Identification of miRNA Profiles in Individual Embryonic Stem Cells

Stem cell biology is a rapidly growing area of research thought to have massive potential for treatment of human disease. Emphasis has been placed on embryonic stem cells (ESCs) because they are totipotent; in other words, they can differentiate into any of the >220 cell types in the adult body. ESCs are derived from the inner cell mass of blastocyst embryos. Their totipotency can be retained in tissue culture, or ESCs in culture can also be directed to mature into specific cell types when given specific differentiation signals.

There is significant evidence that miRNAs are involved in the regulation of stem cell differentiation. Distinct sets of miRNAs have been found exclusively in ESCs or in differentiated adult tissues [1,2]. This research caught the attention of Drs. William Strauss (University of Colorado-Boulder) and Caifu Chen (Research and Development, Applied Biosystems Inc.) who collaborated to investigate whether miRNA expression profiles (signatures) unique to ESCs could be identified. These profiles could be used to characterize and distinguish totipotent ESCs from pluripotent cell types, such as embryoid body (EB) cells, and from terminally-differentiated somatic cells.

To obtain a true miRNA profile from cells at various stages of differentiation, the researchers decided that analyzing individual cells would be the best approach. Clearly this represented a significant technical challenge, but this was considered less important than the biological limitations imposed by the difficulty in culturing ESCs, their tendency to spontaneously differentiate in the absence of feeder cells, and the inherent heterogeneity among EB cells. Dr. Chen recognized this challenge as an opportunity to push the limits of technology under development at Applied Biosystems for a set of miRNA profiling tools that require only very small amounts of RNA for reliable, reproducible, quantitative PCR (see sidebar, "Streamlined miRNA Profiling Using Megaplex™ Primer Pools and TaqMan® MicroRNA Arrays").

70 randomly selected individual cells from a mouse ESC line, day 3 EB cells, and several somatic cell types including 3T3 cells (a well-characterized transformed fibroblast cell line) and primary splenocytes were chosen for miRNA profiling. The cells were lysed by heating to 95°C for 5 minutes in 1X PBS. An early access version of Megaplex™ RT Primers—a pool of 237 stem-loop RT primers from the associated TaqMan MicroRNA Assays—was used to prime reverse transcription of lysates using the TaqMan MicroRNA RT Kit. Since the real-time PCR quantitation step required distributing this minute amount of starting material across 237 independent reactions, it was decided to incorporate a preamplification step. Preamplification preserves the distribution of miRNAs in the starting sample, in particular those miRNAs present at low copy number that would otherwise be diluted out when material was distributed to each independent real-time PCR assay, thereby maintaining overall assay sensitivity. The cDNA was preamplified using a limited number of PCR cycles and an early access version of Megaplex™ PreAmp Primers—a pool of PCR primers from the same 237 TaqMan MicroRNA Assays. Then the preamplification products were subjected to real-time PCR using the 237 individual TaqMan MicroRNA Assays. This workflow enabled single-cell analysis of the expression profiles of most of the murine miRNAs known at the time (miRBase v10, [3]). At the same time, expression data were collected for positive and negative controls, as well as for 21 mRNAs corresponding to transcripts recognized by the stem cell community for their association with ESC differentiation and self-renewal.

Expression of ESC miRNA was generally found to be low in comparison to the pluripotent and terminally differentiated cells (Figure 1). As cells differentiated into EB cells, both the total number of miRNAs and their expression levels increased. By day 3, significant differences in the amounts of miRNA were detected in partially differentiated EB cells compared to their totipotent ESC precursors (Figure 2). One of the goals of these experiments was to evaluate the variability in miRNA profiles among individual cells in a single ESC line. The data indicated that the cells within a given ESC line represent a mixture of undifferentiated cells and those beginning to differentiate, and that a subset of ~6 miRNAs can be used as markers to distinguish these development stages.