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Induced Pluripotent Stem Cells: Proteomics Test

Reading time: 4 – 6 minutes

Stem cells have the ability to continue dividing and renewing for long periods of up to a year or more and transform from their unspecialised structures into specialised cell types such as heart muscle cells or nerve cells. It was news of these properties that created such as buzz in recent years, with the possibility of generating new tissue to replace damaged or unhealthy tissue in the human body.

The initial moral objections to using human embryonic stem cells for experimentation have been expelled by more recent developments that allow stem cells to be produced from human skin fibroblasts, which are the main cells in connective tissue. These so-called induced pluripotent stem cells (iPSCs) also have the ability to change into other cell types and they have allowed research into stem cells to continue unhindered.

Now, iPSCs can be derived via a number of routes but, in all cases, the process is time-consuming and relatively inefficient. As a result, scientists must be able to distinguish between iPSCs and the non-pluripotent stem cells that are also present. This is not a trivial task. One of the more popular approaches, which has better reliability than others, takes about one week and requires the isolation of genetic material.

However, this could be about to change with a new identification procedure developed by scientists in Europe that could cut the analysis time to just a few minutes. This major advance overlooks the genetic approach and takes advantage of proteomics, using a targeted assay based on several core transcription factors which are present in the iPSCs but not the non-pluripotent cells. The main functions of these transcription factors in the cells is to transcribe DNA into RNA.

Peptide biomarker panel

In the new method, a number of different programmed cell lines were used to generate iPSCs. The harvested cells were mixed with stable-isotope-labelled peptides then treated with trypsin. This process broke up the various proteins present into their component peptides, some of which were unique to their parent proteins and can be used to identify them. By monitoring and measuring the abundances of these biomarker peptides, the pluripotency potential of a particular batch was assessed.

A panel of transcription factors was selected based on those that the researchers thought had potential, plus others that were used in the commercial PluriTest kit which is based on genetic testing. Their peptides were analysed by LC/MS with multiple reaction monitoring in experiments which required a short, seven-minute HPLC gradient.

From the chromatograms that were obtained, four proteins were found to be powerful biomarkers for pluripotency. They were POU domain transcription factor, LIN28 homologue A, podocalyxin and transcription factor SOX-2. They were supplemented by a negative marker, the glycoprotein CD44, which is present in fibroblasts but absent in pluripotent cells.

A principal components analysis showed that the markers could clearly distinguish between the fibroblasts and the rest of the cells. In addition, the transition from undifferentiated cells to embryoid bodies after 14 days was clearly demonstrated.

The other potential proteins which were included in the original panel were not detected in the samples, even though they were regarded as strong candidates. Their absence remained unexplained but the four transcription factors and the negative marker provide a strong group for distinguishing fibroblasts from iPSCs.

Rapid and inexpensive test

The same samples from the mass spectrometric analysis were also analysed using the PluriTest kit and a second genetics-based kit called the ScoreCard assay. Although comparisons between tests based on proteomics and genetics are notoriously challenging due to the poor correlation of proteins and mRNA, a high degree of correlation was observed between the tests. The accuracy of the mass spectrometric results was demonstrated by blind testing, which correctly classified iPSCs and fibroblasts in seven samples.

The principal advantage of the new technique is speed. The enzymatic digestion of the proteins and the subsequent purification can be carried out automatically in 96-well plates and the whole procedure can be completed within one day. The actual LC/MS run takes just seven minutes. Both of the genetic tests used here take about one week.

A second key advantage is cost. If a suitable triple quadrupole mass spectrometer is already installed in a lab, the team estimated the running cost per sample to be less than $20. Compare this to the average costs of $150 and $175 for the PluriTest and ScoreCard, respectively. The mass spectrometric test has the potential to evolve into a diagnostic test.

Source: Spectroscopy NOW