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  • To develop an effective cell based


    To develop an effective cell-based therapy against neurodegenerative disorders of the CNS, efficient recruitment of the cells into the CNS is essential. Previous findings suggest that particular bone marrow derived cells are able to cross the blood–brain barrier (Lebson et al., 2010; Simard and Rivest, 2004). To evaluate the migration of the iPS-ML into the CNS, we examined the effect of intravenous, intraperitoneal, and intracerebroventricular injection of iPS-ML into 5XFAD mice. To our disappointment, the iPS-ML injected via these routes did not efficiently infiltrate into Silmitasertib parenchyma and failed to reduce the amyloid burden. A possible reason of the failure to efficiently migrate in brain tissue may be the lack of CC chemokine receptor-2 (CCR2) expression in the iPS-ML (data not shown). To analyze the in vivo effect of iPS-ML/NEP2, we directly administered iPS-ML into the brain. To this end, we stereotaxically inserted microinjection tubes into the hippocampus of 5XFAD mice and transplanted iPS-ML through this tube. The hippocampus plays a major role in cognitive dysfunction of AD, and the 5XFAD hippocampus is one of the regions of the brain where Aβ plaques accumulate. iPS-ML transplanted by this procedure migrated to the brain parenchyma adjacent to the area of the tube insertion (Figs. 5B, C). In vivo, transplantation of iPS-ML/NEP2 into the hippocampus of 3–4-month old 5XFAD/scid mice significantly diminished the levels of soluble Aβ1–42 in the brain ISF compared to the control Ringer\'s solution injection (Fig. 6). The reduction of Aβ was not significant when non-modified iPS-ML were transplanted. Therefore, the reduction of Aβ was caused by the secretion of NEP2 from the iPS-ML, but not phagocytosis of Aβ by the iPS-ML. Our intrahippocampus transplantation of iPS-ML demonstrated short-term and focal remote effects of the IPS-ML; only where the cells were transplanted. Furthermore we could not examine the therapeutic effect of cognitive function, because the mice were weakened by probe implantation. Future studies will be aimed at exploring whether iPS-ML are effective in preventing cognitive decline and neuronal damage in other AD models. In addition, to develop this technique as a therapy for AD, delivery of iPS-ML into the brain by systemic administration is necessary. To examine the chromosomal alteration of iPS-ML, iPS-ML cultured for 6weeks after the introduction of proliferating factors were subjected to karyotype analysis. As shown in Supplemental Fig. S1, some karyotype abnormalities were detected in this analysis. For application of iPS-ML to clinical cellular therapy, we should resolve the issue of genetic instability of iPS-ML. To generate iPS-ML, our current method uses cMYC, BMI1 plus MDM2 to induce proliferation of iPS-MC. Among the introduced factors, MDM2 is involved in degradation of p53 protein as the E3 ubiquitin ligase (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997). Forced expression of MDM2 in iPS-ML may cause complete loss of p53 function and result in the genetic instability of iPS-ML. Although co-introduction of MDM2 enhanced the proliferation rate of iPS-ML, this factor is not absolutely necessary for the establishment of iPS-ML, as previously reported (Haruta et al., 2013; Koba et al., 2013). Omission of MDM2 in the generation of iPS-ML may be one way to improve the genetic stability of iPS-ML.
    Acknowledgments The plasmids used for preparing recombinant lentivirus, pCSII-EF, pCMV-VSV-G-RSV-Rev, and pCAG-HIVgp were kindly provided by Dr. H. Miyoshi (RIKEN BioResource Center). cDNAs for human BMI1 and EZH2 were provided by RIKEN BRC which is participating in National Bio-Resources Project of the MEXT, Japan. This work was supported in part by a Grant-in-Aid No. 23659158 from MEXT, Japan, a Research Grant for Intractable Diseases from Ministry of Health and Welfare, Japan, and a grant from Japan Science and Technology Agency (JST).