“The 88th of Stem Cell Biology and Regenerative Medicine Forum”
Date：Feb 22nd (Mon) 2016
Time : 13:00 〜 14:30
Place：Auditorium in 1st Building------------------------------------------------------------------------------------------------------------------------------------（Internal Speaker）
13:00-13:30 Lin Huan-Ting(Division of Stem Cell Processing The Institute of Medical
Science The University of Tokyo)
An assessment of the effects of ectopic gp91phox expression in XCGDiPSC-derived neutrophils（External Speaker）
13:30-14:30 Yoshihiro Takihara (Department of Stem Cell Biology, Research Institute ofRadiation Biology and Medicine, Hiroshima University)
Geminin, a molecular switch turning on and off quiescence or blood cell
production in hematopoietic stem cells-------------------------------------------------------------------------------------------------------------------------------------

Hosted by Center for Stem Cell Biology and Regenerative Medicine---Information--＊ Please register attendance at the reception desk.
＊ Next forum（the89th）will be held on Mar 10th 18:00〜19:30.
＊ Please contact tatsu-m@ims.u-tokyo.ac.jp, for Forum speaker recommendations

An assessment of the effects of ectopic gp91phox expression in XCGDiPSC-derived neutrophils
Lin Huan-Ting (Division of Stem Cell Processing The Institute of Medical
ScienceThe University of Tokyo)
Disease modeling using patient autologous iPSCs as an exploratory platform has
promising applications in elucidating pathophysiological mechanisms andmethodological development. X-linked chronic granulomatous disease (XCGD) is
unique amongst monogenetic primary immunodeficiency diseases since theappearance of functional neutrophils in the peripheral blood following
hematopoietic stem cell gene therapy is only transient. One possibility is theoccurrence of detrimental effects secondary to ectopic gp91phox expression in
neutrophils, which has not been demonstrated under formal experimental settings.
Since hematopoiesis occurs largely in the bone marrow, modeling XCGD using
iPSCs allows the process of differentiation to be studied intensely in vitro. In thisstudy, alpharetroviral vectors carrying a ubiquitous promoter were used to drive the“ectopic” expression of codon optimized gp91phox cDNA. In the mature fractionof neutrophils differentiated from transduced XCGD-iPSCs, cellular recovery interms of gp91phox expression and reactive oxygen species production wasabruptly lost before cells had fully differentiated. Most critically, ectopic gp91phox
expression could be identified clearly in the developing fraction of the transducedgroups, which appeared to correspond with reduced cell viability. It is possible that
this impedes further differentiation of developing neutrophils. Therefore affording
cellular protection from the detrimental effects of ectopic gp91phox expression
may improve XCGD clinical outcomes.

Geminin, a molecular switch turning on and off quiescence or blood cell
production in hematopoietic stem cells
Yoshihiro Takihara (Department of Stem Cell Biology, Research Institute ofRadiation Biology and Medicine, Hiroshima University)
Hematopoietic stem cell (HSC) transplantation therapy has provided an epochal
clinical outcome. Currently three kinds of different cellular sources, bone marrowcells, peripheral blood stem cells and umbilical cord blood, are available for thetransplantation. A technology for expanding HSCs ex vivo could not only advance
HSC transplantation therapy but may pave a way to further application of HSCs foradvanced immunotherapy and gene therapy. There developed has, however, beenno practically available technology for the expansion. Since hematopoietic
cytokines and niche molecules have been shown not to be enough for theexpansion, we have focused on the cell intrinsic factors. By utilizing biochemical
as well as genetic approaches, we previously demonstrated that Polycomb-groupcomplex 1 and Hoxb4/Hoxa9 regulate HSCs through the ubiquitin proteasome
system-mediated direct regulation of Geminin protein and proposed a hypothesisthat Geminin acts as a key regulator for determining cell fate of HSCs, i.e., cellularquiescence, self-renewal and differentiation. Geminin negatively regulates DNA
replication and chromatin remodeling through the direct interaction with Cdt1 andBrahma/Brg1, respectively. We then generated a recombinant Geminin protein
fused with a membrane translocating motif (MTM) of FGF4, which was designatedas cell-penetrating (CP-) Geminin. We have demonstrated that amount as well as
timing of Geminin expression in NIH 3T3 cells was altered by direct transductionof a CP-Geminin protein. Biologically, CP-Geminin transduction controlled thecell cycle in NIH 3T3 cells, i.e., CP-Geminin efficiently suppressed G0/G1 toS-phase transition to keep cellular quiescence. We currently aim at regulating
activity as well as expression of Geminin to perform detailed verification of ourhypothesis described above and further to generate a new strategy for manipulating
the HSC activity.