(spoiler) AAMC OG B/B Passage 5: (mature vs embryonic fibroblast)

[autumn123]

My question isn't tied to a specific question, just understanding this one piece from the method. I'm confused about whether the cells they isolated are mature fibroblast or embryonic fibroblast?? Does it seem like there's a discrepancy between the text and the figure? Judging from the answer key and the passage text, I think they are studying mature fibroblast. However, Figure 1 is showing "isolation and expansion of embryonic fibroblasts". Passage attached. Thank you so much!

How I infer that it must be mature fibroblast:

1. * The passage says: "it has been shown that fibroblasts can be reprogrammed to a pluripotent state with a combination of transcription factors. These results have caused scientists to question whether specific transcription factors could induce other defined somatic cell fates and not just an undifferentiated state."
2. * A portion of the answer key says: "fibroblasts were isolated from postnatal mice, no embryonic cells were used in the experiment, which involves the conversion of one adult cell type into another adult cell type."

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[treeclimbingmonkey]

Ok, so basically this passage is talking about an experiment about reprogramming fibroblasts (type of cell) to a pluripotent state using specific combinations of transcription factors.
So the experiment is as follows:
From mice embryos, the experimenter isolated neural lineage specific cells which expressed the gene Tuj1. This could be observed through green fluorescence which signalled that the Tuj1 is active. Furthermore, this Tuj1 gene, is specific to neural fibroblast cells. The stuff about GABA seems like extraneous information but might be better to keep it in mind, in case you get a question on it.

Next, the experimenter took 5 transcription factors (for inducing the pluripotent state) and added it to the cell to see if the gene Tuj1 would be expressed less commonly amongst the original Tuj-1 positive cells. Remember, since Tuj1 is specific to ONLY neural lineage cells, fibroblast cells that stop expressing Tuj1 in the experiment would have reverted to the pluripotent state in the presence of the transcription factors tested. The conditions the experiment actually tests, are the: all 5 transcription factors, 4 transcription factors with Asc11 missing, 4 transcription factors with Brn2 missing, 4 transcription factors with Myt11 missing, 4 transcription factors with Zic1 missing, 4 transcription factors with Olig2 missing. Basically they are varying the combination of transcription factors, to see if anything change in terms of Tuj1 expression.
From the graph we can observe a significant drop in expression of Tuj-1 in fibroblasts in Asc11 negative conditions.

In terms of the question, you are asking...
There is no discrepancy. The authors are studying mature fibroblasts are the differentiated fibroblasts. In this experiment, those are the original neural lineage specific embryonic fibroblast that express Tuj1.
What we are trying to do in this experiment is convert mature (embryonic) fibroblasts to pluripotent (embryonic fibroblasts) that express all tissues, not just neural tissues. This is the aim that is trying to be accomplishing using transcription factors.

So Tuj1 IS an early marker of neural differentiation, since only cells that are differentiated specific to the neural lineage express Tuj1.

Remember in embryonic stem cells this is the order of differentiation:

totipotent -> pluripotent (endoderm, ectoderm, mesoderm) -> multipotent (this is the "neural specific mature fibroblast" in this experiment). You are trying to induce pluripotent cells from multipotent cells in this experiment (working backwards).

Differentiation in general is achieved through a variety of way one of which is hypothesize to be by transcription factors, and regulation/activity of specific genes in each embryonic cell. The researchers are trying to determine if differentiation can be reversed simililarly by the administration of specific transcription factor combinations.

I know it is a lot but I hope it helps. Here is an image too.

 

[autumn123]

Thank you!!