The efficiency of cell fusion-based reprogramming is affected by the somatic cell type and the in vitro age of somatic cells.

Cell fusion is one approach that has been used to demonstrate nuclear reprogramming of somatic cells to a pluripotent-like state and is a useful tool for screening factors involved in reprogramming. Recent cell fusion studies reported that the overexpression of Nanog and SalI could improve the efficiency of reprogramming, whereas AID was shown to be essential for DNA demethylation and initiation of reprogramming. The aim of this study was to investigate factors affecting the reprogramming efficiency following cell fusion. We conducted fusions of mouse embryonic stem cells (ESCs) with somatic cells carrying a GFP transgene under control of the Oct4 promoter (Oct4-GFP), which is normally repressed in nonpluripotent cells. The effect of somatic cell type on the reprogramming efficiency was investigated using Oct4-GFP expression as an indicator. Different somatic cell types were tested including mesenchymal stem cells (MSCs), adipose tissue-derived cells (ADCs), neural stem cells (NSCs), and these were compared with the mouse embryonic fibroblast (mEF) standard. The reprogramming efficiencies differed greatly, with mEFs (0.477 ± 0.003%) and MSCs (0.313 ± 0.003%) showing highest efficiencies while NSCs (0.023 ± 0.014%), and ADCs (0.006 ± 0.006%) had significantly lower reprogramming efficiencies (p < 0.05). The differences in the reprogramming efficiencies observed could be in part explained by the in vitro age of the somatic cells used. We demonstrated that the reprogramming efficiency of early passage mEFs was significantly higher compared with late passage mEFs (0.330 ± 0.166% vs. 0.021 ± 0.011%, p < 0.05), suggesting that senescence can affect reprogramming potential. In summary, this study shows that different somatic cell types do not have equivalent potential to be reprogrammed following fusion with ESCs. Furthermore, the in vitro age of somatic cells can also affect the reprogrammability of somatic cells. These findings constitute an important consideration for reprogramming studies.

Comparison of reprogramming ability of mouse ES and iPS cells measured by somatic cell fusion.

The ectopic expression of the key transcription factors Oct4, Sox2, c-Myc, and Klf-4 have been shown to reprogram somatic cells to a pluripotent state. In turn these induced pluripotent stem (iPS) cells, like embryonic stem (ES) cells, have been shown to be able to reprogram somatic cells by cell fusion. In this study we compare the differences and similarities between ES and iPS cells measured by somatic cell fusion to somatic cells harboring an Oct4-GFP transgene. We found that iPS cells were just as potent as ES cells at reprogramming the somatic genome as measured by Oct4-GFP reactivation. The resulting ES-somatic and iPS-somatic cell hybrids were characterized for expression of key pluripotency genes, immunostaining for Oct4, SSEA-1, and the ability to differentiate into cell types representative of the three germ layers. In addition to restoring pluripotency to the somatic genome following cell fusion, the telomere maintenance mechanisms of both the ES and iPS cells were found to be dominant in the resulting ES-somatic and iPS-somatic cell hybrids, resulting in the lengthening of the somatic telomeres following cellular reprogramming. Therefore this study supports the view that iPS cells can be virtually indistinguishable from ES cells, even with regard to their reprogramming ability.

Reprogramming of somatic cells after fusion with induced pluripotent stem cells and nuclear transfer embryonic stem cells.

In this study we examine whether a somatic cell, once returned to a pluripotent state, gains the ability to reprogram other somatic cells. We reprogrammed mouse embryonic fibroblasts by viral induction of oct4, sox2, c-myc, and klf-4 genes. Upon fusion of the resulting iPS cells with somatic cells harboring an Oct4-GFP transgene we observed, GFP expression along with activation of Oct4 from the somatic genome, expression of key pluripotency genes, and positive immunostaining for Oct4, SSEA-1, and alkaline phosphatase. The iPS-somatic hybrids had the ability to differentiate into cell types indicative of the three germ layers and were able to localize to the inner cell mass of aggregated embryos. Furthermore, ntES cells were used as fusion partners to generate hybrids, which were also confirmed to be reprogrammed to a pluripotent state. These results demonstrate that once a somatic cell nucleus is reprogrammed, it acquires the capacity and potency to reprogram other somatic cells by cell fusion and shares this functional property with normal embryonic stem (ES) cells.

The efficient generation of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells.

Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 +/- 2.62%, ADCs 70.61 +/- 15.4%, NSCs, 68.72 +/- 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 +/- 4.10 colonies, 0.13 +/- 0.06%) and ADCs (118.20 +/- 38.28 colonies, 1.14 +/- 0.77%) when compared with the mEF control (3.17 +/- 0.29 colonies, 0.03 +/- 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Somatic cell nuclear transfer: pros and cons.

Even though the technique of mammalian SCNT is just over a decade old it has already resulted in numerous significant advances. Despite the recent advances in the reprogramming field, SCNT remains the bench-mark for the generation of both genetically unmodified autologous pluripotent stem cells for transplantation and for the production of cloned animals. In this review we will discuss the pros and cons of SCNT, drawing comparisons with other reprogramming methods.