Phenotypic and Functional Characterization of Smooth Muscle-Like Cells Derived from Multipotent Adult Progenitor Cells (MAPCs).

Smooth muscle cells (SMC) play a key role in vascular development and morphogenesis of blood vessels defined by a noncontractile, or synthetic SMC phenotype. Mature SMCs respond to vascular injury/repair by phenotypic modulation to a synthetic state, and hence are also involved in various clinical disorders including atherosclerosis and cancer. We have induced relatively homogenous differentiation of SMC-like cells from multipotent adult progenitor cells (MAPC).

MAPCs have been isolated from murine, rat and human bone marrow. MAPCs can differentiate into mesodermal, neuroectodermal, and endodermal cell types. Here we report that serum free medium supplemented with PDGF-BB and TGF-b1 allows differentiation of rat MAPCs to a population of cells expressing smooth muscle specific markers, as determined by Q-RT-PCR (a-SMC actin, calponin, SM-22a, myocardin, and smooth muscle heavy chain myosin),and immunohistochemistry (a-SMC actin staining in >90% cells and calponin staining in ~50% of cells, co-localization localized with defined stress fibers). Such SMCs can subsequently be passed for at least 15 passages (26 doublings) in the presence of FCS and PDGF-BB and TGF-b1, without loss of phenotypic characteristics. RT-PCR analysis for genes of other mesodermal lineages, including cardiac muscle and endothelium expressed genes (Nkx, Mef-2D, PCAM, and vWF) was negative.

To characterize the function of the SMC-like cell population, calcium handling and in-vitro remodeling properties were studied. Passage 4 SMC-like cells responded to endothelin-1, norepinepherine, oxytocin, arg-vasopressin, and bradykinin with an increase in intracellular calcium ion concentration, consistent with a smooth muscle phenotype. To assess the remodeling potential, passage 4 cells were entrapped into fibrin matrix hemispheres as previously described for neonatal rat SMC. After 5 weeks in culture, hydroxyproline assays quantified total collagen as (86.8 +/− 9.3 ug) approximately 50% the amount reported using neonatal SMCs. In addition, the remodeled hemispheres demonstrated defined mechanical properties (tensile strength= 0.20 MPa, modulus = 0.65 MPa), similar to what we have previously observed when neonatal rat SMCs are entrapped in fibrin disks. Masson Trichrome staining demonstrates areas of tissue organization characterized by fibrils and collagen production. While further characterization of this population is needed to determine their developmental plasticity, the data is suggestive of a vascular SMC-like cell population derived from MAPCs that demonstrates functional smooth muscle characteristics and could be used to tissue engineer blood vessels.