Progenitor Cells Derived From Traumatically Injured Muscle Tissue Exhibit Limited Osteogenic Differentiation Potential

Wesley M. Jackson1, Amber B. Aragon1,2, Steven M. Koehler1, Jeffrey R. Giuliani1,2, Leon J. Nesti1,2, Rocky S. Tuan1
1Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, Bethesda, MD; 2Department
of Orthopaedics and Rehabilitation, Walter Reed Army Medical Center, Washington, DC
jacksonw2@mail.nih.gov

Introduction: Skeletal muscle contains progenitor cells that participate in

wound healing following orthopaedic injuries [1]. Dysregulation of these cells may
be sufficient to initiate pathologies such as heterotopic ossification (HO) [2],
which is characterized by the formation of mature bone in soft tissues and is a fre-
quent complication of orthopaedic trauma [3]. We have recently developed a
method of harvesting progenitor cells from injured muscle that demonstrate histo-
logical evidence of differentiation into multiple lineages [4]. Better characteriza-
tion of these progenitor cells may help elucidate their role in tissue regeneration
and disease. The objective of this study was to compare the osteogenic potential of
progenitor cells derived from traumatically injured muscle to bone marrow-derived
mesenchymal stem cells (MSCs). Our specific aims were (1) to determine whether
the phenotype of the progenitor cells was consistent with MSCs, (2) to verify the
differentiation potential of the muscle-derived progenitor cells, and (3) to compare
the osteogenic gene expression of muscle-derived progenitor cells to bone marrow-
derived MSCs.
Materials and Methods: Muscle debridements were obtained at WRAMC
from patients with time-of-war orthopaedic trauma. The muscle tissue was digest-
ed with collagenase, and the progenitor cells were isolated based on their adhesion
characteristics with tissue culture plastic (TCP). MSCs were aspirated with bone
marrow from femoral heads and isolated by differential plating on TCP. At each
passage, cells were stained with antibodies conjugated with phytoerythrin (PE) and
raised against cell surface antigens that are characteristic of hematopoietic stem
cells (HSCs: CD14, CD34 and CD45) and MSCs (CD44, CD49e, CD73, CD90 Surface epitope profile of muscle-derived progenitor cells (MPCs). The fluorescence intensity
and CD105) [5], their surface epitope profile was analyzed by flow cytometry, and of the positive markers was normalized to that of CD73. The CD90/CD73 ratios were
the expression levels of the positive markers were compared by normalizing the flu- reduced by a factor of 10.
orescence intensities to that of CD73. During the third passage, the cells were cul-
tured under induction conditions for osteogenesis, adipogenesis and chondrogene-
sis. After 21 days, the cells were either stained for markers of differentiation (osteo-
genesis: alkaline phosphatase, ALP, and alizarin red; adipogenesis: oil red O; chon-
drogenesis: alcian blue) or lysed to extract the RNA. Differentiation was analyzed
based on the expression of lineage dependent genes using RT-PCR (osteogenesis:
Runx2; adipogenesis: PPARγ2; chondrogenesis: Sox9). Osteogenic potential was
determined by quantifying the expression of Runx2, collagen type 1 (Col1), ALP
and osteocalcin (OC) using real-time RT-PCR.
Results: The progenitor cells were positive for surface markers characteristic of
MSCs, and negative for markers characteristic of HSCs (Fig 1). The ratio of
CD105/CD73 was significantly greater (p=0.01) for the muscle-derived progeni- Expression of osteogenic genes normalized to the expression under expansion conditions.
tor cells during the first passage than MSCs (one-way ANOVA with multiple Discussion: Based on their phenotype, the progenitor cells derived from
comparisons, n=36), although the expression of CD105 appeared to decrease with injured muscle may have originated from a population of MSCs. However, the
subsequent population doublings. There was no significant difference in the levels expression levels of CD105 at early time-points were statistically different than
of all other markers normalized to CD73 between the progenitor cells at any pas- that of bone-marrow derived MSCs. The differences in phenotype and osteogenic
sage and MSCs. After 21 days under differentiation conditions, histological stain- gene expression may be attributed the tissue of origin or a result of the wound heal-
ing and expression of lineage dependent genes indicated that the muscle-derived ing and inflammation responses in the traumatized muscle. The enhanced expres-
progenitor cells had differentiated into osteoblasts, adipocytes and chondrocytes. sion of CD105 (endoglin) on the muscle-derived progenitor cells will be of partic-
There was no significant difference in the up-regulation of Runx2, Col1 or ALP ular interest in future studies, since it regulates the sensitivity of TGF-β receptors
in muscle-derived progenitor cells under osteogenic induction compared to MSCs. [6], and increased expression levels of CD105 may alter the process of osteogene-
However, the expression level of osteocalcin (OC) was significantly depressed sis. These results suggest that the progenitor cells in traumatic muscle may play a
(p<0.001) compared to MSCs (t-test, n=6; Fig 2). role in wound healing and disease processes involving cells from mesenchymal lin-
eages. However, the osteogenic differentiation of these cells appears limited com-
pared to bone-marrow derived MSCs, indicating that other factors may be neces-
sary for the formation of HO following traumatic orthopaedic injury.
References: 1. Peault et al. (2007) Mol Therapy 15:867; 2. Kaplan et al. (2004)
JAAOS 12:116; 3.Potter et al. (2007) J Bone Joint Surg Am 89:476; 4. Nesti et al.
(2007) ORS Abstracts #463; 5. Chamberlain et al. (2007) Stem Cells [Epub ahead of
print]; 6. Duff et al. (2003) FASEB J 22:6557.
Acknowledgements: Supported by a grant from the Military Amputee
Research Program at WRAMC (PO5-A011) and by the Intramural Research
Program at the NIH, NIAMS (Z01 AR41131).

Poster No. 828 • 54th Annual Meeting of the Orthopaedic Research Society