7.342: Week 6 Questions

Post discussion, questions, or comments about the Week 6 course material here.

Amber
Wichterle et al.

What is figure 6a showing?

Chambers et al.

In figure 6a they choose four days as the amount of time to test if Nanog prevents differentiation. Is this too a short a time to see if Nanog really maintains undifferentiated cells?

How did 'sonic hedgehog' get its name?

Elizabeth
Chambers et al.

I really liked how they set up their experiment with LIF, hLIF-05, and nanog. In Figure 5D, why did they use Cytokine as a control?

Wichterle et al.

I think it’s interesting that they sited the Terada paper twice (Terada et al. Nature 2002). I don’t actually understand why they did. They introduce their research saying that it is known that some cell populations fuse with somatic cells, but then they claim that through their research this is not necessarily true…. “These findings provide evidence against somatic cell fusion.” The paper then immediately changes focus. If they know that the Terada paper specifically was looking at bone marrow cells, why discuss this at all?

Georgi
Wichterle et al.

Why do they graft mouse ESC-derived cells into chick embryos? Is it so they can distunguish between graft6ed and embryonal cells and how does this influences the differentiation of the cells?

Chambers et al.

Is it known how the expression of Nanog is regulated? What are the targets regulated by Nanog and what's their role in stem cell self-renewal?

Holly
Wichterle et al:

How does fluoresecence- activated cell sorting work?

Chambers et al:

How might knowledge of Nanog be utilized? Could it be used to maintain a supply of embryonic stem cells (thereby avoiding the need to have lots of embryos to fulfill a therapeutic requirement)? Would it not be adequate just to use LIF for this purpose?

Kathy
Chambers: It's interesting that Nanog requires presence of Oct4 for function, and yet Oct4 induces differentiation. If Nanog really works by countering Oct4 as the authors suggest, then shouldn't we see Nanog-like behavior in just Oct4-/- cells? How is nanog itself regulated?

Wichterle: The finding that ESC-derived MNs can populate the spinal cord and form networks after being grafted back in vivo presents many medical possibilities, so I agree with Manpreet that it would've been cool to see if those MNs were also functional. Have any clinical trials been done in humans in the 4 years since this paper was published?

Manpreet
Wichterle et al:

Throughout the text of the paper, they state 'data not shown'. What checks are put in place to verify the data they choose not to show?

It would have been good to see a further experiment showing that the ES derived spinal MNs are functional (i.e. showing stimulation of the MNs results in muscle contraction.)

Chambers et al:

Figure 2, they write about B2 elements. "B2 elements are expressed at high levels in embryonic cells". What are B2 elements?

In figure 5C, they show that when nanog is overexpressed, and the cells are cultured with LIF, around 90% of colonies remain undifferentiated. Is there a time limit on this effect? Could this be used to maintain a permanent population of undifferentiated ES stem cells for an organism?

Zak
Wichterle, et al.:

They say that the embryoid bodies were implanted into the chick embryos at development "stage 15-17." At that point in development, what stage of development and differentiation are the chick's own motor neurons in? Have they received their bursts of retinoic acid and Shh yet, or are they still pluripotent stem cells? If they had implanted the EBs at a later stage of development, would the grafts have been as successful? Successfully grafting into a still plastic embryo is one thing, but the more clinically useful goal of grafting into a fully developed adult may be harder.

Chambers, et al.: What organism evolved the Cre system for excising DNA, and what was its purpose? When the research team started out and synthesized all the plasmids for their screen, were they already planning ahead and including the Cre recognition sites in all of the tested plasmids? Or did they identify Nanog first (with the screen), and then synthesize a single plasmid with the Nanog gene and the Cre sites?