Scientists from Malaysian stem cell firm Stempeutics and Malaysia's Institute for Medical Research have developed a multiplex PCR method for characterizing human embryonic stem cells and their differentiated progenies.
The technique holds promise as a tool to determine spontaneous differentiation of hESCs and induced pluripotent stem cells during their routine maintenance, and may eventually serve as a way to monitor the purity and authenticity of such cells in regenerative medicine and drug screening applications, according to the researchers.
In a paper published online this month ahead of print in the Journal of Biomolecular Screening, corresponding author Satish Totey, former chief scientific officer at Stempeutics, and colleagues describe the evolution of their method, which they developed due to a dearth of rapid, inexpensive, and simple techniques to identify spontaneous differentiation in stem cells being propagated in Stempeutics' regenerative medicine studies.
According to Totey — now president and CEO of Indian stem cell R&D firm Advanced Neuroscience Allies — and colleagues, "despite standardized culture conditions, spontaneous differentiation in hESC cultures is very common, rendering their maintenance and expansion technically challenging."
The researchers add that it is "undeniable that routine characterization of hESC lines is crucial to avoid compromising the integrity of results," and cite PCR using "novel stage-specific genes that distinguish between hESC and [embryoid bodies]" as one of the most common and effective methods of characterizing pluripotent stem cells.
This is primarily because the number of genes that can be considered reliable markers for undifferentiated hESCs is "relatively concise" and "well-documented;" while differentiated cells can be identified by "a number of lineage- and tissue-specific gene markers," the authors add.
The group had previously described a method of characterizing hESCs that combined a slew of techniques, including PCR, immunochemistry, karyotyping, human leukocyte antigen and short tandem repeat analyses, telomerase assays, teratoma formation in SCID mice, real-time PCR, and focused cDNA microarray, miRNA, and mitochondrial DNA analyses.
However, in an effort to save time and money, they focused on whittling down this rather extensive list to only single-reaction multiplex PCR to semi-quantitatively evaluate hESC proliferation during ex vivo expansion.
By comparing relative mRNA levels of a set of 15 markers in hESC and embryoid body samples, they were able to successfully discriminate between undifferentiated hESCs and their differentiated derivatives, according to the paper.
They standardized their method using the HUES-7 cell line, and further validated the technique with another independent hESC line, HUES-9, as well as with a human teratocarcinoma cell line and mouse embryonic fibroblast cells.
All gene primers used in the study had already been optimized using hESCs and their derivatives. For the multiplexed PCR analysis, the researchers obtained TaqMan probes and primers from Life Technologies' Applied Biosystems business. They extracted total RNA from undifferentiated hESCs and EBS and reverse transcribed using Superscript II from Life Tech's Invitrogen business; and finally, they performed PCR analysis on the ABI Prism 7900HT sequence detection system.
According to the researchers, multiplex PCR provides several advantages over standard uniplex PCR. For example, standard PCR commonly includes false negatives due to reaction failure or false positives due to contamination, the researchers wrote.
In multiplex PCR, however, false negatives are often revealed because each amplicon provides an internal control for other amplified fragments. In addition, the quality of the template may be determined more efficiently in multiplex than in single-locus PCR; and the expense of reagents and preparation time is much less in multiplex PCR than in systems involving several single PCR reactions, according to the researchers.
The researchers evaluated several permutations and combinations of primers to simultaneously amplify target sequences, eventually settling on four separate sets of markers: One indicating ESC pluripotency; and three associated with the formation of ecto-, meso-, and endoderm germ layers. Using their method, the researchers found a high concordance in gene expression profiles and patterns between the different cells tested.
The researchers noted in their paper a number of factors that can influence their experimental protocol and should be carefully considered by researchers wishing to replicate their procedure. These included careful attention to annealing temperature, the number of PCR cycles used, amount of cDNA template, and primer concentration.
"After several attempts with various combinations of relevant gene primers and exhaustive regime of troubleshooting, we successfully developed a [multiplex] PCR assay that allowed simultaneous and specific detection of a candidate set of markers regularly used for hESC characterization," the researchers wrote.
"Being simple, efficient, and cost-effective, semiquantitative multiplex RT-PCR analysis may materialize as a convenient option for hESC characterization;" and its affordability in particular may make the method attractive "in countries with limited economic resources and highly skilled expertise," they added.
It is unclear whether Stempeutics or Advanced Neuroscience Allies are now using the technique as part of their routine stem cell maintenance and propagation. Company representatives could not be reached for comment.