Laboratory: Stem Cell Biology

PREVIOUS PROJECTS

Oxidative stress-induced apoptosis of endothelial cells (Burlacu A. et al., Cell and Tissue Research, 2001)
In vitro differentiation of adult bone marrow stem cells into cardiomyocytes (Burlacu A. et al., Eur J Cell Biol, 2008

CURRENT PROJECTS

Experimental procedure to achieve different grades of myocardial infarct in mice

Goal: to set up a method for inducing myocardial infarction in mouse by ligation of left coronary artery (LCA).
The results revealed that LCA ligation induced significant ST height elevation (204 vs. 14 μV) and QTc prolongation (136 vs. 76 ms).
Both parameters were rapidly normalized on reperfusion as demonstrated by ECG, which validated the proper ligation and reperfusion (Fig. 1). A direct correlation between the extent of ischemia and the infarct size has also been established (Fig. 2).
The model will be employed to study the mechanisms involved in the progression of heart failure, including the fibrous scarring and cardiomyocyte loss, which are imperative for the development of new therapies

Angiogenic properties of human endothelial progenitor cells (EPC) under hypoxic conditions

Aim: to evaluate the behaviour of EPCs in the ischemic environment faced after transplantation into the infarcted myocardium.

Thus far, the results showed that hypoxia did not significantly modified the viability of human EPCs and induced an increased VEGF secreted level. On contrary, secreted level of SDF-1 decreased under hypoxic conditions; however, in comparison to mature endothelial cells, hypoxic EPCs still secreted larger amounts of SDF. The angiogenic properties of EPC were similar when cells were grown under normoxic or hypoxic conditions (Fig. 3). Therefore, these conditions will be retained for further use to evaluate the potential of EPCs to promote angiogenesis in interaction with the infarcted myocardium.

Paracrine effects of stem cell-secreted factors on ischemic myocardium

Goal: to evaluate the potential of factors secreted by stem cells to promote tissue repair by sustaining endothelial cell (EC) adhesion and proliferation and conferring protection against apoptosis.
The results obtained thus far showed that factors secreted by mesenchymal stem cells conditioned medium (MSC-CM) supported adhesion, but not proliferation, of ECs in vitro.
On the contrary, factors secreted by endothelial progenitor cells (EPC) had complementary effects to MSC-CM. Thus, EPC-CM supported EC proliferation but did not sustain EC adhesion.
Combining the MSC-CM and EPC-CM promoted both EC adhesion and proliferation (Fig. 4), suggesting that factors secreted by multiple stem/progenitor cell populations can better stimulate cardiac regeneration after myocardial infarction.

The effect of 5-azacytidine: evidence for alteration of the multipotent ability of mesenchymal stem cells

Aim: to evaluate the stemness potential of mesenchymal stem cells (MSC) after the treatment with the demethylation agent 5-azacytidine, which is a general inducer of cardiac differentiation of stem cells.
Our results showed that MSC retained their multipotent capacity after one pulse with 5-azacytidine, whereas additional pulses restricted the differentiation potential with concomitant increased ability to accomplish chondrogenic commitment, only (Fig. 5).

In the light of our findings, it is likely that a unique exposure of MSC to 5-azacytidine prior to their transplantation into the injured myocardium is helpful because the cells would be still multipotent at the time of transplantation, but in the same time more amenable by the subsequent differentiation stimuli. Following this priming step, the cell differentiation process could be completed in vivo by the factors within the cardiac environment. These aspects remain to be experimentally confirmed.

Isolation and cardiac differentiation of mouse embryonic stem cells

Aim: to derive the embryonic stem (ES) cell lines from RAP mice and evaluate the impact of size and aggregation conditions of embryoid bodies (EBs) on the efficiency of ES cell differentiation into CMC.
The results showed that the newly derived ES cells expressed pluripotency markers and induced teratomas in vivo (Fig. 6). We have also found that EBs produced by aggregating ES cells generated contractile tissue formation in a direct correlation with the initial number of cells. The presence of knock-out serum replacement (KO-SR) during ES cell aggregation resulted in less compacted EBs than those produced in fetal bovine serum (FBS) and augmented ES cell differentiation into CMC (Fig. 7).
We concluded that cardiac differentiation of ES cells is dependent on the size and compaction degree of EBs and the presence of KO-SR during EB initiation may lead to improved cardiogenic differentiation of ES cells.

Apoptosis in ischemic and post-ischemic cardiomyocytes (CMC)

Goal: to determine the main inducer of CMC apoptosis in ischemia-reperfusion setting. To this aim, CMC were exposed to either 30-minute ischemia followed by reperfusion or to 25-hydroxycholesterol (HC).
The results showed that both ischemia-reperfusion and exogenous oxidants increased the Bax/Bcl-2 ratio, a favourable event for the apoptotic process. Apoptosis was not observed in ischemic CMC in the absence of reperfusion.

On the contrary, HC promoted CMC apoptosis by Caspase-3-dependent mechanism that involved the transcriptional activation of the pro-apoptotic protein Bax and post-translational degradation of anti-apoptotic Bcl-2 protein (Fig. 8).
Based on these results, we concluded that CMC apoptosis is not induced by ischemia per se, but rather by the oxidants from the surrounding microenvironment at the time of reperfusion.