Yasuhito Tokumoto

Comparison of efficiency of terminal differentiation of oligodendrocytes from induced pluripotent stem cells versus embryonic stem cells in vitro.

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), and defects in these cells can result in the loss of CNS functions. Although oligodendrocyte progenitor cells transplantation therapy is an effective cure for such symptoms, there is no readily available source of these cells. Recent studies have described the generation of induced pluripotent stem cells (iPS cells) from somatic cells, leading to anticipation of this technique as a novel therapeutic tool in regenerative medicine. In this study, we evaluated the ability of iPS cells derived from mouse embryonic fibroblasts to differentiate into oligodendrocytes and compared this with the differential ability of mouse embryonic stem cells (ES cells). Experiments using an in vitro oligodendrocyte differentiation protocol that was optimized to ES cells demonstrated that 2.3% of iPS cells differentiated into O4(+) oligodendrocytes compared with 24.0% of ES cells. However, the rate of induction of A2B5(+) oligodendrocyte precursor cell (OPC) was similar for both iPS-derived cells and ES-derived cells (14.1% and 12.6%, respectively). These findings suggest that some intracellular factors in iPS cells inhibit the terminal differentiation of oligodendrocytes from the OPC stage.

Induction of oligodendrocyte differentiation from adult human fibroblast-derived induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) prepared from somatic cells might become a novel therapeutic tool in regenerative medicine, especially for the central nervous system (CNS). In this study, we attempted to induce O4-positive (O4+) oligodendrocytes from adult human fibroblast-derived iPSCs in vitro. We used two adult human iPSC cell lines, 201B7 and 253G1. 201B7 was induced by four-gene transduction (oct4, sox2, klf4, c-myc), and 253G1 was induced by three-gene transduction (oct4, sox2, klf4). We treated these cells with two in vitro oligodendrocyte-directed differentiation protocols that were optimized for human embryonic stem cells. One protocol used platelet-derived growth factor as the major mitogen for oligodendrocyte lineage cells, and the other protocol used epidermal growth factor (EGF) as the mitogen. Although the differentiation efficiency was low (less than 0.01%), we could induce O4+ oligodendrocytes from 253G1 cells using the EGF-dependent differentiation protocol. This is the first report of the in vitro induction of oligodendrocytes differentiation from human iPSCs.

Immunopanning selection of A2B5-positive cells increased the differentiation efficiency of induced pluripotent stem cells into oligodendrocytes

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), and defects in these cells can result in CNS dysfunction. Although oligodendrocyte precursor cell (OPC) transplantation therapy is an effective cure for several disorders, there is no readily available source of these cells. Recent studies have described the generation of induced pluripotent stem cell (iPSC) from somatic cells, leading to speculation that this technique might become a novel therapeutic tool in regenerative medicine. In a previous study, we were able to produce O4 positive (O4(+)) oligodendrocytes from mouse iPSC in vitro. Unfortunately, the efficiency of differentiation achieved was relatively low (2.3%). In the current study, we improved the differentiation efficiency using a mouse monoclonal antibody (A2B5) to select cells of oligodendrocyte lineage. During in vitro differentiation, we purified A2B5-positive (A2B5(+)) cells by immunopanning from a mixed culture of iPSC-derived cells. This procedure increased the differentiation efficiency of O4(+) oligodendrocytes to 43.5%. We also examined the expression of myelin basic protein (MBP), a marker of mature oligodendrocytes. After 21 days of terminal differentiation, 62.3% of iPSC-derived O4(+) oligodendrocytes expressed MBP.

Parameter optimization by using differential elimination: A general approach for introducing constraints into objective functions

BackgroundThe investigation of network dynamics is a major issue in systems and synthetic biology. One of the essential steps in a dynamics investigation is the parameter estimation in the model that expresses biological phenomena. Indeed, various techniques for parameter optimization have been devised and implemented in both free and commercial software. While the computational time for parameter estimation has been greatly reduced, due to improvements in calculation algorithms and the advent of high performance computers, the accuracy of parameter estimation has not been addressed.ResultsWe propose a new approach for parameter optimization by using differential elimination, to estimate kinetic parameter values with a high degree of accuracy. First, we utilize differential elimination, which is an algebraic approach for rewriting a system of differential equations into another equivalent system, to derive the constraints between kinetic parameters from differential equations. Second, we estimate the kinetic parameters introducing these constraints into an objective function, in addition to the error function of the square difference between the measured and estimated data, in the standard parameter optimization method. To evaluate the ability of our method, we performed a simulation study by using the objective function with and without the newly developed constraints: the parameters in two models of linear and non-linear equations, under the assumption that only one molecule in each model can be measured, were estimated by using a genetic algorithm (GA) and particle swarm optimization (PSO). As a result, the introduction of new constraints was dramatically effective: the GA and PSO with new constraints could successfully estimate the kinetic parameters in the simulated models, with a high degree of accuracy, while the conventional GA and PSO methods without them frequently failed.ConclusionsThe introduction of new constraints in an objective function by using differential elimination resulted in the drastic improvement of the estimation accuracy in parameter optimization methods. The performance of our approach was illustrated by simulations of the parameter optimization for two models of linear and non-linear equations, which included unmeasured molecules, by two types of optimization techniques. As a result, our method is a promising development in parameter optimization.

Caspase inhibitors increase the rate of recovery of neural stem/progenitor cells from post-mortem rat brains stored at room temperature

To match the demand of regenerative medicine for nerve system, collection of stem cells from the post-mortem body is one of the most practical ways. In this study, the storage condition of the post-mortem body was examined. We prepared neural stem/progenitor cells (NSPCs) from post-mortem rat brains stored at different temperatures. When brains were stored at 4 degrees C, for one week, we were able to obtain neurospheres (a spheroid body containing NSPCs) by stimulation of cells with epidermal growth factor (EGF). Incremental increases in storage temperature decreased the rate of appearance of neurospheres. Within 48 h at 15 degrees C, 24 h at 25 degrees C, in both condition, we were able to recover NSPCs from post-mortem rat brains. At 15 degrees C, 90% of neurosphere-forming activity was lost within 24 h. However, even after 24 h at 25 degrees C, 2% neurosphere-forming activity remained. After 6 h of death, there was very little difference between the rates of NSPC recovery at 4 degrees C and 25 degrees C. Addition of caspase inhibitors to both the rat brain storage solution and the NSPC culture medium increased the rate of neurosphere-forming activity. In particular, an inhibitor of caspase-8 activity increased the NSPC recovery rate approximately three-fold, with no accompanying detrimental effects on neural differentiation in vitro.

Overexpression of cyclin dependent kinase inhibitor P27/Kip1 increases oligodendrocyte differentiation from induced pluripotent stem cells

Cell transplantation therapy with oligodendrocyte precursor cells (OPCs) is a promising and effective treatment for diseases involving demyelination in the central nervous system (CNS). In previous studies, we succeeded in producing O4+ oligodendrocytes (OLs) from mouse- and human-induced pluripotent stem cells (iPSCs) in vitro; however, the efficiency of differentiation into OLs was lower for iPSCs than that for embryonic stem cells (ESCs). To clarify the cause of this difference, we compared the expression of proteins that contribute to OL differentiation in mouse iPSC-derived cells and in mouse ESC-derived cells. The results showed that the expression levels of cyclin dependent kinase inhibitor P27/Kip1, mitogen-activated protein kinase (MAPK) JNK3, and transcription factor Mash1 were lower in iPSC-derived cells. In contrast, the expression levels of MAPK P38α, P38γ, and thyroid hormone receptor β1 were higher in iPSC-derived cells. We attempted to compensate for the expression changes in P27/Kip1 protein and Mash1 protein in iPSC-derived cells through retrovirus vector-mediated gene expression. Although the overexpression of Mash1 had no effect, the overexpression of P27/Kip1 increased the differentiation efficiency of iPSC-derived cells into O4+ OLs.

TRAIL inhibited the cyclic AMP responsible element mediated gene expression.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) not only causes apoptotic cell death in tumor cells, but also activates some transcription factors and affects several other cellular functions. In this study, we observed the effect of administration of TRAIL on gene expression downstream of the cyclic AMP responsive element (CRE) enhancer by using the signal transduction reporter cis-element plasmid pCRE-d2EGFP. Western blotting showed that after administration of TRAIL, the expression level of reporter protein d2EGFP was down-regulated in NIH3T3 cells. To confirm the TRAIL-induced down-regulation of CRE enhancer controlled gene expression, DNA Chip time series analysis of the intrinsic genes expressed in NIH3T3 cells was carried out. As a result, the expression levels of six genes, which have CRE sequence in their promoter region, were slightly down-regulated within three hours after administration of TRAIL.