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RESEARCH ARTICLE

Engineered human mesenchymal stem cells: a

novel platform for skeletal cell mediated gene

therapy

Gadi Turgeman

Debbie D. Pittman

Ralph Mu

ller

Basan Gowda Kurkalli

Shuanhu Zhou

Gadi Pelled

Amos Peyser

Yoram Zilberman

Ioannis K. Moutsatsos

Dan Gazit

Molecular Pathology Laboratory,

Hebrew University-Hadassah Medical

and Gene Therapy Center, PO Box

12272, Jerusalem, Israel

Genetics Institute, Cambridge, MA

02140, USA

Institute of Biomedical Engineering,

ETH and University of Zu

rich, 8044

rich, Switzerland

Orthopedic Surgery and Orthopedic

Oncology, Hadassah-Hebrew

University Medical Center, PO Box

12000, Jerusalem, Israel

*Correspondence to: D. Gazit,

Molecular Pathology Laboratory,

Hebrew University-Hadassah

Medical and Gene Therapy Center,

PO Box 12272, Jerusalem, Israel.

E-mail: dgaz@huji.ac.il

Received: 15 September 2000

Revised: 2 January 2001

Accepted: 19 January 2001

Abstract

Background Human mesenchymal stem cells (hMSCs) are pluripotent cells

that can differentiate to various mesenchymal cell types. Recently, a method

to isolate hMSCs from bone marrow and expand them in culture was

described. Here we report on the use of hMSCs as a platform for gene therapy

aimed at bone lesions.

Methods Bone marrow derived hMSCs were expanded in culture and

infected with recombinant adenoviral vector encoding the osteogenic factor,

human BMP-2. The osteogenic potential of genetically engineered hMSCs was

assessed in vitro and in vivo.

Results Genetically engineered hMSCs displayed enhanced proliferation

and osteogenic differentiation in culture. In vivo, transplanted genetically

engineered hMSCs were able to engraft and form bone and cartilage in

ectopic sites, and regenerate bone defects (non-union fractures) in mice

radius bone. Importantly, the same results were obtained with hMSCs

isolated from a patient suffering from osteoporosis.

Conclusions hMSCs represent a novel platform for skeletal gene therapy

and the present results suggest that they can be genetically engineered to

express desired therapeutic proteins inducing speciﬁc differentiation path-

ways. Moreover, hMSCs obtained from osteoporotic patients can restore their

osteogenic activity following human BMP-2 gene transduction, an important

ﬁnding in the future planning of gene therapy treatment for osteoporosis.

Keywords human mesenchymal stem cell; gene therapy; bone formation;

bone regeneration; rhBMP-2; adenovirus

Introduction

In planning gene therapy strategies for tissue repair and regeneration, two

options need to be considered: those of direct gene delivery in vivo via viral or

non-viral vectors of desired therapeutic genes and of ex vivo cell mediated

gene therapy. Ex vivo cell mediated gene therapy allows speciﬁc transduction

conditions in vitro, but relies on engraftment of cells in vivo in order to obtain

efﬁcient gene delivery. We have previously hypothesized that mesenchymal

stem cells (MSCs) as the transgene vehicle will have a better therapeutic

potential for bone regeneration compared with other cell types [1]. We have

shown that MSCs genetically engineered to express recombinant human

BMP-2 gene have the ability to induce bone regeneration not only via

paracrine secretion of rhBMP-2, but also by an autocrine effect on the stem

cells promoting their osteogenic differentiation [1].

THE JOURNAL OF GENE MEDICINE

J Gene Med 2001; 3: 240–251.

DOI: 10.1002/ jgm.181

Human bone marrow derived mesenchymal stem cells

(hMSCs) represent a pluripotent mesenchymal population

of cells that also serve as precursors for osteoprogenitor

cells which are the main cellular mediators for bone

formation [2–4]. hMSCs are relatively easy to isolate and

expand in culture [3,4]. In vitro, hMSCs can differentiate

along the osteogenic pathway and induce ectopic bone

formation upon transplantation in vivo into ectopic sites

and segmental bone defects [5–9]. Treating hMSCs with

rhBMP-2 protein can markedly increase osteogenic

differentiation in culture, evidenced by expression of

osteogenic marker genes [10–12].

Marrow derived MSCs were seriously considered as

vehicles for cell therapy and for gene therapy. As vehicles

for gene therapy, non-human MSCs were transduced

in vitro to express genes (human factor IX and growth

hormone) for systemic delivery of the transgene product,

by expressing the transgene in the bone marrow

environment [13–17]. Alternatively, non-human MSCs

were used for local delivery of transgenes with rhBMP-2

for repair of bone segmental defect [18] and with L-DOPA

in the brain of a rat model of Parkinson’s disease [19].

It has been suggested that MSCs could be genetically

engineered for the treatment of bone-related diseases

such as osteogenesis imperfecta and osteoporosis [2].

hMSCs were shown to be effectively transduced with

retroviral vectors to express green ﬂuorescence protein

marker gene [20] and tyrosine hydroxylase gene encod-

ing for the corresponding enzyme necessary for the

production of L-DOPA [19]. Adenoviral vectors were also

shown to effectively transduce hMSCs to express lacZ

marker gene [21]. Moreover, hMSCs transduced ex vivo

with a retroviral vector encoding the human factor VIII

gene were shown to engraft into the spleen and express

human factor VIII in vivo [22]. The aim of the present

study was to establish a novel platform for skeletal gene

therapy based on engineered hMSCs. Speciﬁcally, the aim

was to enhance the osteogenic potential of hMSCs in vitro

and in vivo by rhBMP-2 gene transfer using adenoviral

vector. We investigated the effects of rhBMP-2 expression

on differentiation and proliferation of hMSCs in vitro,on

ectopic bone formation and on bone segmental defect

(non-union fracture) regeneration in vivo using engi-

neered hMSCs.

Materials and methods

Construction of adenoviral vectors

Adenoviral vectors were prepared as described previously

[23]. The present study emplyed recombinant replication

defective type 5 adenovirus, with deleted E1a, E1b and

partial E3 viral genome regions. Constructs of human

BMP-2 cDNA and the b-galactosidase gene (LacZ) under

the control of CMV promoter were incorporated into the

viral vectors to create the two adenoviral vectors,

Ad-BMP-2 and Ad-lacZ. Viruses were stored at x80uC

in 10% phosphate buffered saline (PBS).

hMSC isolation and infection with

adenoviral vectors

hMSCs were isolated from explants of human bone

marrow surgical waste (approved by the Helsinki Com-

mittee Board of the Hadassah Medical Center, Jerusalem,

Israel) and expanded in vitro. Isolation of hMSCs was

performed as described previously [3]. Brieﬂy, 10 ml

marrow aspirates were collected into a tube with 6000 U

heparin, washed with PBS, and recovered cells were

collected by centrifugation at 900 g. Collected cells were

then loaded onto Percoll solution (density 1.073 g/ml).

Cell separation was accomplished by centrifugation at

1100 g (30 min at 20uC). Nucleated cells collected were

washed twice with PBS and then cultured and sub-

cultured in Dulbecco’s minimal essential medium

(DMEM) (low glucose) supplemented with 10% fetal

calf serum (FCS). hMSCs were infected in vitro at 80%

conﬂuence with recombinant adenoviruses encoding

rhBMP-2 (Ad-BMP-2) and LacZ (Ad-LacZ) at multiplicity

of infection (MOI) of 100, 2 h in PBS after which the

medium was added for 3 days. Efﬁciency of infection

was estimated post-infection with Ad-LacZ using X-gal

staining. X-gal histochemical staining was performed as

follows: cells were ﬁxed with 0.25% glutaralde-

hyde, 0.1 M NaPO

(pH 8.3), 5 mM ethylen glycol-bis

(b-aminoethyl ether) (EGTA) and 2 mM MgCl

for

30 min. Cells were then washed three times (with

0.1 M NaPO

, 2 mM MgCl

, 0.1% deoxycholate, 0.2%

Nonident P40) and stained with X-gal solution (1 mg/

ml), 5 mM K

Fe(CN)

, 5 mM K

Fe(CN)

e3H

O, 0.1 M

NaPO

, 2 mM MgCl

, 0.1% deoxycholate, 0.2% Nonident

P40, in the dark at room temperature (RT) overnight.

For infecting hMSCs with both Ad-BMP-2 and Ad-LacZ,

cells were grown and infected in the same conditions

described above with both adenoviral vectors at a MOI of

50 for each of the viral vectors.

In vitro

analysis of rhBMP-2 expression

by RT-PCR

hMSCs infected with Ad-BMP-2 or Ad-LacZ (MOI 100)

were grown in culture and RNA was harvested at Days 4

and 10 after infection. RNA was isolated using TRIzol

Reagent (Life Technologies, Grand Island, NY, USA)

according to the manufacturer’s protocol. Reverse tran-

scriptase-polymerase chain reaction (RT-PCR) was mod-

iﬁed from a procedure described previously [24]. RhBMP-2

primers were a kind gift from B. Sibley (Genetics

Institute, Cambridge, MA, USA) and were designed

according to the rhBMP-2 cDNA sequence [25]: forward:

5k-CATCCCAGCCCTCTGAC-3k, reverse: 5k-CTTTCCCACC

TGCTTGCA-3k. The PCR of hBMP-2 was performed for 30

cycles at 94uC for 1 min, annealing at 50uC for 1 min, and

72uC for 1 min in a MJ Minicycler. Human glyceraldehyde

3 phosphat dehydrogenase (GAPDH) as an internal

control, forward: 5k-TGATGACATCAAGAAGGTGAAG-3k;

reverse: 5k-TCCTTGGAGGCCATGTGGGCCAT-3k. The PCR

Human Mesenchymal Stem Cells in Gene Therapy 241

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