It is possible to direct the differentiation of insulin-secreting beta-cells from iPS cells in a petri dish using signaling molecules that mimic normal embryonic development. In the diagram below, molecules labelled in yellow are inducers. Major genes expressed at each step of the differentiation process are also listed. For example, iPS cells are marked by OCT4 and SOX2 expression and treating them with soluble BMP4 for four days will induce endoderm cells expressing SOX17 and FOXA2.

BMP4 FGF Wnt3a EGE han iPS cells Endoderm Beta-cells Endocrine progenitor SHH Pancreatic Endoderm SHH Sox9 GP2 Hnf4 FoxA2 Sva

i. Based on the expression pattern of the genes listed, is Nkx6.1 a cell type specific gene?

ii.  Why do you think addition of SHH on day 4 result in the formation of HNF4A/FoxA2 positive hepatic progenitors whereas the same molecule added on day 8 produced Sox9/GP2 Exocrine progenitors?

iii. It is day 4 of your differentiation experiment to produce beta-cells and you realize you do not have any Wnt3a left in your laboratory. Since it takes 6-8 weeks to get cell culture reagents to Turkey, your friend suggests using one of the alternative Wnt pathway modulator molecules found in your freezer. Which of the following would you add to your culture plate on day 8. Explain.

CHIR99021 – An inhibitor of GSK3-beta

IWP4 – Inhibits secretion of Wnt ligands from the cell

DKK1 – An inhibitor of LRP

XAV939 – A tankyrase inhibitor that increases Axin levels

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