Tissue Engineering and Regenerative Medicine

TISSUE ENGINEERING AND REGENERATIVE MEDICINE

a
Division of Obstetrics and
Gynecology, bCenter for
Hematology and Regenerative
Pre- and Postnatal Transplantation of Fetal
Medicine, dDepartment of Mesenchymal Stem Cells in Osteogenesis
Women’s and Children’s Health and
Neuro-pediatric Unit, and
m
Imperfecta: A Two-Center Experience
Hematology Center, Karolinska
Institutet and Karolinska University CECILIA GÖTHERSTRÖM,a,b MAGNUS WESTGREN,a S.W. STEVEN SHAW,c EVA ÅSTRÖM,d
Hospital, Stockholm, Sweden; ARIJIT BISWAS,e PETER H. BYERS,f CITRA N.Z. MATTAR,e GAIL E. GRAHAM,g JAHAN TASLIMI,h
c
Department of Obstetrics and
UWE EWALD,i NICHOLAS M. FISK,j ALLEN E.J. YEOH,k JU-LI LIN,l PO-JEN CHENG,c
Gynecology and lDivision of Medical
Genetics, Department of Pediatrics, MAHESH CHOOLANI,e KATARINA LE BLANC,b,m JERRY K.Y. CHANe,n,o
Chang Gung Memorial Hospital at
Key Words. Mesenchymal stem cells x Mesenchymal stromal cells x Cell therapy x
Linkou and Chang Gung University Osteogenesis imperfecta x Prenatal transplantation x In utero transplantation
College of Medicine, Taoyuan,
Taiwan; eExperimental Fetal
Medicine Group, Department of ABSTRACT
Obstetrics and Gynecology, and
k Osteogenesis imperfecta (OI) can be recognized prenatally with ultrasound. Transplantation of
Department of Pediatrics, Yong mesenchymal stem cells (MSCs) has the potential to ameliorate skeletal damage. We report the
Loo Lin School of Medicine and clinical course of two patients with OI who received prenatal human fetal MSC (hfMSC) trans-
National University of Singapore, plantation and postnatal boosting with same-donor MSCs. We have previously reported on pre-
Singapore; fDepartments of natal transplantation for OI type III. This patient was retransplanted with 2.8 3 106 same-donor
Pathology and Medicine (Medical MSCs per kilogram at 8 years of age, resulting in low-level engraftment in bone and improved
Genetics), University of linear growth, mobility, and fracture incidence. An infant with an identical mutation who did
Washington, Seattle, Washington, not receive MSC therapy succumbed at 5 months despite postnatal bisphosphonate therapy.
USA; gDepartment of Genetics, A second fetus with OI type IV was also transplanted with 30 3 106 hfMSCs per kilogram at
Children’s Hospital of Eastern 31 weeks of gestation and did not suffer any new fractures for the remainder of the pregnancy
or during infancy. The patient followed her normal growth velocity until 13 months of age, at
Ontario and Department of
which time longitudinal length plateaued. A postnatal infusion of 10 3 106 MSCs per kilogram
Pediatrics, University of Ottawa, from the same donor was performed at 19 months of age, resulting in resumption of her growth
Ottawa, Canada; hOrthopedic Unit, trajectory. Neither patient demonstrated alloreactivity toward the donor hfMSCs or manifested
Uppsala University Hospital, any evidence of toxicities after transplantation. Our findings suggest that prenatal transplanta-
Uppsala, Sweden; iWomen’s and tion of allogeneic hfMSCs in OI appears safe and is of likely clinical benefit and that retransplan-
Children’s Health, Uppsala tation with same-donor cells is feasible. However, the limited experience to date means that it is
University, Uppsala, Sweden; not possible to be conclusive and that further studies are required. STEM CELLS TRANSLATIONAL
j
Centre for Clinical Research, MEDICINE 2014;3:255–264
University of Queensland, Brisbane,
Australia; nDepartment of
Reproductive Medicine, Women’s INTRODUCTION for more than half of all recognized affected
and Children’s Hospital, Singapore; individuals. OI type III, the most severe form
o Osteogenesis imperfecta (OI) is a genetically in children who survive the neonatal period,
Cancer and Stem Cell Biology,
and clinically heterogeneous disorder in which has a prevalence of approximately 1–2 per
Duke-National University of
the major clinical manifestations are atypical 100,000 and is usually caused by de novo mu-
Singapore Graduate Medical
School, Singapore skeletal development, osteopenia, multiple tations [2] in type I collagen genes; however,
painful fractures, and short stature [1]. In more phenocopies occur among those with some re-
Correspondence: Cecilia Götherström, than 90% of affected individuals, the causative cessively inherited forms of OI. Currently, there
Ph.D., Division of Obstetrics and mutations lie in the two genes that encode the
Gynecology, K57, Karolinska University
is no cure for OI, although use of bisphospho-
Hospital Huddinge, SE-141 86 chains of the major protein of the bone, type I nates has some symptomatic but not cura-
Stockholm, Sweden. Telephone: 46-8- collagen, COL1A1 and COL1A2. In the most tive therapeutic effect, and the longer-term
585 81161; E-Mail: Cecilia. severely affected individuals, the mutations effects of early intervention remain undeter-
[email protected] result in production of abnormal type I col- mined [3, 4].
Received April 30, 2013; accepted for lagen molecules [1]. The effects of these Mesenchymal stem cells (MSCs) are multi-
publication September 23, 2013; first changes begin during fetal life and often potent cells that are readily isolated and
published online in SCTM EXPRESS present with abnormal fetal skeletal devel- culture expanded and can be induced to dif-
December 16, 2013. opment, shortened long bones, and multiple ferentiate into osteoblasts, chondrocytes, or
©AlphaMed Press fractures that can be identified during rou- adipocytes [5]. MSCs have a low immunogenic
tine prenatal ultrasonography. The prevalence profile and typically are not rejected during
http://dx.doi.org/ of OI is approximately 6–7 per 100,000 [2]. OI allogeneic transplantation paradigms [6, 7], al-
10.5966/sctm.2013-0090 type I and IV, the two mildest forms, account though engraftment may not be long lived [8].

STEM CELLS TRANSLATIONAL MEDICINE 2014;3:255–264 www.StemCellsTM.com ©AlphaMed Press 2014

256 MSC Transplantation for Improvement of OI

MSCs are generally considered safe for transplantation when Stockholm, Sweden (428/01, 2006/308-31/2, 2005/867-31/3)
grown under normal culture conditions, and there are no reports and Singapore (D/10/247). Informed written consent was
of malignant transformation or generation of ectopic tissues obtained from all participants in this study.
[9–12]. The first clinical transplantation of MSCs was performed
more than 15 years ago, and adult MSCs have now been used clin- Patients
ically in hundreds of patients without adverse effect [13, 14].
The potential of MSC transplantation for OI was demonstrated This paper presents two infants in whom prenatal and postnatal
initially in the oim mouse, in which allogeneic transplanted wild- transplantation were performed; patients A and B were trans-
type donor MSCs homed to the bones, where they contributed planted in Sweden and Singapore, respectively (Table 1). In addi-
to the osteoprogenitor pool, with improvement in collagen content tion, we present patient C from Canada, with a mutation identical
and mineralization [15]. This was followed by a clinical trial in which to that of patient A but who had not received a transplantation.
children with severe OI type III were treated with an infusion of al-
logeneic MSCs. This was reported to result in increased skeletal MSC Isolation and Culture
mineralization and growth velocity, despite a low level of engraft- MSCs were isolated from two male fetal livers (from 7 weeks and
ment (∼1%) [8]. The mechanisms by which such a small population 3 days and 10 weeks of gestation). Cells were analyzed for HIV
of cells could confer benefit [16, 17] were uncertain, and an effect antigen, human T-lymphotropic virus types 1 and 2, and hepatitis
of nonadherent bone marrow cells cannot be excluded [18]. B and C and were cultured as described previously in good
Stem cells derived from the fetus present some favorable char- manufacturing practice-qualified premises at Karolinska Institu-
acteristics over adult sources. Although human fetal MSCs (hfMSCs) tet [21]. Briefly, the fetal liver was mechanically disrupted by pas-
are phenotypically similar and nonimmunogenic [19–21] like their sage through a 100-mm nylon filter, and the cell suspension
adult counterparts, they have higher proliferative capacity and lon- diluted to a final concentration of 107 cells per milliliter. Mononu-
ger telomeres and differentiate more readily into bone, skeletal clear cells were collected by gradient centrifugation and sus-
muscle, and oligodendrocytes [22–26]. These characteristics suggest pended in Dulbecco’s modified Eagle’s medium-low glucose
that they may have a greater utility in cellular therapy. (DMEM-LG; Invitrogen, Carlsbad, CA, http://www.invitrogen.
Because the damage in severe OI begins early in fetal life and com) supplemented with 10% fetal bovine serum (FBS; Hyclone;
may lead to perinatal lethality, a good case can be made for pre- Invitrogen) approved for clinical cultures of MSCs and 100 IU/ml
natal MSC transplantation, to treat the fetus before additional penicillin and 100 mg/ml streptomycin (Invitrogen). Cells were
pathology occurs. Other arguments that favor a fetal approach plated at 1.6 3 105 cells per cm2 in culture flasks and maintained
include higher engraftment rates reported after prenatal fetal at 37°C in a humidified environment containing 5% carbon diox-
stem cell transplantation; the small size of the fetus, which allows ide. After 3 days, nonadherent cells were discarded, and the me-
higher proportionate cell doses to be delivered; the right-to-left dium was replaced every 3–4 days thereafter. Before confluence,
heart shunting that enhances systemic distribution of the cells; the cells were detached by treatment with 0.05% trypsin and
and the relative naiveté of the immune system, which permits 0.53 mM ethylenediaminetetraacetic acid (Invitrogen) and
the development of immune tolerance toward donor cells replated at a density of 4 3 103 cells per square centimeter in
[27–30]. We have previously found that prenatal transplantation culture flasks. After subsequent passaging, the cells were frozen
in the oim mouse with hfMSCs resulted in significant engraftment in DMEM-LG, 10% AB plasma, 2 IU/ml heparin (Leo Pharma A/S,
to bone (∼5%), with restitution of bone histology toward normal, Ballerup, Denmark, http://www.leo-pharma.com), 100 IU/ml
reduction in fracture frequency [31], and measurable expression penicillin, 100 mg/ml streptomycin, and 10% dimethyl sulfoxide
of normal human type I collagen [32]. Recently, Panaroni et al. (WAK-Chemie Medical GmbH, Steinbach, Germany, http://www.
demonstrated that prenatal allogeneic bone marrow transplanta- wak-chemie.net), and aliquots were assayed for sterility, myco-
tion in a perinatally lethal, dominant model of OI resulted in in- plasma, and cell characteristics (surface expression of proteins,
creased perinatal survival, bone mineralization, and architecture. trilineage differentiation ability, and karyotype).
In this model, donor cells accounted for 20% of the total colla-
gen I content of bone, despite an engraftment rate of only 2%
[33]. Enzyme-Linked Immunosorbent Assay for Detection of
Ten years ago, we reported that prenatal hfMSC transplanta- Antibodies Against FBS
tion in a fetus with OI type III resulted in engraftment in bone that Enzyme-linked immunosorbent assay was performed to detect
could be detected at 9 months of age [34]. We now describe the antibodies directed against FBS used in the expansion of
same patient’s long-term clinical course with a secondary trans- hfMSCs, as described previously [35]. Briefly, 96-well plates
plantation of same-donor hfMSCs at 8 years of age. We also pres- were coated with 1% FBS, washed and incubated with recipient
ent a case with the same mutation as this patient but without serum, and washed and then incubated with alkaline
transplantation. In addition, we describe an infant with OI type phosphatase-conjugated polyclonal anti-human IgG antibodies
IV that underwent prenatal and postnatal hfMSC transplantation. (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.
com). Diethanolamine with addition of p-nitrophenol phosphate
(Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com)
MATERIALS AND METHODS was used as the color development substrate solution. Plates
were assayed at 405 nm in a spectrophotometer. The positive
Ethics and Institutional Research Board Approval control was serum from a male with known high titers of anti-
Derivation of good manufacturing practice MSCs from human FBS antibodies, and the negative control consisted of PBS-
first trimester fetal liver and its prenatal and postnatal trans- Tween. Optical density values of more than 1.0 were deemed
plantation was approved by the ethical review boards in positive.

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Götherström, Westgren, Shaw et al. 257

Table 1. Details of the three cases of osteogenesis imperfecta

Patient Mutation Phenotype Prenatal transplantation Postnatal transplantation Outcome
A COL1A2, c.3008G.A; OI type III 6.5 3 106 hfMSCs at 31 wk 42 3 106 at 8 yr and 2 mo No new fractures, improved
p.Gly1003Asp; Gly913Asp (∼5 3 106/kg) (∼2.8 3 106/kg) growth velocity
in the triple-helical domain
B COL1A2, c.659G.A; OI type IV 40 3 106 hfMSCs at 31 wk 88 3 106 at 19 mo and 11 days No new fractures, improved
p.Gly220Asp; Gly130Asp in (∼30 3 106/kg) (10 3 106/kg) growth velocity
the triple-helical domain
C COL1A2, c.3008G.A; OI type II/III None None Deceased at 5 mo of age
p.Gly1003Asp; Gly913Asp
in the triple-helical domain
Patients A and B received both prenatal and postnatal hfMSC transplantation, and patient C was not transplanted.
Abbreviations: hfMSC, human fetal mesenchymal stem cell; OI, osteogenesis imperfecta.

Transplantation Patient A blood were prepared by centrifugation on a Ficoll gradient (Lym-

The prenatal transplantation procedure was reported previously phoprep; Nycomed Pharma, Asker, Norway, http://www.
[34]. For the postnatal transplantation of hfMSCs in Sweden (patient nycomed.no). To investigate whether the donor hfMSCs induced
A), male hfMSCs derived from 10-week gestation fetal liver from the an allograft reaction in the patient, triplicate samples of 1 3
same donor as the first transplantation were thawed and cultured as 105 lymphocytes were cultured with 10,000 irradiated donor or
described previously for one passage, followed by 2 days in 10% hu- control MSCs in 0.2 ml RPMI 1640 medium supplemented with
man AB plasma with 2 IU/ml heparin in DMEM-LG and 100 IU/ml 20 mM HEPES, 100 IU/ml penicillin, 100 mg/ml streptomycin,
penicillin and 100 mg/ml streptomycin. The cells were then frozen, 20 mM L-glutamine (Invitrogen), and 10% pooled human AB se-
and sterility and hfMSC characteristics were analyzed as described. rum in 96-well plates. The cultures were incubated at 37°C
On the day of infusion, the cells were thawed and counted, and vi- in humidified 5% carbon dioxide air for 6 days. On day 5, 1 mCi/ml
ability was evaluated. The syringe used for infusion was heparinized [3][H]-thymidine (GE Healthcare Life Sciences, Little Chalfont, U.K.,
with 100 IU/ml heparin. Then, 42 3 106 hfMSCs (2.8 3 106 per kilo- http://www.gelifesciences.com) was added. After 24 hours, the
gram) at passage 5 in 21 ml saline containing 10% human AB plasma cells were harvested on a glass-fiber filter (PerkinElmer, Waltham,
was slowly infused in patient A through her intravenous line 5 days MA, http://www.perkinelmer.com), using a semiautomatic har-
after surgery for bilateral femoral osteotomy and replacement of vesting machine (Harvester 96; Tomtec, Hamden, CT, http://
rods at 8 years and 2 months of age. www.tomtec.com/). Radioactivity was determined as counts
per minute with an Intertechnique b-counter (PerkinElmer).
Transplantation Patient B
Fluorescence In Situ Hybridization
For transplantation of hfMSCs in Singapore (patient B), male
hfMSCs (7 weeks and 3 days of gestation) were thawed and cul- Fluorescence in situ hybridization (FISH) was performed on speci-
tured at 1 3 104 cells/cm2 for one to three passages in 10% FBS mens obtained before and after postnatal transplantation, as pre-
(Sigma-Aldrich) in DMEM-LG and harvested as described previ- viously described [5]. At 6 years and 1 month of age, when patient
ously. The hfMSCs were obtained from the tissue bank for fetal A underwent osteotomy to reposition the femurs and to replace
transplantation at Karolinska. 40 3 106 hfMSCs (∼30 3 106 hfMSCs her intramedullary rods, opportunistic samples of bone (proximal
per kilogram of fetal weight) at passage 7 suspended in 8 ml of femur), muscle, and skin were taken. Another section of dis-
warmed saline were used for the prenatal transplantation, and carded bone (bone overgrowth, trochanter major, ventral rim
88 3 106 of the same donor’s hfMSCs (10 3 106 MSCs per kilogram) of the proximal plate of the hip) was acquired 9 months after post-
at passage 9 suspended in 15 ml of warmed saline supplemented natal transplantation, at 8 years and 11 months of age, in a proce-
with 500 IU heparin were used for the postnatal transplantation. dure to remove the plates used in the previous surgery.
Samples were fixed in 4% formalin, embedded in paraffin, sec-
Genotyping tioned in 4-mm slices, and dewaxed before preparation for
FISH with a tissue pretreatment kit (MP Biomedicals, Illkirch
The COL1A2 mutation in patient A was determined previously Cedex, France, http://www.mpbio.com). Several sets of FISH
[34]. Analysis of the sequence of the COL1A1 and COL1A2 genes probes were used: CEPH satellite probes for the centromeric
in DNA from patients B and C was performed following amplifica- regions of X and Y (Abbott-Vysis, Abbott Park, IL, http://www.
tion of 16 segments of genomic DNA that encompassed all of the abbottmolecular.com), X/Y Poseidon SE probes (BioNordikaBerg-
coding sequences and flanking intronic domains of COL1A1 and man AS, Oslo, Norway, http://www.bionordika.no), or probes for
23 fragments of the larger COL1A2 gene. Amplification primers the total human genome X (red) and Y (green) (Abbott-Vysis). The
and conditions and sequencing primers and conditions are DNA in the slides and in the probes were simultaneously dena-
available on request to the authors (Collagen Diagnostic Labo- tured in a HYBrite (Abbott-Vysis) at 72°C and hybridized at 37°C
ratory, Seattle, WA, http://www.pathology.washington.edu/ for 16 hours. After washing, the slides were counterstained with
clinical/collagen/). 49,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame,
CA, http://www.vectorlabs.com) to detect cell nuclei and then
Immune Response Assay scanned in a Nikon microscope (Tokyo, Japan, http://www.
Blood samples for mixed lymphocyte cultures were collected nikon.com) and documented with the CytoVision image system
from patients A and B before pre- and postnatal transplantation. (Leica, Heerbrugg, Switzerland, http://www.leica.com), and XY
Briefly, lymphocytes from umbilical cord blood or peripheral and XX cells were counted.

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258 MSC Transplantation for Improvement of OI

Figure 1. Immunological reaction toward donor cells. MLC was performed to evaluate patient A’s and patient B’s immunological reactions
toward donor cells. Overall, 10,000 hfMSCs or 100,000 allogeneic peripheral blood lymphocytes were cocultured with 100,000 patient periph-
eral blood lymphocytes. (A): Patient A before postnatal transplantation, 8 years after prenatal transplantation. (B): Patient B before prenatal
transplantation. (C): Patient B at birth, 7 weeks after prenatal transplantation. In all cases, an immune response was detected against allogeneic
lymphocytes (MLC) but not against donor hfMSCs. Data are reported as mean 6 SD of triplicate experiments. Abbreviations: BG, background;
hfMSC, human fetal mesenchymal stem cell; MLC, mixed lymphocyte culture.

Polymerase Chain Reaction HIV antigen, human T-lymphotropic virus types 1 and 2, hepatitis
Quantitative real-time polymerase chain reaction (PCR) analysis B and C, anaerobic and aerobic bacteria, and mycoplasma and had
of tissues acquired from patient A at 6 years and 1 month of a normal karyotype (data not shown).
age was performed. DNA was extracted from the bone using
the protocol described by Svensson et al. [36] and from the skin, Clinical Course and Experimental Findings: Patient A
muscle, and MSCs isolated from the bone marrow and bone using We describe the initial outcome of the prenatal transplantation
the salting-out method [37]. Chimerism analysis was performed performed for patient A [5], demonstrating evidence of osteo-
using a biallelic genetic system to distinguish the patient from blastic differentiation of donor cells in a bone biopsy at 9 months
the donor [38]. PCR analysis was performed on the ABI 7000 Se- of age. Although the molecular analysis predicted a severe OI phe-
quence Detection System (Applied Biosystems, Foster City, CA, notype based on the nature and location of the mutation in the
http://www.appliedbiosystems.com) using TaqMan technology. COL1A2 gene (c.3008G.A; p.Gly1003Asp; Gly913Asp in the
The sensitivity of the method to detect a minor population is 1 triple-helical domain) (Table 1), she had a better-than-expected
in 10,000. clinical course. Patient A grew along her own growth velocity
curves for weight and height, albeit at 5 standard deviations
(SD) below the mean, and had three fractures but no complica-
RESULTS tions or pain for the first 2 years of life, as previously reported
The details of genetic mutation, type of OI, donor cells trans- [34]. She was treated with intermittent intravenous pamidronate
planted, response, and outcome for the three cases of OI are sum- infusion beginning at 4 months of age for postnatally acquired
marized in Table 1. vertebral compression fractures.
Between the ages of 2 years and 8 years and 2 months, patient
A suffered multiple complications; 9 fractures (5 femoral, 2 clavic-
MSC Characteristics ular, 1 shoulder, and 1 skull) and 11 vertebral compression frac-
Harvested hfMSCs composed a single phenotypic population by tures have been confirmed, and another 4 long-bone fractures
flow cytometry, being positive for CD29, CD44, CD73, CD105, and 1 vertebral compression fracture were suspected clinically.
and CD166; intermediate for human leukocyte antigen (HLA) class She developed scoliosis and was treated with a brace beginning
I antigens; and negative for HLA class II antigens and for the he- at 6 years of age. During surgery at 6 years and 1 month of age
matopoietic and endothelial markers CD14, CD31, CD34, and for rod replacement in her long bones, opportunistic biopsies
CD45 (data not shown). The cells differentiated along the adipo- of the bone, bone marrow, muscle, skin, and isolated MSCs from
genic, chondrogenic, and osteogenic lineages, as described previ- the bone and bone marrow showed no evidence of donor cells by
ously (data not shown) [21]. The cell cultures were negative for either XY FISH or SRY by quantitative PCR (data not shown).

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Götherström, Westgren, Shaw et al. 259

Figure 2. Growth curves for patients A and B. (A): Over the 2 years following postnatal transplantation (arrows), patient A’s linear growth
improved from 26.5 to 26 standard deviations, and she is almost following her own growth velocity centiles. (B): Patient B’s growth followed
a line just under the 3rd centile until 12 months of age, where it plateaued. Her growth resumed at just under the 3rd centile after postnatal
transplantation of same-donor human fetal mesenchymal stem cells (arrow). Length is given in centimeters, weight in kilograms.

A decision to give a second transplant of hfMSCs from the A system. Her ability to walk improved, and she is able to walk for
same donor to patient A was made on the following basis: (a) 1,000 meters without difficulties. She was able to start dance clas-
the limited duration of the beneficial effects of MSC infusion that ses, increased her participation in gymnastics at school, and could
have been shown in the postnatal setting [8], (b) the absence of play modified indoor hockey. Analysis of a bone sample acquired 9
donor cells in the bone at 6 years of age, and (c) the continued months after the postnatal transplantation at 8 years and 11
occurrence of spontaneous fractures and reduced linear growth months of age showed low levels (0.003%) of donor cell engraftment
(25 SD at 2 years of age to 26.5 SD at 8 years of age). A pretrans- by Y-chromosomal FISH (Fig. 3).
plantation workup showed the absence of antibodies directed to-
ward HLA class I and II, IgG and IgM, or FBS (data not shown).
There was no alloreactivity of the patient’s lymphocytes toward Clinical Course and Experimental Findings: Patient B
the donor hfMSCs (Fig. 1A). Five days after bilateral femur osteot- The second patient was a female fetus identified at 26 weeks of ges-
omy and rerodding, 42 3 106 male hfMSCs (2.8 3 106 per kilo- tation in a 31-year-old woman. The fetus’s short long bones (,5th
gram) were infused intravenously without any adverse early or centile) and multiple fresh and healing fractures were identified at
late reactions. the Chang Gung Memorial Hospital in Taiwan. The mother was re-
During the 2 years after the postnatal transplantation, patient A ferred to the National University Hospital in Singapore for an allo-
did not develop any new fractures. Her linear growth followed her geneic prenatal transplantation at 31 weeks of gestation, and
previous growth (from 26.5 SD to 26 SD; Fig. 2A). Dual-energy x- repeat ultrasound confirmed the presence of short long bones
ray absorptiometry (DXA; QDR 4500 system; Hologic, Bedford, MA, and multiple fresh healing and healed fractures (Fig. 4A).
http://www.hologic.com) revealed a lumbar spine (L1–L4) skeletal The fetus was visualized on ultrasound, and the placenta and
mineralization of 0.539 g/cm2 (Z score of 20.3 of age-matched con- intrahepatic vein were mapped. After administering terbutaline
trols before retransplantation at 7 years and 9 months of age), 0.558 to the mother for uterine relaxation, a 22G needle was used to
g/cm2 (Z score of 20.4 of age-matched controls 1 year after retrans- deliver a dose of intrahepatic fetal pancuronium (0.18 mg), fol-
plantation at 9 years of age), and 0.584 g/cm2 (Z score of 20.5 of lowed by cannulation of the intrahepatic vein. Eight hundred
age-matched controls at 10 years of age), using Hologic Discovery microliters of fetal blood was aspirated before 40 3 106 male

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260 MSC Transplantation for Improvement of OI

At 19 months and 11 days of age, patient B underwent a

postnatal intravenous infusion of 88 3 106 same-donor hfMSCs
(10 3 106 hfMSCs per kilogram) without any acute adverse
event. The child was assessed and then discharged from the
hospital the following day. Thereafter, her growth velocity im-
proved, she continued to grow just below the 3rd centile (Fig. 2B),
and she started to walk shortly after the transplantation.

Clinical Course and Experimental Findings: Patient C

Patient C was born at 38 weeks of gestation in 2009 to a healthy
22-year-old woman in Canada. The newborn boy weighed 2155 g
(,5 SD) with a length of 39 cm (,5 SD). Physical examination
Figure 3. Detection of male cells in sections from a bone biopsy revealed the presence of a very large anterior fontanelle, soft
taken after postnatal transplantation. Fluorescence in situ hybridiza- skull, bitemporal narrowing, blue sclera, superiorly narrow chest
tion analysis of 4-mm single sections for X and Y chromosomes using
a-satellite probes for the centromeric regions. The bone sample was with reduced internipple distance (-2 SD), bilateral inguinal her-
taken 9 months after postnatal transplantation at 8 years and 11 nias, bilateral hydroceles, short limbs with notable rhizomelic in-
months of age from patient A. X chromosomes (red dots) and Y chro- volvement, positional abnormalities of the limbs (elbows, hips,
mosomes (green dots) can be recognized in 49,6-diamidino-2-phenyl- knees, and feet were all held in flexion), bowed femurs, adducted
indole-stained cell nuclei (blue). In total, 4 Y-chromosome-positive thumbs, and bilateral talipes equinovarus. Skeletal survey dem-
cells were detected among 60,000 cells. The arrows indicate cells con-
taining Y chromosomes. Original magnification, 3100. onstrated severe and diffuse osteopenia, prominent fontanelles,
multiple Wormian bones, multiple bilateral healing rib fractures
sustained prenatally, and compression fractures of T11, T12, and
hfMSCs in 8 ml of warmed saline were infused over 1 minute L1 with a related thoracolumbar kyphosis. The boy suffered from
under direct ultrasound visualization (∼30 3 106 hfMSCs per multiple fractures of his humeri, femurs, tibias and fibulas, and
kilogram). The fetal heart rate and umbilical artery Doppler left ulna and shortening, widening, and deformity of both humeri,
waveforms were normal before and 5 minutes after transplanta- with all his lower-extremity long bones having the typical accor-
tion. Similarly, cardiographic monitoring before and 1 hour after dion appearance of severe OI.
infusion was uneventful. Testing for alloreactivity toward donor The infant was started on calcium/vitamin D3 and zoledronic
hfMSCs performed on peripheral blood lymphocytes retrieved acid (0.0125 mg/kg) treatment on day 6 of life. At day 9, a DXA
from fetal blood prior to transplantation was negative (Fig. 1B). scan of the lumbar spine at L2–L4 was 0.133 g/cm2, and whole-
Genotyping of the fetus (patient B) identified a mutation in body bone mineral density was 0.450 g/cm2. New fractures
the COL1A2 gene at exon 14 (c.659G.A; p.Gly220Asp, Gly130Asp of the left humerus resulting in a widened, irregular, and
in the triple-helical domain) consistent with OI type IV. Because accordion-like shape secondary to multiple fractures, together
there was a familial history of an affected first cousin with a pre- with fractures of the left radius, left acromion, and right tibia,
sumptive diagnosis of OI and both the patient’s father and pater- and compression fractures of T2, T3, T6, and L2 were seen on se-
nal grandmother had short stature and a history of multiple rial radiology performed on days 25, 27, and 37 of life.
fractures, genotyping of her family pedigree showed the same He received a second dose of zoledronate (0.025 mg/kg) at
mutation in her father, her paternal uncle and first cousin, and 12 weeks of age, and at 16 weeks, lumbar spine bone mineral
her paternal grandmother, consistent with an autosomal domi- density at L2–L4 assessed by a DXA scan had improved to 0.146
nant mode of inheritance (Fig. 5). g/cm2 (an increase of 14%) and whole-body bone mineral den-
After prenatal transplantation, the fetus was followed up by sity had improved to 0.458 g/cm2 (an increase of 1%–2%). The
serial weekly ultrasound at a tertiary maternal-fetal medicine infant was discharged home following this treatment but was
unit, and no new fractures were identified over the ensuing 7 readmitted at 19 weeks of age when he succumbed 1 week later
weeks. The child was delivered at 38 weeks and 3 days of gesta- to respiratory failure secondary to pneumonia. Postmortem ex-
tion by elective cesarean section. She weighed 2.54 kg and was in amination was declined.
good condition. Quantitative PCR of umbilical cord blood, umbil- Genotyping of patient C identified the same dominant muta-
ical cord, and placenta did not yield any Y-chromosome signals tion seen in patient A in COL1A2 (c.3008G.A; p.Gly1003Asp;
(sensitivity of 1 in 500; data not shown). There was no alloreactiv- Gly913Asp in the triple-helical domain), which confirmed the di-
ity of patient B’s umbilical cord blood-derived lymphocytes to- agnosis of severe OI (Table 1). Analysis of the type I collagen pro-
ward the donor cells at the time of birth (Fig. 1C). duced by cultured fibroblasts from a skin biopsy (Collagen
A full-body skeletal survey at birth confirmed the presence of Diagnostic Laboratory) was compatible with the mutation and
multiple healed fractures (Fig. 4B), and the neonate was started demonstrated that two populations of molecules were produced.
on bisphosphonate therapy from 1 month of age. A DXA scan of One population was normal and the other contained markedly
the lumbar spine confirmed poor mineralization, with a Z score of overmodified chains with slow mobility, and the molecules were
–7.3 (0.245 g/cm2) neonatally, which gradually increased to –0.9 poorly secreted (data not shown).
(0.365 g/cm2) at 13 months of age. Over the course of her first 12
months, she grew at just below the 3rd centile for length along her
DISCUSSION
own centile lines, with no new fractures detected. Because her
linear growth stopped just after 12 months of age, a decision Short-term therapeutic effects of MSC transplantation in OI have
was reached to perform a postnatal hfMSC transplantation. been reported previously [39, 40]. We present the long-term

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Götherström, Westgren, Shaw et al. 261

Figure 4. Skeletal examination of patient B. (A): Fetal ultrasound examination at 26 weeks showed the presence of short long bones and mul-
tiple fresh healing and healed fractures (arrows). (B): A full-body radiological skeletal survey at birth confirmed the occurrence of healed frac-
tures, indicated by arrows.

follow-up after prenatal and postnatal transplantation of HLA- hfMSCs in the fetal circulation bypasses the pulmonary vascula-
mismatched hfMSCs in two children with OI. The follow-up period ture through the patent foramen-ovale, leading to enhanced en-
of this study is 3–10 years after prenatal transplantation and 2–2.5 graftment downstream of the arterial tree. This is in contrast to
years after postnatal transplantation. We extensively investigated postnatal infusion, for which pulmonary trapping of the cells fol-
the patients’ responses toward the infused donor cells after prenatal lowed by redistribution to the body has been described [41, 42].
transplantation and before postnatal transplantation. There was no Second, there could still be an immune response toward the in-
evidence of immune reaction toward the donor cells. These data fused cells, although we found no evidence of any adaptive cellu-
suggest that infusion of allogeneic hfMSCs does not cause any acute lar response toward the donor cells. Last, specific cellular
or chronic toxicity, underpinning their safety in a prenatal setting. adhesion molecules present on hfMSCs [43] may regulate more
The cell dose is a critical parameter in cell transplantation be- efficient homing to fetal bones than in the postnatal setting.
cause it may be related to therapeutic efficacy, but a high cell dose In patient A, the postnatal infusion was associated with the
may cause toxicity. In our study, the cell doses infused varied be- lack of new fractures over a 2-year period and improved growth
tween 5–30 3 106 per kilogram at prenatal transplantation and velocity and mobility. In patient B, prenatal infusion was followed
2.8–10 3 106 per kilogram at postnatal transplantation. The cell by the absence of new fractures in the ensuing 7 weeks of fetal
doses were largely based on the number of cells harvested on the and 12 months of postnatal life, and the postnatal infusion was
day of transplantation. The higher cell doses did not cause any ad- associated with resumption of her stalled growth and improved
verse events. However, it is unclear from our data and those from mobility. In addition, the disparate clinical course of patient A
others whether a higher cell dose is more efficacious. In patient A, compared with patient C, who shared an identical genetic muta-
a low engraftment rate of 0.003% in bone was found after post- tion resulting in severe OI, points to at least partial efficacy of the
natal transplantation in contrast to the 7.4% donor engraftment prenatal hfMSC transplantation in ameliorating the severe OI
level seen after prenatal transplantation. There could be a number phenotype. Although both patients A and B received concurrent
of explanations for this observation. First, systemic infusion of bisphosphonate therapy, the improvement after a second

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262 MSC Transplantation for Improvement of OI

Figure 5. Pedigree tree of patient B’s family history of mutation indicating osteogenesis imperfecta. Genotyping of patient B’s family showed
the same mutation in the patient’s father, first uncle, first cousin, and paternal grandmother, suggesting an autosomal dominant mode of in-
heritance. Abbreviations: Ht, height; y.o., years old.

transplantation and the poor outcome of patient C despite lethal OI in their offspring). However, a recent study suggests that
bisphosphonate therapy also suggest that there was a beneficial MSCs do not affect collagen production in OI but merely improve
effect of hfMSC transplantation. We are aware that the genotype the growth velocity via paracrine effects independent of engraft-
alone may not completely predict phenotype and severity of OI. ment [18]. This may account for the transient therapeutic effects
In patient B’s family, there was a heredity pattern consistent seen with MSC transplantation for OI or, indeed, for other condi-
with an autosomal dominant form of OI. Her father, paternal tions. In this sense, it might act as a complementary treatment
cousin, and paternal grandmother were of short stature and had strategy to bisphosphonate therapy because these drugs exhibit in-
experienced multiple fractures during childhood. Although this complete effects on longitudinal bone growth [44, 45].
pointed toward a milder OI phenotype, the decision for prenatal Prenatal transplantation followed by postnatal transplanta-
hfMSC transplantation was made based on the continued evolu- tion appears to be both efficacious and safe in the context of
tion of new fractures during antenatal surveillance and the persis- OI. The lack of long-term engraftment and transient clinical im-
tent shortness of long bones. These factors were balanced against provement after transplantation suggests that further refine-
the relative safety of a prenatal transplantation in a hospital that ments in selection of the appropriate stem cell type and
routinely carries out similar interventions. Although we did not ob- preparation will be required for sustained therapeutic benefits.
tain any bone biopsies from patient B to confirm donor cell engraft- Presently, a multiple-transplantation strategy may be required
ment, the lack of new prenatal and postnatal fractures together to optimize skeletal growth and development during fetal life
with the resumption of growth velocity argues for a beneficial ef- and childhood for maximal therapeutic benefit.
fect of hfMSC transplantation on skeletal development. Finally, fetal stem cell transplantation for OI is still a highly ex-
We previously reported that prenatal hfMSC transplantation perimental therapy. From experience in other fields of stem cell
resulted in donor cell engraftment and site-specific differentia- therapy, we are aware of the importance of coordination and co-
tion in bone at 9 months of age [34]. Extensive analysis of tissue herence between different centers interested in advancing this
samples from the same patient (patient A) showed no donor cell development. We advocate different research groups in this area
engraftment at 6 years of age, but at 9 months after postnatal joining forces to develop common programs that include guide-
transplantation, donor cells were detected in a bone sample. It lines for isolation of MSCs, transplantation strategies, and uni-
is likely that the low level engraftment seen was from the post- form measurements during the post-transplantation period.
natal transplantation, although it would be impossible to verify Considering the complexity of this field, we see this approach
this, given the lack of additional marking of same-donor cells. as the only realistic way to proceed if we are to accurately eval-
The placental tissues from patient B showed no signs of donor cell uate the potential of this treatment strategy in children with OI.
engraftment, but because donor cells have been detected only in
the bone of OI patients, we would not necessarily expect engraft-
CONCLUSION
ment of donor cells in other tissues.
Clinically, the level of engraftment observed in this study has Prenatal and postnatal transplantation of allogeneic hfMSCs in OI
been low, between 7.4% and 0.003% in the bone after prenatal is safe and probably clinically efficacious. To date, however, there
and postnatal transplantation, respectively. However, low-level en- is only limited clinical experience, and further studies are needed.
graftment of 1%–2% has been shown clinically to improve severe OI
in both animal models and in patients [8, 31, 33]. The underlying
ACKNOWLEDGMENTS
understanding is that even low engraftment levels could produce
mosaicism that would allow local production of normal bone and We acknowledge Dr. Anders Götherström for work on bone
potentially convert a severe OI phenotype to one less severe (as DNA extraction, Monika Jansson for fluorescence in situ hybrid-
recognized in parents who are mosaic for a mutation that produces ization, Dr. Mehmet Uzunel for polymerase chain reaction,

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Götherström, Westgren, Shaw et al. 263

Dr. Mark Chong and Lay Geok Tan for cell culture and expansion, diagnosis and antenatal care and postnatal follow-up (patient
Dr. Sherry Ho for polymerase chain reaction and karyotyping, B), final approval of manuscript; E.A.: provision of study material
and Dr. James H.P. Hui for current good manufacturing practice (patient A), postnatal follow-up, collection and/or assembly of
laboratory cell cultures. This study was financed by the Swedish data, data analysis and interpretation, manuscript writing, final
Society for Medical Research and Vinnova (2010-00501). J.K.Y.C. approval of manuscript; P.H.B. and G.E.G.: provision of study ma-
and M.C. received salary support from the Ministry of Health’s terial or patients, collection and/or assembly of data, data anal-
National Medical Research Council’s Clinician Scientist Awards ysis and interpretation, manuscript writing, final approval of
(CSA/009/2012, CSA/012/2009, and CSA/043/2012). manuscript; J.T. and U.E.: provision of study material (patient
A), postnatal follow-up, final approval of manuscript; N.M.F.: data
AUTHOR CONTRIBUTIONS analysis and interpretation, manuscript writing, final approval of
manuscript; A.B. and C.N.Z.M.: prenatal transplantation (patient
C.G. and J.K.Y.C.: conception and design, financial support, provi-
B), final approval of manuscript; A.E.J.Y.: postnatal transplanta-
sion of study material or patients, collection and/or assembly of
tion (patient B), final approval of manuscript.
data, data analysis and interpretation, manuscript writing, final
approval of manuscript; M.W., M.C., and K.L.B.: conception and
design, financial support, provision of study material or patients,
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
data analysis and interpretation, manuscript writing, final ap-
proval of manuscript; S.W.S.S., P.-J.C., and J.-L.L.: prenatal The authors indicate no potential conflicts of interest.

engraftment and no ectopic tissue formation. dystrophic mdx mouse. STEM CELLS 2007;25:
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