Development of quantitative PCR methods to analyse neural progenitor cell culture state

Elsa Abranches, Analeah O’Neill, Matthew J. Robertson, David V. Schaffer, and Joaquim M. S. Cabral

Biotechnol. Appl. Biochem. (2006) 44, 1-8

Stem cells have great potential for use in tissue engineering since they are able to differentiate into various specialized types of cells. But in order for them to be helpful we must be able to grow them up immature (undifferentiated) and then control their differentiation into the specific cell lineage needed for the application of interest. Being able to quantify the cell differentiation in a cell culture is therefore very important.

There are a few existing methods for quantifying cell differentiation. One of these is immunostaining in which slides of cells are stained and the number of cells of each phenotype is counted manually. This technique is very laborious and time consuming and cannot be readily automated because of complex images due to elaborate cell morphologies and surface markers. Accurate quantification is also difficult using this method especially if the cells are very dense or have extensive cellular processes. In this paper a quantitative reverse transcription polymerase chain reaction (qRT-PCR) method is developed to improve quantification of cell differentiation. This faster method accurately quantifies mRNA to monitor expression of markers that are specific to different cell types. The paper also discusses how this method was applied to a study on how conditions of cell culture effect the cell differentiation.

So what is qRT-PCR? Let’s start with RT-PCR…

Reverse transcription polymerase chain reaction (RT-PCR) is a very useful technique for measuring gene expression. It works by amplifying a specific sequence of RNA even if it has a low copy number. This allows the investigator to see what is going on in the cell. For example, if you insert a plasmid encoding a protein into a strain of bacteria and you want to find out if the bacteria are actually producing the protein then you can use RT-PCR to see if the mRNA for your protein is present in the cell.

So how does RT-PCR work? Your mRNA is first copied to cDNA via reverse transcriptase. The temperature is then raised so that the two strands of the cDNA separate. Then the temperature is lowered to allow specific primers to anneal to the single stranded DNA. Again the temperature is raised (though not as high as before) and Taq DNA polymerase extends the DNA from the primers. Then the temperature is further increased to separate the DNA. At this point there are four strands of cDNA (from the two we started with). And the process continues to repeat itself a number of times (30-40 cycles) increasing the number of strands of DNA each time. The products can then be analyzed using gel electrophoresis.

RT-PCR is useful for detecting the presence of a specific RNA molecule but it is not good for quantifying the abundance of the molecule. There is a newer method that allows for quantification in addition to amplification which is called quantitative RT-PCR or qRT-PCR (also known as real time PCR). As we saw above, in each cycle the amount of DNA doubles, but at some point during the 30-40 cycles a plateau is reached where the amount of DNA is no longer doubling with each cycle. With RT-PCR you don’t know when this plateau was reached so you don’t know when the DNA stopped doubling with each cycle and therefore don’t know how much of your RNA of interest was in your original sample.

In qRT-PCR a dye (in this case SYBR Green) is used that has very high fluorescence when it binds with double stranded DNA and low fluorescence with single stranded. This allows you to track the growth of the amount of DNA. Comparison of the amount of fluorescence of your sample at a certain cycle number to that of a control containing a known initial quantity of RNA allows you to quantify the amount of RNA in your original sample.

In this paper a method of qRT-PCR is developed for use in quantifying the cell differentiation of neural progenitor cells. Under the proper conditions, neural stem cells are able to undergo extended proliferation while remaining undifferentiated or they may be differentiated into the three major neural lineages becoming astrocytes, oligodendrocytes, or neurons. It is believed that there are also shorter-term neural precursor cells called neural progenitor cells present in the central nervous system in addition to the neural stem cells. In this paper cell differentiation of neural progenitor cells from the hippocampus of adult female rats is investigated under proliferation and differentiation conditions.

Neural progenitors were cultured for 2 weeks in serum with retinoic acid which promotes neuronal differentiation (differentiation conditions). The cultures had markers for astrocytes, oligodendrocytes, and neurons. Over the 2 weeks the total RNA was isolated from samples of the cell cultures. qRT-PCR was used to measure the levels of gene expression markers in these samples. On day 0 (proliferation conditions) and day 8 immunostaining was also used to quantify differentiation. Immunostaining was not done at the conclusion of the 2 weeks because the cell density was too great.

What was found is that the level of nestin (associated with undifferentiated cells) increased for the first 4 days while the cell population increased and then after that the level decreased as the cells differentiated. The expression of the astroctye marker steadily increased through the 2 weeks showing that the fraction of cells differentiating into astrocytes increases with time. Neuron expression also increased whereas oligodendrocytes initially increased until overtaken by the growing populations of astrocytes and neurons. The qRT-PCR measurements were consistent with those obtained via immunostaining.

The other study that was done using this qRT-PCR method was to monitor the effect of cell culture feeding schedule on cell differentiation. Undifferentiated cells were cultured for 4 days with one culture being fed on day 2 and the other remaining unfed. It was found that the unfed cells started differentiating and the fed cells did not. Therefore it was determined that fresh medium is required to keep cells in the undifferentiated state.

From being exposed to immunostaining in class it is easy to see how laborious it would be to use this method to quantify cell differentiation in a culture, especially one containing cells that differentiate into more than one type of specialized cell. I think this paper is important because it shows how the qRT-PCR results were consistent with those from immunostaining, which is the standard method used for studying cell phenotype. Also, I chose this paper because it provides a couple examples for applications of qRT-PCR.