STEM CELL BIOLOGY
We could show that all of four carriers tested were able to transfect
MSCs with mRNA encoding CXCR4 very efciently. Receptor
expression was again compared to that obtained by pDNA delivery,
and revealing that the number of transfected cells was considerably
higher with mRNA than with pDNA, except when DOTAP/DOPE
was used as a carrier. The expression of CXCR4 was still detectable
48 hours post transfection, i.e. long enough to be sufcient for the
MSCs to migrate to the target tissue.
262. Genetic Modication of Neural Stem Cells
Ravi K. Chandra.
Pharmacy, Acharya & B.M. Reddy College of Pharmacy,
Bangalore, Karnataka, India.
Neural stem cells (NSCs) are the main vehicle for genetic and
molecular therapies in the central nervous system (CNS). The
sustainability of NSCs has been ensured through genetic manipulation
both in vitro and in vivo. NSC lines have also been immortalized
and controlled for cell growth in similar fashion. Their potential to
differentiate and their genetic plasticity make them the modality of
choice for cellular transplantation. After transplantation, NSCs also
exhibit inherent long-distance migratory capabilities and a remarkable
capacity to integrate into brain structures. This makes NSCs the
ideal candidate for delivery and expression of therapeutic genes.
Finally, the imaging possibilities with NSC transplants are endless,
and they will be a pivotal component to safe and effective human
transplantation. This paper provides an overview on NSCs and the
various methods in which they have been genetically manipulated
for biological investigation.
263. APOA-1 Is a Novel Marker of Erythroid Cell
Maturation from Hematopoietic Stem Cells in Mice
and Humans
Tomoko Inoue,1 Daisuke Sugiyama,2 Ryo Kurita,1 Tatsuo
Oikawa,1 Kasem Kulkeaw,2 Hirotaka Kawano,1 Yoshie Miura,1
Michiyo Okada,1 Youko Suehiro,1 Atsushi Takahashi,1 Tomotoshi
Marumoto,1 Hiroyuki Inoue,1 Norio Komatsu,3 Kenzaburo Tani.1
1
Department of Molecular Genetics, Medical Institute of
Bioregulation, Kyushu University, Fukuoka, Japan; 2Department of
Hematopoietic Stem Cells, SSP Stem Cell Unit, Kyushu University,
Fukuoka, Japan; 3Department of Transfusion Medicine and Stem
Cell Regulation, Juntendo University School of Medicine, Tokyo,
Japan.
The mechanism that regulates terminal erythroid cell maturation
from hematopoietic stem cells is poorly defined. Therefore,
identifying genes and surface markers that are restricted to specic
stages of erythroid maturation will further our understanding of
erythropoiesis. To identify genes expressed at discrete stages of
erythroid development, we screened for genes that contributed to
the proliferation and maturation of erythropoietin (EPO)-dependent
UT-7/EPO cells. After transducing erythroid cells with a human fetal
liver (FL)-derived lentiviral cDNA library and culturing the cells in
the absence of EPO, we identied 17 candidate genes that supported
erythroid colony formation. In addition, the mouse homologues
of these candidate genes were identied and their expression was
examined in E12.5 erythroid populations by qRT-PCR. The expression
of candidate erythroid marker was also assessed at the protein level
by immunohistochemistry and ELISA. Our study demonstrated that
expression level of Apoa-1 gene, an apolipoprotein family member,
was signicantly increased as hematopoietic stem cells differentiate
into terminal differentiated erythroid cells of mouse FL. Apoa-1
protein was more abundant in differentiated erythroid cells than
hematopoietic stem and progenitor cells in mouse FL by ELISA.
Moreover, the expression of APOA-1 gene was detected in terminal
differentiated erythroid cells of human peripheral blood. We conclude
S100
that APOA-1 is a novel marker of terminal erythroid maturation
from hematopoietic stem cells in mice and humans. APOA-1 can
potentially be used to identify mature erythrocytes in combination
with Ter119 antigen or glycophorinA antigen from in vitro cultured
erythroid cell sources such as ES or iPS cells. Also, gene transfer of
APOA-1to hematopoietic stem cells derived from ES/iPS cells may
be helpful for ex vitro expansion of red cells from the standpoint of
regenerative medicine. Now, we are trying to investigate whether
APOA-1 plays pivotal roles in erythroid cell maturation.
264. A Stem Cell Gene Therapy Approach for
Enhancing the Efcacy of Breast Cancer Therapy
by Trastuzumab (Herceptin)
Ines Beyer,1 Jonas Persson,1 ZongYi Li,1 Ying Liu,1 Roma Yumul,1
Andre Lieber.1
1
Medical Genetics, University of Washington, Seattle, WA.
Trastuzumab is an essential targeted anticancer drug, which is
the rst-line treatment of Her2/neu-overexpressing breast cancer.
Unfortunately, a signicant number of Her2/neu-positive patients
do not respond to treatment with trastuzumab-containing regimens.
Mechanisms for resistance include obstacles preventing trastuzumab
binding to its target – Her2/neu. In breast cancer, tumor cells are
often surrounded by tumor stroma consisting of stroma cells, tumor-
associated macrophages and matrix proteins such as collagen or
laminin. We observed extensive extracellular matrix and intercellular
junctions in breast cancer patients and xenograft models, which block
access to Her2/neu and, potentially, the intratumoral dissemination
of trastuzumab after systemic application. Recently, we established a
new stem cell gene therapy approach for breast cancer therapy. This
approach is based on the ex vivo modication of hemapoietic stem
cells, which, after transplantation, home in on the tumor and deliver
therapeutic transgenes to the tumor stroma in models with transplanted
mouse and human tumors (Blood; 2009; 113:5423-33). Here we
tested whether this approach could be used to transiently degrade
tumor stroma and facilitate trastuzumab therapy in a xenograft model
for breast cancer. To degrade tumor stroma proteins we utilized the
peptide hormone relaxin. In in vitro studies, we demonstrated that the
expression of relaxin in the Her2/neu-positive breast cancer cell line
BT474-M1 improved the therapeutic effect of trastuzumab. For in vivo
studies, we established tumors by the injection of BT474-M1 cells into
the mammary fat pad of CB17/SCID/beige mice. We demonstrated
that these tumors were massively inltrated by mouse CD45+ and
F4/80 positive macrophages. Towards the testing of a combination
of our stem cell gene therapy with trastuzumab therapy, we showed
that intraperitoneal trastuzumab injection delayed the growth of
BT474-M1 tumors. To test a stem cell gene therapy-based approach,
we transduced syngeneic mouse bone marrow cells with insulated
SIN lentivirus vectors expressing relaxin under tight doxycyclin
(Dox) control and transplanted them into sub-lethally irradiated
mice. After engraftment of transplanted cells in the bone marrow
and BT474-M1 tumor establishment, mice will be treated with Dox
and trastuzumab. Results will be presented. We expect a signicant
prolongation of survival in mice that received trastuzumab therapy and
Dox, compared to mice that were treated with trastuzumab without
Dox or control mice.
Molecular Therapy Volume 18, Supplement 1, May 2010
Copyright © The American Society of Gene & Cell Therapy