Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors
Embryonic stem cells fused with nuclear contents, or said nuclear contents transferred into oocytes result in differentiated cells programmed to behave like embryonic cells. The study demonstrated by Takahashi and Yamanaka introduce factors Oct3/4, Sox2, c-Myc, and Klf4 under the conditions of embryonic stem cells to induce the creation of pluripotent stem cells from the mouse embryonic/adult fibroblasts. Consequently, the iPS (induced pluripotent cells) displayed the characteristics of embryonic stem cells and expressed the same cell marker genes. The scientists discovered through their study that pluripotent stem cells could be generated from fibroblast cultures with a few alterations.
ES cells can grow indefinitely and maintain the ability to be pluripotent, indicating that they can differentiate into cells from the ectoderm, endoderm and mesoderm. ES cells can, therefore, treat a multitude of diseases, ranging from Parkinson’s to spinal cord injuries and diabetes. ES cells, however, are not as easily accessible due to their controversial status ethically, and because there have been cases of tissue rejection following transplantation. These problems that have arisen with the use of ES cells can be avoided if the patient’s cells provide pluripotent cells. The somatic cells can become pluripotent cells through either transfer of nuclear contents to oocytes, or by fusing with embryonic stem cells, which suggests that these ES cells can provide somatic cells with pluripotency. The scientists hypothesized in their study that the factors with importance in ES cells remain the same in somatic cell pluripotency, as transcription factors such as Oct3/4, Sox2, and Nanog play the same role in both types of cells. However, there are genes that have been seen to encourage proliferation in ES cells exclusively. The study was performed to examine whether the factors would behave the same way in somatic pluripotent cells as they do in ES cells, using Oct3/4, Sox2, c-Myc, and Klf4 and iPS from mouse embryonic/adult fibroblast cultures.
The scientists performing this study initially selected 24 genes, which induce pluripotency in somatic cells, and examined whether these genes would play a similar role in ES cells with regards to maintaining their cell identity. To test the pluripotency of the various genes, the scientists developed an assay system, as displayed by Figure 1A, which showed resistance to G418 (asserting pluripotency). Figure 1 shows the generation of iPS cells from MEF (mouse embryonic fibroblasts) with the 24 genes through the (A) strategy testing the 24 candidate factors (by resistance to them). Figure 1B shows the G418 resistant colonies after 16 days following transduction with the 24 factors, which showed that no single gene was enough to activate the Fcx15 locus. However, the petri dish on the right of Figure 1B shows that all of the 24 genes together were able to build resistance to G418. Figure 1C demonstrates that out of the 12 clones, 5 displayed similar morphology to embryonic stem cells. From the repeated experiment, scientists found 29 G418-resistant colonies, so they selected 6 of those colonies, 4 of which also displayed ES cell-like morphology/proliferation as seen in Figure 1D. The proliferation rate (doubling time of the cells) was comparable to that of the embryonic stem cells. These cells exhibiting these similarities were referred to as iPS-MEF24 since they are pluripotent stem cells induced from MEFs using 24 gene factors. Figure 1E shows that the PCR analysis yielded results similar to what was expected – the cell markers for the iPS-MEF24 cells were the same as in the ES cells. Finally, Figure 1F shows through bisulfate sequencing that the Fbx15 and Nanog factors were demethylated, while the Oct3/4 was methylated, indicating that there was a combination of the 24 factors tested that demonstrated the same behavior in the iPS-MEF24 cells as in the ES cells.
In order to figure out which of the 24 factors were most important to maintaining the ES cell identity, some factors were withdrawn to allow scientists to examine the effects of the withdrawal. Figure 2A displays a graph with the information for how each of the 24 genes affects the resistant colony number through the time periods of 10 and 16 days. Consequently, scientists identified 10 of the factors that affected the colony growth most significantly (absence resulted in no colony growth) and used these 10 factors to examine colony growth. The result of this was that more resistant cell colonies were formed than in the combination of the 24 factors. Next, the withdrawal of these 10 selected factors was examined (Figure 2B), and it was seen that the resistant colonies did not form in the absence of Oct3/4 or Klf4, few formed in the absence of Sox2 and non-ES-cell-like morphology resulted from the absence of c-Myc. These were the factors that were seen to be most important in the creation of pluripotent somatic cells. The combination of these four important factors resulted in colony formation in the same quantity as found when there was a combination of the initial 10 factors, as shown by Figure 2C, showing that iPS cells can be formed from the four main factors.
Next, various combinations of these four factors were examined, and the combination of Oct3/4, Klf4 and c-Myc resulted in the formation of 54 G418 resistant colonies, and 6 of these colonies were selected. The morphology of these colonies were unlike the previous colonies examined, and showed a rough surface (Figure 2D), suggesting that while this combination can activate the Fbx15 locus, the change seen is different from the previously examined cells. To see whether cell marker genes were displayed in iPS cells, RT-PCR was performed, as seen in Figure 3A. The combination of the 10 factors, and the combination of the 4 factors demonstrated similar cell markers to ES cells and because they also were positive for alkaline phosphatase and SSEA-1 (Figure 3D), it was seen that they were the most similar to ES cells. Figure 3C showed that the CpG dinucleotides remained at least partially methylated in the iPS cells. Various factors were not activated in the combination of iPS-MEF3 clones, showing a different morphology completely from the primary two combinations. With DNA microarrays, scientists compared the gene expressions of ES cells, iPS cells and Fbx15 MEFs, revealing that iPS were clustered closer to ES cells, but not with fibroblasts, and identifying the genes that were upregulated (more cell component) in the ES and iPS cells. Some genes were seen to be more efficiently upregulated in the iPS-MEF4/iPS-MEF10/ES cells than iPS-MEF3 cells, and others were more upregulated in ES cells than in the iPS cells, asserting that while iPS cells are very similar to ES cells, they are not identical.
Through teratoma formation (Figure 5A), iPS cell pluripotency was examined and cell proliferation resulting in tumors were found in 5 iPS-MEF10, 3 iPS-MEF4, 1 iPS-MEF4wt, and 6iPS-MEF3 clones. Two of the iPS-MEF10, two of the iPS-MEF4 and the iPS-MEF4wt clone displayed differentiation through all of the germ layers. The differentiation of neural and muscle tissues was determined with immunostaining, as shown in Figure 5B. Ultimately, this demonstrated that most of the iPS-MEF10 and iPS-MEF4 clones would exhibit pluripotency. Figure 5C shows that iPS-MEF10, iPS-MEF4, and iPS-MEF3 formed embryoid bodies in dishes, but only iPS-MEF10 and iPS-MEF4 cells initiated differentiation. The immunostaining in Figure 5D detected the cells positive for markers in all three germ layers, but there was no differentiation in iPS-MEF3, so iPS-MEF4 and iPS-MEF10 were confirmed to be pluripotent.
The four factors were then introduced into tail-tip fibroblasts of Fbx15 mice, and a few resistant colonies were found, so iPS cells could be established from these. Additionally, these four factors were introduced to TTFs (tail-tip fibroblasts) from a 12-week-old female Fbx15 mouse. 13 resistance colonies were obtained and 6 clones for which iPS cells could be established were selected. Another iPS-TTFgfp4 was established, and these cells were seen to be very similar to ES cells, and showed the same cell marker genes, as seen in Figure 6A and Figure 6B. Figure 6C shows the 18 embryos obtained showing the contribution of the iPS cells, which ultimately contributed to all three germ layers, as shown in Figure 6D. All data demonstrated that the four factors selected could induce pluripotency.
Furthermore, scientists examined the expression of the factors in the iPS cells, and found that Oct3/4 and Sox2 were lower in the iPS cells than they were in ES cells, but the amount of expression from the four factors in total was greater than in ES cells. However, the Western blot analyses exhibited that the protein amounts in the four factors were similar to the protein amounts in ES cells. Figure 7A/B show the protein amounts relative to those in ES cells, and it was seen that the p53 levels in the iPS cells were less than those in ES cells. Following in vitro differentiation, the mRNA expression of Oct3/4 and Sox2 decreased, though remaining higher than that of ES cells. Each iPS clone had a different transgene integration pattern, as seen in Figure 7C. The karyotyping analysis revealed that some clones displayed a normal karyotype, while others displayed kayotypes of 39XO, 40XO+10, and 40Xi(X) (Figure 7D). Figure 7E shows that the iPS cells could not stay undifferentiated in the absence of feeder cells. All of these results prove that iPS cells are not contaminated with ES cells.
The four factors were shown to create pluripotent cells that would behave the same way as ES cells, through many different results. Consequently, this study indicates that embryonic stem cells, which have been known to raise many questions and concerns ethically, or even scientifically (if the patient’s body rejects them), can be replaced with somatic pluripotent cells, engineered to behave like ES cells. This is a step toward finding out if a human’s somatic cells can behave the same way as the mouse used in the experiment. If a human cell can be engineered, it will have great implications for the future of stem cell research, and controlling pluripotency.
There’s something at the bottom of this well I’d like you to look at.
Did Gary tell you to write this?