Dear Editor,

The study of Shenje et al, recently published in your Journal (April 2008, Vol 3 (4) e1929), aims essentially to demonstrate the un-adequacy of mice adult cardiac explants derived cells to undergo to differentiate in vivo, with an arbitrary jumping to the suggestion of considering caution about using cells from this source (cardiac explants) in human clinical trial.
We understand that the research in this field is highly competitive and frequently science is considered as second or third order criteria respect to economic implications.
However, criticisms directed to a scientific study are un-acceptable if they are referred to different experimental conditions. In particular, if to verify the goodness of a protocol, changes are introduced, these have to be carefully justified and explained. In absence of these clarifications, the risk is that the criticisms are referred to something different from the original study (as highlighted by reviewer 3).

Some of the results coming from the GFP analysis, need to be discussed. Figures 5d-f show the presence of GFP (which is real and not autofluorescence because of the immunoreactivity). The authors claim that GFP fluorescence is phagocitosis-dependent, however the PCR conditions are at least confounding. In fact:

1. ZEG amplification is over saturated, thus the DNA amount in the different lanes is unknown. On the other hand, the GFP amplification is poor. In particularly, the sample coming from the fresh explant shows an over ZEG-saturation associated to a low GFP signal: that is strange, taking into account that, in this sample, most of the cells should have their GFP in the active form. At this point it is not surprising that cultured cells show poor if any amplification. With GFP amplification’s efficiency so low, it’s at least fantascientific that the cells in culture should give rise to a much lower, if any, amplification.
How many are the fluorescent foci in one dish, considering that they are about 40 in total from one heart? With a so lower amplification there are several doubts that it should be possible to obtain the GFP amplification from one or two fluorescent foci, in the presence of a high number of other cells. In other words, A PCR analysis of all the fluorescent foci pool should be performed.

2. GFP positivity is strong. Even if we consider that’s due to phagocitosis, it’s at least surprising that its expression persists in those cells (that should be a clone) for several days or weeks after the presumed engulfment, unless these cells are actually macrophages in the first days of the explant’s life. If that is the case, they could have phagocitised some GFP but they should absolutely correspond to the foci obtained with the published procedure, even several weeks after the initial seeding of the explants.

We strongly believe that, before claiming that a procedure is inappropriate, the correct scientific method should be applied, otherwise there is only the risk to consistently slowing an advancement in cardiac regenerative biotechnology.

The possibility to clinically translate the ex vivo isolation and expansion of progenitor cells from cardiac biopsy (that is cells with a spontaneous cardiac commitment), should really open a breakthrough in the cardiac regenerative therapy