response in the brain compared with allogeneic grafts.
[show abstract][hide abstract] ABSTRACT: Parkinson's disease (PD) is one of the candidate diseases for cell transplantation therapy, since successful clinical experiments have accumulated using human fetal tissue grafting for PD patients. Although some grafted PD patients have shown drastic improvements, several issues still remain with regard to using human fetal tissue. This review highlights the recent advances in stem cell technology toward clinical applications using human pluripotent stem cells. In particular, pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells (iPSCs), are the focus as a source of cell transplantation therapy that can be used instead of human fetal tissues. Additionally, efficient methods for stem cell maintenance and differentiation have been developed and improved toward the clinical transition. These advances in the basic technologies have helped accelerate the realization of regenerative medicine. We also review the current topics regarding disease modeling and drug screening using iPSC technology. Finally, we also describe the future prospects of these stem cell research fields toward clinical application.
[show abstract][hide abstract] ABSTRACT: Although transplanted pluripotent stem cell-derived neurons can contribute to functional recovery in animal models of Parkinson's disease, the risk of tumor formation hinders clinical applications of this approach. Removing undifferentiated cells from the donor population is critical to reduce tumorigenesis. Moreover, immature neural progenitors in transplants can proliferate unpredictably, resulting in neural overgrowth and long-term risks of compressing the surrounding host tissue. Because Notch signaling plays a role in maintaining the multipotency and proliferative capacity of neural progenitors, we used γ-secretase inhibitors (GSIs) to dampen Notch signaling in human-induced pluripotent stem cell-derived neural progenitors before transplantation and examined the effects on the growth of proliferative grafts. We observed a marked reduction in the percentage of dividing cells and increased neuronal maturation in GSI-treated samples in vitro. Next, grafts were transplanted into the striata of nonobese diabetic/severe combined immune deficiency mice. Histological analyses performed 8 weeks after the operation showed that grafts pretreated with GSIs-N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester or compound E-were significantly smaller than control samples. Immunohistologic analyses revealed that briefly treating the donor population with GSIs not only reduced the graft volume, but also altered the composition of the graft; control grafts showed neural overgrowth with numerous PAX6(+) and Ki67(+) neural rosettes, whereas GSI-treated samples developed into mature neuronal grafts containing primarily Tubβ3(+) cells. These results suggest that pretreating potentially proliferative progenitors with GSIs may improve the safety of cell replacement therapies using pluripotent stem cells.
Stem cells and development 09/2012; · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a late-onset, fatal disorder in which the motor neurons degenerate. The discovery of new drugs for treating ALS has been hampered by a lack of access to motor neurons from ALS patients and appropriate disease models. We generate motor neurons from induced pluripotent stem cells (iPSCs) from familial ALS patients, who carry mutations in Tar DNA binding protein-43 (TDP-43). ALS patient-specific iPSC-derived motor neurons formed cytosolic aggregates similar to those seen in postmortem tissue from ALS patients and exhibited shorter neurites as seen in a zebrafish model of ALS. The ALS motor neurons were characterized by increased mutant TDP-43 protein in a detergent-insoluble form bound to a spliceosomal factor SNRPB2. Expression array analyses detected small increases in the expression of genes involved in RNA metabolism and decreases in the expression of genes encoding cytoskeletal proteins. We examined four chemical compounds and found that a histone acetyltransferase inhibitor called anacardic acid rescued the abnormal ALS motor neuron phenotype. These findings suggest that motor neurons generated from ALS patient-derived iPSCs may provide a useful tool for elucidating ALS disease pathogenesis and for screening drug candidates.
Science translational medicine 08/2012; 4(145):145ra104. · 10.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Brain research 03/2012; 1459:52-60. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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 Parkinson's 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.
[show abstract][hide abstract] ABSTRACT: Parkinson's disease has been so far commonly treated with medication therapy. Although the medication works effectively in the initial phase, it turns out to be less effective at the later stage of the disease. Recently, induced pluripotent stem (iPS) cells have attracted much attention because of their potential to cure diseases such as Parkinson's disease. Due to the accumulating clinical experiences of cell transplantation procedures with aborted fetal tissues, Parkinson's disease has become one of the most promising targets for the clinical application of this iPS cell technology. In this review, we will summarize the ongoing research in the field of iPS cells and Parkinson's disease. The method for establishing iPS cells has advanced rapidly that can be applied in the clinical stage in terms of avoiding the use of viral vectors, xenogenic materials, etc. The differentiation protocol to derive the dopamine neurons from iPS cells has also been improved. However, several issues, such as the risk of tumor formation and the poor survival of the grafted dopamine neurons in vivo remain to be solved before these cells can be used in the clinical settings. Other than cell transplantations, iPS cell technology can also provide a valuable platform for disease analysis and drug development with in vitro systems of human cells. Several lines of iPS cells have already been established from Parkinson's disease patients with either sporadic or genetic background. For patients to achieve maximum benefits of this technology, further research must be conducted in both fields, that is, cell transplantation and the disease modeling with patient-derived iPS cells.
Brain and nerve = Shinkei kenkyū no shinpo 01/2012; 64(1):29-37.