351 in vitro induction of the hematopoietic progenitorstem cells from human ES cells

351 In Vitro Induction of the Hematopoietic Progenitor/Stem Cells from Human ES Cells Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S136 STEM CEL[.]

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

S136

STEM CELL THERAPIES I

350 Identifi cation and Characterization of

Chondrogenic Progenitor Cells in Adult Skeletal

Muscle

Guangheng Li, Bo Zheng, Laura B Meszaros, Joseph B Vella,

Karin A Corsi, Arvydas Usas, Tomoyuki Matsumoto, Johnny

Huard

Stem Cell Research Center, Children’s Hospital of Pittsburgh,

Department of Orthopaedic Surgery, University of Pittsburgh,

Pittsburgh.

Introduction: When ectotopic bone formation is induced in skeletal

muscle by the addition of bone morphogenic protein (BMP), a

chondrogenic phase is typically observed This suggests that there

exists chondrogenic cells in skeletal muscle Previously, multipotent

cells have been isolated from skeletal muscle vasculature (e.g

pericytes, myoendothelial cells) In the present study, we sought to

identify these chondrogenic cells and to determine if endothelial cells

participate in chondrogenesis METHODS: Transgenic mice with

LacZ expressing endothelial cells (FVB/N-Tg(TIE2-lacZ)182Sato/J)

were induced to generate ectopic bone in skeletal muscle by injection

of AAV-BMP4 Muscle derived cells (MDCs) were then isolated from

Fischer rats and sorted by fl ow cytometry (FACS) and evaluated for

their chondrogenic and myogenic potential using immunostaining

(desmin, vimentin and MyoD) RESULTS: 1 Endothelial cells (blue)

in skeletal muscle vasculature were not observed to participate in the

chondrogenic phase

Legend: a AAV-BMP4 virus was injected into the gluteofemoral

muscle pocket of a FVB/N-Tg (TIE2-lacZ)182Sato/J mouse b X-gal

staining showed lacZ positive endothelial cells stained in blue c

lacZ positive cells were not observed in the newly formed cartilage

tissue at 17 days following injection of AAV-BMP4 virus d No lacZ

positive cells were observed with higher magnifi cation 2 MDCs

contain myogenic cells as well as chondrogenic cells that may arise

from peri- and endomysium

Legend: (a) Surface marker profi le of freshly isolated MDCs (b) Phenotypic characterization of MDCs (c) Chondrogenic assay demonstrates positive chondrogenic differentiation of MDCs (d, e) Typical chondrocytes which occupy lacunae can be observed in the MDCs pellet DISCUSSION: Despite the isolation of multipotent cells from muscle vasculature our results suggest that endothelial cells do not participate in the chondrogenic phase of ectopic bone formation However, chondrogenic cells have previously been isolated from rat MDCs, particularly the fi brogenic cells residing in the muscle fascia (FDCs) (ORS 2007 abstract) In the present study, FACS sorting and subsequent characterization of MDCs demonstrated that MDCs contain at least two populations of cells, one that is chondrogenic and one that is myogenic These chondrogenic cells are the likely candidate that participates in the chondrogenic phase of ectopic bone formation

Progenitor/Stem Cells from Human ES Cells

Ryo Kurita, Rui Kageyama, Yoshie Miura, Takafumi Hiramoto, Tomoko Yokoo, Michiyo Okada, Atsushi Takahashi, Hiroyuki Inoue, Kenzaburo Tani

Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

The human embryonic stem (ES) cells are considered to differentiate to three lineage cells in vitro and in vivo as mouse

ES cells However, the best condition to induce these cells into the target cells/tissues and the safety of these differentiated cells/tissues remains unknown We have recently reported that the lentivirally transduced ES cells of common marmoset, small nonhuman primate, differentiated effi ciently into hematopoietic progenitor cells in vitro without the requirement of stromal cells In this study, we examined whether the lentivirally transduction of tal1/scl induced human ES cells into hematopoietic progenitor cells in vitro, too First of all,

we determined the optimal culture condition to induce multilineage hematopoietic progenitor cells from human ES cells of hES-1 cells based on the expressions of Brachyury, Flk1 and CD34 Then we established four human ES cell lines lentivirally expressing tal1/ scl gene The expression of hematopoietic cell markers including CD34, CD235a and CD133 was significantly increased in the embryoid bodies derived from tal1/scl-expressing ES cells cultured

in the optimal growth factor condition The number of hematopoietic

Molecular Therapy Volume 17, Supplement 1, May 2009

STEM CELL THERAPIES I

progenitor cells increased in two tal1/scl-expressing ES cell lines

determined by colony-forming assay In vivo hematopoiesis of the

tal1/scl-expressing ES cells in immune defi cient mice is now under

investigation Our results suggested that the combination of growth

factor and tal1/scl gene transduction is highly effective to induce the

hematopoietic stem cells from human ES cells at least in vitro

Stem Cells Using Helper Virus-Free Packaged

HSV-1 Amplicons

Yasser M El-Sherbini,1 Mark Stevenson,1 Leonard W Seymour,1

Richard W Wade-Martins.2

1 Clinical Pharmacology, University of Oxford, Oxford, United

Kingdom; 2 Department of Physiology, Anatomy and Genetics,

University of Oxford, Oxford, United Kingdom.

Bone marrow mesenchymal stem cells (MSCs) have recently

received much attention as a promising cellular delivery vehicle

due to the ease of genetic manipulation and in vitro culturing The

multipotency of MSCs also makes them promising candidates

for regenerative medicine applications In the present study we

evaluate the effi ciency of genetically modifying MSCs using large

carrying capacity (152 Kb) herpes simplex type 1 (HSV-1) based

amplicons In the present study we have used four amplicons,

pHSVCMVGL, pHSVEF1GL, pHSVPUBGL and pHSVCA9GL

pHSVCMVGL is a DNA plasmid containing the HSV-1 packaging

signal (pac) and HSV-1 origin of replication (oriS); it also expresses

enhanced green fl uorescence protein (EGFP) under the control

of the HSV-1 immediate early promoter (IE4/5) and luciferase

under the control of the immediate early cytomegalovirus (CMV)

promoter pHSVEF1GL, pHSVPUBGL and pHSVCA9GL have been

modifi ed from the pHSVCMVGL where the CMV promoter driving

luciferase is replaced by the human elongation factor promoter (EF1),

Polyubiquitin promoter and Carbonic anhydrase hypoxia specifi c

promoter (CA9) respectively The amplicons successfully transduced

MSCs with a transduction effi ciency of 6% at MOI 10; however the

effi ciency of transduction was increased up to 30% at the same MOI

by applying a centrifugation force during the transduction The ability

of the vector to deliver a functional gene was also proven by strong

expression from the EGFP and luciferase reporter genes within cells

post transduction The levels of luciferase expression driven by the

four promoters varied accordingly EF1, PUB and CA9 was nearly

one tenth the expression from CMV However the EF1 promoter

showed as much as twice the expression from PUB On the other hand

CA9 hypoxic promoter didn’t show any sign of upregulation when

hypoxic condition (5% oxgen) was applied comparable to normoxic

conditions The transduction with HSV-1 amplicons altered neither

the morphological features nor the phenotypic surface markers of

the transduced MSCs as shown by fl uorescence microscopy and

fl ow cytometry respectively The transduction also did not affect

the differentiation plasticity of the engineered cells as we were

able to differentiate the transduced cells into adipocytes This study

demonstrates the possibilities of using extrachromosomal vectors with

large carrying capacity to modify MSCs, thereby avoiding deleterious

affect associated with integrating vectors

Selective Gene Therapy for Breast Cancer Metastases

Donghong Zhao,1 Joseph Najbauer,1 Elizabeth Garcia,1 Marianne

Z Metz,1 Carlotta A Glackin,1 Seung U Kim,2 Karen S Aboody.1

1 City of Hope National Medical Center and Beckman Research Institute, Duarte, CA; 2 University of British Columbia, Vancouver,

BC, Canada.

Metastases to multiple organs are the primary cause of mortality

in breast cancer patients It is estimated that ∼80% of women who die as a result of breast cancer have lymph node, lung, bone or brain metastases Current therapies cannot effectively eradicate breast cancer after metastasis to distant organs, with a poorer prognosis if bone and brain metastases are present In addition, chemotherapy doses are limited by toxicity to normal tissues Neural stem cells (NSCs) have the potential to overcome obstacles that limit current chemotherapy strategies by targeting therapeutic agents specifi cally

to primary and metastatic breast tumor sites—thus, increasing the therapeutic index of a given drug In effect, NSCs offer a platform for tumor-localized chemotherapy production, thereby decreasing

associated toxicities to normal tissues Here, we report a novel in vivo

study that exploits the inherent tumor-tropism of NSCs (HB1.F3.CD,

an established human NSC line) to effectively target breast tumor metastases in multiple organs, including liver, lung, lymph nodes, and femur This study was done in human breast cancer (MDA-MB-231, MCF-7) immunodeficient mouse models, employing Xenogen imaging and histochemical analysis to show formation of breast tumor metastases in multiple organs, including bone Our data demonstrate that NSCs localize to malignant cells throughout the body, with a greater preference for metastatic tumor sites in the liver, bone marrow, lung and lymph node, as compared to the primary mammary fat pad site, and with minimal NSC localization to normal tissues We also investigated the tropism of NSCs to various human breast cancer

lines in vitro When we compared the migration of NSCs to different

invasive breast cancer cell lines by Boyden chamber migration assays,

we found that NSCs displayed a 3–4-fold increase in migration to highly invasive breast cancer cells (MDA-MB-231, MDA-MB-468, Hs578T) as compared to less invasive breast cancer cells (MCF-7, T47D, ZR75, SKBR3) These data showed that the potency of the NSC-tumor tropism strongly correlated with the invasiveness of the

breast cancer cells in vitro To identify the cytokines responsible for

attraction of NSCs to breast cancer cells, we screened 79 cytokines for their expression level in these invasive breast cancer cell lines Conditioned media were analyzed using a cytokine antibody array and demonstrated that IL-6 and IL-8 were highly expressed in the most invasive breast cancer cells Neutralizing antibodies against IL-6 inhibited 50% of the NSC migration to tumor-conditioned media, suggesting that IL-6 is an important cytokine mediating this selective, tumor-directed NSC migration, but that other factors are also involved This NSC-mediated therapeutic approach provides a novel tumor-localized strategy to eradicate breast tumor metastases and has signifi cant clinical implications, especially for patients with advanced metastatic disease, who are refractory to currently available treatments