Improved Derivation Efficiency and Pluripotency of Stem Cells from the Refractory Inbred C57BL-6 Mouse Strain by Small MoleculesReportar como inadecuado

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The ability of small molecules to maintain self-renewal and to inhibit differentiation of pluripotent stem cells has been well-demonstrated. Two widely used molecules are PD 98059 PD, an inhibitor of extracellular-signal-regulated kinase 1 ERK, and SC1 Pluripotin, which inhibits the RasGAP and ERK pathways. However, no studies have been conducted to compare their effects on the pluripotency and derivation of embryonic stem ES cells from inbred mice C57BL-6, an important mouse strain frequently used to model behavior, cognitive functions, immune system, and metabolic disorders in humans and also the first mouse strain chosen to be sequenced for its entire genome. We found significantly increased derivation efficiency of ES cells from in vivo fertilized embryos fES of C57BL-6 with the use of PD 71.4% over the control of 35.3%. Because fES and ES from cloned embryos ntES are not distinguishable in transcription or translation profiles, we used ntES cells to compare the effect of small molecules on their in vitro characteristics, in vitro differentiation ability, and the ability to generate full-term ntES-4N pups by tetraploid complementation. NtES cells exhibited typical ES characteristics and up-regulated Sox2 expression in media with either small-molecule. Higher rates of full term ntES-4N pup were generated by the supplementation of PD or SC1. We obtained the highest efficiency of ntES-4N pup generation ever reported from this strain by supplementing ES medium with SC1. Lastly, we compared the pluripotency of fES, ntES and induced pluripotent stem iPS cells of C57BL-6 background using the tetraploid complementation assay. A significant increase in implantation sites and the number of full-term pups were obtained when fES, ntES, and iPS cells were cultured with SC1 compared to the control ES medium. In conclusion, supplementing ES cell culture medium with PD and SC1 increases the derivation efficiency and pluripotency, respectively, of stem cells derived from the refractory inbred C57BL-6 strain.

Autor: Chih-Jen Lin, Tomokazu Amano, Yong Tang, Xiuchun Tian



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