Infoscience

Student project

Characterization of the paracrine stimulation potential of human CAP cells for cardiac repair

According to the last WHO numbers, cardiac diseases remain the first cause of death in the world. Cell therapy represents a promising approach to restore heart function. Cardiac derived adherent proliferation (CAP) cells were shown to support the heart regeneration by unexplained mechanisms when injected in a mouse myocarditis model. Not differentiating themselves in cardiomyocytes and expressing cellular markers for angiogenesis, they are now supposed to have a paracrine effect on heart resident cell populations. In the present work, this paracrine effect had to be characterized on a model cell line. A paracrine effect test system based the P19Cl6 teratocarcinoma cell line had to be established and used to carry out a first screening of paracrine interactions on CAP cells and mesenchymal stem cells (MSC) from bone marrow as reference. Initially, critical parameter of the test system had to be adjusted. Specific marker must be identified and their detection had to be proven possible in the designed test system. Suitable serum lots (k,g) have been identified on the base of growth rates and cardiac differentiation potential (beating activity, Gata-4, Myl2, Mef2c gene expression) using standard protocol (ɑMEM, 1% DMSO). Pretreatment of P19Cl6 with 10 [micro]M 5-azacytidine substantially raised the response to known potent cardiomyogenic inductors (DMSO, Oxytocin). Beating activity was remarkedly elevated, as well as Gata4 (5 to 300 fold), Myl2 (2 to 300 fold), and MEF2C (3 to 4 fold) expression induced compared to non-treated culture. Troponin I ELISA-(2 to 5 fold) and Connexin 43 imunnohistochemistry confirmed the reliable induction of cardiac differentiation in pretreated culture. Based on those results, the test system was conducted using 5-azacytidine pretreated P19Cl6 cells and explored their reactions to different direct (cell to cell in coculture) and indirect (secreted factors in preconditioned medium) interactions with CAP cells or MSC. Since no reference setup existed, several coculture compositions with fixed total cell number (10-90%) or fixed P19Cl6 cell number (10-1000%) were tested, as well as exposition to differently conditioned media (24h, 72h) in different dilutions (100%, 50%, 33%). Beating activity was almost never observed. However, CAP cell experiments (n=6) showed a significant increase in P19Cl6 GATA-4 expression compared to control P19Cl6 (3.3 fold). This expression was also increased in MSC analyses (n=4) but in small amount (1.8 fold). Screening of CAP and MSC interactions led to the identification of active/effective compositions and dilutions. In addition, aza pretreatment proved itself enhancing the sensitivity of the test system. To sum up, the results suggested that CAP cells as wells as MSC have a paracrine stimulation potential and gave starting points for deeper analyses.

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