In conclusion the Olfm IRES

In conclusion, the Olfm4-IRES-eGFPCreERT2 allele described here provides a tool, separate from Lgr5, that can be used to further characterize intestinal stem cells.
Experimental Procedures
Acknowledgments We thank Hugo Snippert for analysis of the Olfm4 animals and Valentina Sasselli for a critical reading of the manuscript. J.S. was supported by Cancer Genomics Centre II, Molecular Mechanisms.
Introduction Amyotrophic lateral sclerosis (ALS) is a disorder of motor neurons (MNs) that is characterized by their relatively rapid degeneration, resulting in progressive muscle weakness and respiratory failure (Bruijn et al., 2004). Approximately 90%–95% of ALS cases are sporadic in nature, with 20% of the remaining familial cases linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene. Transgenic mice and rats carrying ALS-associated mutant human SOD1 genes (mSOD1) recapitulate many features of the human disease (Gurney et al., 1994). Despite the relative selectivity of MN loss in ALS, studies in mSOD1 rodent and tissue culture models show nonneuronal (glial) cell involvement in the disease process (Boillée et al., 2006; Yamanaka et al., 2008). Astrocytes in particular are hypothesized to play a role in both mSOD1 and sporadic forms of ALS (Haidet-Phillips et al., 2011; Howland et al., 2002; Papadeas et al., 2011). Regardless of whether astrocyte dysfunction is a cause of the disease or a consequence of neuronal death, altered astrocyte physiology results in further susceptibility to MN loss (Boillée et al., 2006). Targeted enrichment of normal astrocytes in mSOD1 rat spinal cord via intraspinal transplantation of rodent glial-restricted progenitors promoted focal MN protection, delayed decline in respiratory function, and extended disease progression (Xu et al., 2011). Various kinds of A-1210477 have been investigated for transplantation studies (Corti et al., 2004; Garbuzova-Davis et al., 2008; Iwanami et al., 2005; Piccini et al., 1999). Neuronal cells are probably the most relevant cell type for ALS treatment, but such cells suffer from a limited supply, ethical issues, and/or invasive harvest from human donors. On the other hand, human induced pluripotent stem cells (hiPSCs) can be obtained from a donor less invasively and can be expanded indefinitely in vitro. In this context, here we established a differentiation protocol of glial-rich neural progenitors (GRNPs) from hiPSCs and investigated the potential of hiPSC-derived glial-rich neural progenitors (hiPSC-GRNPs) as a cell source for intraspinal transplantation therapy of ALS.
Results
Discussion Here, we describe that transplantation of human iPSC-derived GRNPs produced astrocytes in vivo and prolonged the survival period of mSOD1 mice. We used hiPSC-GRNPs for testing the efficacy in mSOD1 mice, because replacement therapy using astrocytes from rodent glial-restricted progenitors in the cervical spinal cord of ALS rodent models is already well established (Xu et al., 2011). We showed that glial cells represent a potential target of ALS therapy. However, we observed transient improvement of lower limb function, as shown in Figure 2C, and a similar previous study failed to show improvement in the rescue of clinical manifestations and neuronal survival by transplantation of human-derived glial-restricted progenitor cells from 17- to 24-week fetus into SOD1 transgenic mouse spinal cord despite the survival and proliferation of exogenous astrocytes (Lepore et al., 2011). Although the transient improvement in our study might have stemmed from neuroprotective effects of the transplanted cells only in the lumbar region, with a possible broader effect of neurotrophic factors on other regions or behavioral alteration for food intake, as previously discussed (Table S1), we comprehensively compared the two studies as well as others regarding lumbar transplantation in terms of a number of aspects (Table S1), speculating that there were differences in graft type, transplantation condition, and/or transplantation timing. Regarding the timing of transplantation, the survival improvement in our study might have resulted in attenuation of the glial contribution to the disease pathogenesis at an early symptomatic stage (Boillée et al., 2006; Yamanaka et al., 2008). Regarding the cell injection site, instead of the cervical cord, we injected the cells into the lumbar spinal cords of ALS model mice, which resulted in improved clinical scores of lower limbs. These data supported the possibility of targeting not only the cervical cord but also the lumbar spinal cord in ALS clinical trials, depending on the symptoms to be treated. Following previous transplantation research (Table S1), we selected PBS, which is a vehicle solution for grafts, as control agent of transplantation. Dead cells or fibroblasts can be appropriate control agents but may also possibly secrete various factors. Furthermore, previously an extensive study showed that, as a control agent, there was no significant difference among vehicle solution, dead cells, and fibroblasts (Lepore et al., 2008).