Inducing pluripotency
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Inducing pluripotency
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[post_date] => 2024-12-23 18:03:16
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[post_content] => Practice Passage (Question 1-6)
*This passage is the property of Khan Academy and has been reformatted into an AAMC-style interface in their entirety by MedLife Mastery. MedLife Mastery does not endorse and is not an affiliate of Khan Academy.
The progression from pluripotent to somatic cells typically occurs through a process called differentiation, where pluripotent cells undergo specific genetic and epigenetic changes that restrict their potential and lead them to develop into specialized somatic cells with distinct functions and characteristics. However, recent research indicates that genetic factors can induce pluripotency in somatic cells.
To explore this, researchers developed an assay system in which the induction of the pluripotent state could be detected as resistance to the antibiotic G418. They inserted a βgeo cassette (a fusion of the β-galactosidase and neomycin resistance genes) into the mouse Fbx15 gene, which is specifically expressed in mouse embryonic stem (ES) cells. ES cells homozygous for the βgeo knockin construct (Fbx15βᵍᵉᵒ/βᵍᵉᵒ) were resistant to extremely high concentrations of G418 (up to 12 mg/ml), whereas somatic cells derived from Fbx15βᵍᵉᵒ/βᵍᵉᵒ mice were sensitive to a normal concentration of G418 (0.3 mg/ml).
Experiment 1
Researchers introduced 24 candidate genes into mouse embryonic fibroblasts (MEFs) from Fbx15βᵍᵉᵒ/βᵍᵉᵒ embryos by retroviral transduction. To determine which of the 24 candidates were critical, they examined the effect of withdrawal of individual factors from the pool of transduced candidate genes on the formation of G418-resistant colonies. Results are shown in Figure 1.
Figure 1 Colony numbers at day 10 and 16 post transduction
Experiment 2
Researchers identified the 10 factors whose individual withdrawal from the bulk transduction pool resulted in no colony formation 10 days after transduction and fewer colonies 16 days after transduction in Experiment 1. They then examined the formation of colonies after withdrawal of individual factors from this 10-factor pool transduced into MEFs. They also compared results to MEFs transduced with all 24 of the original genes. Results are shown in Figure 2.
Figure 2 Colony numbers at day 16 post transduction
Experiment 3
Researchers analyzed the role of the Oct3/4, Klf4, Sox2, and c-Myc genes, which correspond to factors 14, 20, 15, and 22, respectively. Figure 3 shows the number of G418-resistant colonies at day 16 in cells transduced with these four factors versus all 10 factors analyzed in Experiment 2, as well as the effects of removing one or two of these factors from the pool of four.
Figure 3 Colony numbers at day 16 post transduction
Adapted from K Takahashi and Shinya Yamanaka. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. 2006. https://doi.org/10.1016/j.cell.2006.07.024
[post_title] => Inducing pluripotency
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[question] => Why did researchers insert the βgeo cassette into the Fbx15 gene?
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[answer] => 2
[description] => Reason for Correct Answer:
The purpose of the experiments was to determine the effect of genetic factors on the ability of somatic cells to become pluripotent.
In the graphs, antibiotic resistance to G418 serves as a marker of pluripotency.
Paragraph two says that the gene Fbx15 is specifically expressed in embryonic cells and that the researchers inserted antibiotic resistance genes into it.
The passage also states that βgeo knockin construct inserted into the Fbx15 gene (Fbx15βᵍᵉᵒ/βᵍᵉᵒ ) causes embryonic stem cells to be resistant to high levels of antibiotic but does not have this effect on somatic cells. (Although antibiotics are usually used against bacteria, here it is clear that they are also toxic to cultured somatic mouse cells.)
This all means that the insertion of the cassette into the Fbx15 gene allows for detection of pluripotent cells because it allows antibiotic resistance to only be expressed in pluripotent (stem) cells, as these are the cells that express Fbx15.
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[each_answer] => A. It allowed both pluripotent and somatic cells to gain some level of resistance to G418.
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[each_answer] => B. It allowed G418 resistance to be selectively expressed in pluripotent cells.
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[each_answer] => C. Because the Fbx15 gene normally codes for G418 resistance.
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[each_answer] => D. Because the Fbx15 gene normally induces pluripotency.
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[quiz_unique_key] => 1403770772
[question] => What data likely led researchers to select the four genes they analyzed in Experiment 3?
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[answer] => 3
[description] => Reason for Correct Answer:
The genes analyzed were Oct3/4, Klf4, Sox2, and c-Myc genes, which correspond to factors 14, 20, 15, and 22 shown in the other experiments.
Choice A is incorrect. First, the inclusion of all 4 factors was only compared with the inclusion of 10 factors in Experiment 3, so this comparison would not provide reasoning for performing Experiment 3. In addition, the results of Figure 3 show that the inclusion of the 10 factors from Experiment 2 caused slightly higher antibiotic resistance than only the inclusion of the four select factors.
Choice D is also wrong because the group with the 10 factors had higher antibiotic resistance compared to the group with 24 factors, and these four genes were also included in the 10 factor group. In addition, this comparison would not have helped researchers select the specific genes.
For the remaining answers, look at the specific data in Experiments 1 and 2. Note that the factor-specific data shows the effect of removing an individual factor from the pool of 24 (Experiment 1) or 10 (Experiment 2). Notice the effect of removing factors 14, 20, 15, and 22 in each:
These results show that removal of these factors caused the greatest antibiotic resistance at day 10 in the 24 factor pool (Experiment 1) and day 16 in the 10 factor pool (Experiment 2).
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[each_answer] => A. Inclusion of these four factors resulted in greater antibiotic resistance than inclusion of the combination of the group of 10 or 24 factors studies.
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[each_answer] => B. Inclusion of these factors caused the greatest antibiotic resistance at day 10 in the 24 factor pool and day 16 in the 10 factor pool.
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[each_answer] => C. Removal of these factors caused the greatest antibiotic resistance at day 10 in the 24 factor pool and day 16 in the 10 factor pool.
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[each_answer] => D. Removal of these factors had likely resulted in the decrease in antibiotic resistance between the 24 factor and 10 factor group.
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[quiz_unique_key] => 1403770772
[question] => What data likely led researchers to select the four genes they analyzed in Experiment 3?
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[answer] => 1
[description] => Reason for Correct Answer:
Experiment 3 compares factors Klf4, Sox2, and c-Myc.
Remember that the number of resistant colonies reflects the number of pluripotent cells.
Figure 3 shows the effect of removing each of these factors from the set of 4:
Removal of Klf4 (as well as Oct3/4) had the greatest negative effect on the number of resistant colonies and thus pluripotent cells. It is therefore the most important for induction of pluripotency out of the answer options.
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[each_answer] => A. Klf4
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[each_answer] => B. Sox2
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[each_answer] => C. c-Myc
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[each_answer] => D. Factor 9
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[question] => In a follow up study, induced stem cells resulting from gene transduction were transplanted into the respective healthy adult mice from which the cells were originally harvested, and this grafting resulted in tumor formation. What would indicate that induced stem cells are as effective as regular stem cells?
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[answer] => 2
[description] => Reason for Correct Answer:
Stem cells are known for pluripotency. This is their unique strength in the healing process.
Pluripotency is defined as the potential of a cell to differentiate into many different cell types and tissues.
Being as “effective” as regular stem cells suggests also having this ability.
The induced stem cells being able to create tumors containing tissue from all germ layers suggests that they are very effective stem cells able to generate diverse cell types. On the other hand, their G418 resistance or Oct3/4 gene expression do not suggest anything about their effectiveness as stem cells. Lastly, no immune suppression from the hosts would be a result of the fact that the injected cells were harvested from the same mouse.
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[each_answer] => A. The majority of cell colonies were G418 resistant.
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[each_answer] => B. The tumors contained tissue from all germ layers.
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[each_answer] => C. There was no immune suppression from the host mice.
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[each_answer] => D. The majority of the cells express the Oct3/4 gene.
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[question] => Mesodermal cells are not typically considered pluripotent; instead, they are considered multipotent. Pluripotent cells have the ability to differentiate into cells from all three germ layers (endoderm, mesoderm, and ectoderm), whereas multipotent cells have a more limited differentiation potential, typically giving rise to cell types within a specific germ layer.
Which of the following organs is derived from multipotent mesodermal cells?
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[answer] => 3
[description] => Reason for Correct Answer:
The three germ layers are mesoderm, ectoderm, and endoderm. Each layer gives rise to a number of related tissue/organs.
The ectoderm is involved in the eyes, skin, ears, and gonads.
The endoderm gives rise to the GI tract and respiratory tract.
The mesoderm is involved in the musculoskeletal and circulatory system.
In this instance, the heart is mesodermal in origin.
https://commons.wikimedia.org/wiki/File:419_420_421_Table_04_01_updated.jpg
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[each_answer] => A. Liver
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[each_answer] => B. Neural tube
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[each_answer] => C. Heart
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[each_answer] => D. Skin cells
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[quiz_unique_key] => 1325138223
[question] => Mesodermal cells are not typically considered pluripotent; instead, they are considered multipotent. Pluripotent cells have the ability to differentiate into cells from all three germ layers (endoderm, mesoderm, and ectoderm), whereas multipotent cells have a more limited differentiation potential, typically giving rise to cell types within a specific germ layer.
**According to the data shown, what would scientists expect if an epithelial cell culture expressing none of the other factors were exposed to an excess amount of Klf4?
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[answer] => 4
[description] => Reason for Correct Answer:
This question is asking about the effect of introducing only one factor.
Klf4 is one of the factors tested in Experiment 3.
Experiment 3 shows that in the group of 4 factors tested, the removal of even just 2 of the factors eliminated the number of induced stem cells.
This suggests that the addition of only one of the factors would NOT result in an induction of stem cells.
Note that regards to the tumor suppressors in Choice C – induced stem cells can result in tumors, indicating that their tumor-suppressor count would more likely decrease, if they were in fact induced. (Cancer cells actually become less differentiated and more like stem cells compared to somatic cells.)
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[each_answer] => A. A large increase in induced stem cells
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[each_answer] => B. A large decrease in induce stem cells
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[each_answer] => C. An increase in tumor-suppressor gene count
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[each_answer] => D. No change to the cell culture
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Figure 1
Figure 2
Figure 3
