Abstract
Currently, there are genetic- and chemical-based methods for producing pluripotent stem cells from somatic cells, but all of them are extremely inefficient. However, a simple and efficient technique has recently been reported by Obokata et al (2014a, b) that creates pluripotent stem cells through acid-based treatment of somatic cells. These cells were named stimulus-triggered acquisition of pluripotency (STAP) stem cells. This would be a major game changer in regenerative medicine if the results could be independently replicated. Hence, we isolated CD45 (+) splenocytes from five-day-old Oct4-GFP mice and treated the cells with acidified (pH 5.7) Hank's Balanced Salt Solution (HBSS) for 25 min, using the methods described by Obokata et al 2014c. However, we found that this method did not induce the splenocytes to express the stem cell marker Oct4-GFP when observed under a confocal microscope three to six days after acid treatment. qPCR analysis also confirmed that acid treatment did not induce the splenocytes to express the stemness markers Oct4, Sox2 and Nanog. In addition, we obtained similar results from acid-treated Oct4-GFP lung fibroblasts. In summary, we have not been able to produce STAP stem cells from neonatal splenocytes or lung fibroblasts using the acid-based treatment reported by Obokata et al (2014a, b, c).
Highlights
Takahashi and Yamanaka (2006) reported that it was possible to induce adult fibroblasts into pluripotent stem cells using four factors: Oct3/4, Sox2, c-Myc and Klf4. The creation of these induced pluripotent stem cells and their replication in human somatic cells has been heralded as a major breakthrough in regenerative medicine (Takahashi et al, 2007)
It has been reported that all of the spermatogonia in the testes of this type of transgenic mice were capable of expressing Oct4 (Denn et al, 2008)
It has been reported that mouse induced pluripotent stem (iPS) cells could be generated using a cocktail of seven chemical molecules without any genetic manipulation, with an efficiency of around 0.2% (Hou et al, 2013)
Summary
Takahashi and Yamanaka (2006) reported that it was possible to induce adult fibroblasts into pluripotent stem cells using four factors: Oct3/4, Sox, c-Myc and Klf. The authors reported that bathing somatic cells in a mild acid could reprogram them to become pluripotent stem cells They harvested spleen cells from 1-week-old Oct-4-GFP transgenic mice, and isolated the CD45+ population by flow cytometry. The CD45+ splenocytes were treated with acidified HBSS (pH 5.7) for 25 min at 37°C and maintained in DMEM/F-12 culture medium containing B27 and Leukemia Inhibitory Factor (LIF) for one to seven days This simple procedure activated the Oct promoter two days post-treatment, making the splenocytes express the GFP reporter
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