Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived From Human Embryonic Stem Cells.

Abstract 1451

Poster Board I-474

Mesenchymal stem cells (MSC, aka marrow stromal cells) present a promising tool for cell therapy, and have been shown to contribute to the recovery of tissues in myocardial infarction, stroke, meniscus injury, limb ischemia, and even neurodegenerative disorders. The percentage of engrafted MSC in these studies has been very low in comparison to the recipient tissue cells, suggesting that their efficacy relies upon actions other than differentiation. One theory of tissue repair and regeneration by adult MSC is that the injected stem cells home to the injured area, in particular to hypoxic, apoptotic, or inflamed areas, and release trophic factors that hasten endogenous repair. These secreted bioactive products can suppress the local immune system, enhance angiogenesis, inhibit fibrosis and apoptosis, and stimulate recruitment, retention, proliferation and differentiation of tissue-residing stem cells. Paracrine effects exerted by MSC are distinct from the classical model of direct differentiation of stem cells into the tissue to be regenerated. MSC can, however, directly contribute to the repair of bone, tendon and cartilage. In some cases, where the patient lacks expression of a critical gene product, genetic engineering of the MSC is desired. Using human embryonic stem cells (hESC), the integration site of a vector can be fully characterized and the clones with benign integration sites can be expanded. Homologous recombination is also now feasible for embryonic cells due to increased efficiencies, and clones with vectors targeted for gene correction can be expanded. For this reason we have generated MSC from the well-characterized human embryonic stem cell line H9. H9-derived MSC (H9-MSC) expressed CD105, CD90, CD73 and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR. H9-MSC also lacked expression of the hESC pluripotency markers SSEA-4 and Tra-1-81, which were expressed by the starting H9 line. Additionally, they lacked expression of SSEA-1, an early marker of hESC differentiation. Marrow-derived MSC showed a similar phenotype when analyzed concurrently with H9-MSC. Morphology was similar to adult MSC derived from marrow or adipose tissue. H9-MSC grew more robustly than MSC derived from marrow or adipose tissue, with a rapid doubling time and a failure to display contact inhibition. However, upon reaching maturity the cells did slow to the same rate as bone marrow-derived MSC, and then were finally subject to contact inhibition. Additionally, H9-MSC were injected with matrigel subcutaneously into the flank of immune deficient mice [NOD/SCID/IL2Rg-/- (NSG)] to assess their ability to form tumors due to possible growth abnormalities. No mice that received injections of H9-MSC formed teratoma or other tumors, whereas the parent H9 line robustly forms teratoma. H9-MSC could be robustly differentiated into bone, as shown by alizarin red staining of mineralized plaques after 21 days of culture in standard osteogenic differentiation medium containing 0.2 mM ascorbic acid, 0.1 m M dexamethasone, and 10 mM b-glycerophosphate with media changes every three days. H9-MSC morphology was noticeably different from that of H9-MSC undergoing differentiation as early as day 3 of the differentiation protocol. Finally, cells were tested for their capacity to respond to a hypoxic in vivo environment, using our standard hindlimb ischemia model in immune deficient [NOD/SCID/b2M-/-] mice. H9-MSC were found to have homed to the hypoxic muscle by 48 hours after injection into the bloodstream. In summary we have defined methods for differentiation of hESC into MSC, have defined their characteristics, and in vivo migratory properties. This system could be useful, following further safety studies, for production of large numbers of MSC from embryonic or induced pluripotent stem cells that have been corrected for gene defects by lentiviral vector integration with careful assessment of integration site, or by homologous integration, with subsequent expansion, characterization and banking of the line prior to differentiation into functional mesenchymal stem cells.