Seeking to identify the biochemical triggers that commit stem cells to distinct fates, researchers at the Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital in Toronto assessed the culture conditions that support human embryonic stem cell (hESC) pluripotency. Traditional quantitative reverse transcription-PCR presented researcher Mark Kibschull with challenges: the need to purify RNA prior to RT-qPCR restricted the number of samples that could be processed at one time, and analyzing gene targets for hESC differentiation meant that multiple cell cultures were required to produce sufficient sample for analysis.
Traditional animal-derived media used to grow stem cells often have contaminants, introducing unknown variables that can impact experiment outcomes. These contaminants can also contain pathogens that are harmful to humans if the stem cells are used for therapeutic purposes. Kibschull thus began to investigate the effects of xeno-free growth media on stem cells.
This requires step-by-step examination of all cell culture conditions, from the feeder cells on which the hESCs are grown to eliminating growth factors derived from animals. To do this, Kibschull transfers stem cells from the growth media to specialized plates composed of microwells that force the cells to aggregate into embryoid bodies (EBs)—3-D structures that promote differentiation of the cells. After weeks of culturing the EBs, RNA purification and qPCR are performed. But certain culture conditions were so harsh on the cells that in some instances only 20% of the cells needed for RT-qPCR analysis were left. In fact, this step had to be repeated a quarter of the time. By the end of the process, only a handful of genetic markers could be analyzed from the small amount of RNA obtained. A process was needed that either preserved more of the genetic material or multiplied the number of analyses performed on the isolated RNA.
An improved qPCR workflow for limited samples
Figure 2 – Embryoid bodies viewed under a microscope. The aggregation of stem cells into these 3-D structures induces differentiation.A new workflow allows the analysis of hundreds of gene targets from just a few hundred cells (see Figure 1). Stem cells can be profiled at varying points during differentiation, providing detailed time-courses that can help researchers better understand the impact of culture conditions on differentiation (see Figure 2). This data allows optimization of culture conditions.
The first step in the workflow uses a lysis reagent that eliminates the need for RNA purification, reducing potential loss of sample. The RNA lysate generated by the SingleShot Cell Lysis Kit (Bio-Rad Laboratories, Hercules, Calif.) is free of contaminating genomic DNA and used directly in the RT-qPCR. By lysing embryoid bodies directly and eliminating the RNA purification step, Kibschull dramatically increased his experimental success rate.
Figure 1 – qPCR workflow.Despite this, the analysis was still limited by the amount of cDNA after reverse transcription. Kibschull was still unable to profile all the necessary gene targets to draw meaningful conclusions about the differentiation of the stem cells. The problem was solved by performing target-specific cDNA preamplification with Bio-Rad SsoAdvanced PreAmp Supermix, which is formulated to allow 1000-fold enrichment of up to 100 targets in a single reaction.
Improved qPCR results
With enough material for a complete analysis, Kibschull could move on to performing qPCR. He used the Bio-Rad PrimePCR human embryonic stem cell panel, which contains a preselected set of validated qPCR primer pairs for the analysis of 89 genes involved in hESC differentiation. Prior to using PrimePCR assays, Kibschull designed his own primers, which required extensive in-laboratory validation and troubleshooting when primer designs yielded sub-par PCR efficiency or nonspecific amplification products. Commercially validated primer sets eliminated this tedious and often unpredictable obstacle.
With the PrimePCR hESC panel, Kibschull analyzes markers from many differentiation pathways, rather than only a handful of markers spanning a few representative cell fates. The additional markers allow more thorough characterization of the response of stem cell lines to changes in culture conditions.
Increased throughput
An unexpected benefit is the significant increase in throughput. Kibschull has been able to adopt this workflow into a 96-well format, which allows testing of many more culture conditions and combinations. Downscaling the culture format has the added benefit of reducing the cost associated with culturing the stem cells.
In addition to hESCs, Kibschull studies induced pluripotent stem cells (iPSCs). Use of less sample means that iPSC colonies can be screened earlier in the derivation process. Phenotyping at earlier stages enables determination of which colonies are worth culturing further and which can be discarded, saving time and cost.
Conclusion
Incorporation of an RNA lysis buffer, preamplification supermix and commercially validated qPCR assays into gene expression workflows will benefit scientists in many fields, especially those with limited amounts of sample quantity.
Joshua Fenrich is a product manager for gene expression reagents at Bio-Rad Laboratories, 4000 Alfred Nobel Dr., Hercules, Calif. 94547, U.S.A.; tel.: 510-724- 7000; e-mail: [email protected]; www.bio-rad.com