Abstract 166: Soluble Factors Released By Neonatal Cardiomyocytes Up Regulate C-kit Expression In Adipose Tissue Derived Stem Cells.
It has been shown previously that c-kit+ cells are increased in the heart after infarction. The recruited c-kit+ cells established a proangiogenic milieu in the infarct border zone by increasing VEGF and by reversing the cardiac ratio of angiopoietin-1 to angiopoietin-2. These cells were traced back to a bone marrow origin. We asked whether adipose tissue could also be a potential source of c-kit+ cells and whether c-kit expression can be regulated by soluble factors released by neonatal cardiomyocytes grown under hypoxic condition. Human adipose tissue derived stem cells (ADSC) were isolated and cultured in alpha MEM containing 20% FBS. The ADSCs are plastic adherent mesenchymal stem cells which are positive for CD105, CD44, CD90 and negative for CD34, CD45, CD11b. Cardiomyocytes were isolated from neonatal rat hearts by collagenase treatment using the Neonatal Cardiomyocyte Isolation System (Worthington). The cultured neonatal cardiomyocytes were subjected to serum deprivation and hypoxia for 48 hours and the conditioned medium were collected and concentrated 10 times. Adherent passage 3 human ADSCs were cultured with the conditioned medium (diluted 10x with fresh medium containing 20% FBS) for 72 hours. Controls were incubated with conditioned medium from cardiomyocytes grown under normal condition. We used flow cytometry to determine the percentage of c-kit+ cells in ADSCs. Our data showed that ADSCs cultured with conditioned medium from normal neonatal cardiomyocytes expressed only 1.46±0.49% of c-kit+ cells whereas ADSCs cultured with conditioned medium from neonatal cardiomyocytes grown under serum deprivation and hypoxia condition expressed 4.55±0.61% of c-kit+ cells. The increased c-kit expression was blocked by VEGF receptor Flk-1 antibody (1.81±0.84%) and by MAP inhibitor PD98059 (2.39±0.17%) but not JNK inhibitor II (4.47±0.47%). In conclusion, adipose tissue derived stem cells contain a small percentage of c-kit+ cells which can be up regulated by soluble factors from the conditioned medium of neonatal cardiomyocytes grown in serum free and hypoxia conditions. Our results suggest that VEGF may be involved in c-kit up regulation through ERK-mediated Flk-1 activation.