Application of Multiplex PCR for Characterization of Human Embryonic Stem Cells (hESCs) and Its Differentiated Progenies

Abstract

Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.

Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation

“…Then, 1 µl of cDNA was amplified by PCR using the Abgene 2× PCR master mix (Abgene, Epsom, UK) and appropriate primers (Table II). PCR reaction was performed using the published protocol [Mamidi et al, 2010].…”

Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells

“…We also performed gene expression analysis by RT-PCR using the same set of candidate markers (data not shown) and karyotype analysis (data not shown) to lend support to our initial finding. Moreover, based on our earlier experience (Pal et al 2009;Mamidi et al 2010), we preferentially selected this well-characterized cell line, HUES-7, to study the effect of DMSO.…”

Human embryonic stem cell proliferation and differentiation as parameters to evaluate developmental toxicity

“…1J–S). Based on our earlier experience (Pal et al, 2009; Sivasubramaniyan et al, 2010; Mamidi et al, 2010), we chose two well‐characterized cell lines, HUES‐7 and HUES‐9 (courtesy: Prof Douglas Melton, HHMI) for this study.…”

“…For immunocytochemistry, EBs was plated in two‐well chamber slides (Nunc). Immunostaining was performed as described in our earlier report (Mamidi et al, 2010). Here we have tested for lineage specific markers such as GFAP and AFP (both from Millipore).…”