Let\'s do an experiment. You are supposed to differentiate hematopoietic precurs
ID: 168179 • Letter: L
Question
Let's do an experiment. You are supposed to differentiate hematopoietic precursors from embryonic stem cells in vitro. You have done all your literature searches and are pretty confident that you have to use methylcellulose medium (which is a semisolid medium) and a multi-step differentiation protocol with addition of hematopoietic factors. This is what you have done in the lab:
Step 1. You have formed embryoid bodies in Stemline media and added a cocktail containing BMP-4, VEGF and bFGF. After 48 hours, you added SCF, thrombopoietin and FLT3 ligand.
Step 2. On day 5 of this procedure, you have dissociated your EBs and replated the cells in blast-colony growth media with addition of bFGF and HoxB4 protein.
As per Till and McCulloch, which in vivo assay could you use to characterize whether or not your cells have a hematopoietic potential? Explain how the assay works. (Experimental results not necessary, just what assay works and how it works to determine if cells have HSC potential).
Explanation / Answer
According to Till and Mc Culloch, hematopoesis could be transplanted by cloning the responsible cells.
Background: Inteavenously injected normal human HSC's could repopulate the bone marrow of certain species of mice, this hypothesis led to the development of assay.
Assay Method:
Sublethally irradiated, nonobese diabetic/severe combined immunodeficient rats as hosts(NOD/SCIID) were used as the biomaterial.
In these mice, both human B-lineage lymphopoiesis and myelopoiesis along multiple lineages which were obtained from adult human bone marrow as well as cord blood cells were given.Limiting dilution experiments have been undertaken with both unseparated and highly purified (CD34%D38-) populations of cells were isolated from human cord blood.
The results of these studies indicate that the majority of mice repopulated after six weeks with limiting numbers of human cells (is., transplants that repopulate only a minority of recipients) contain both lymphoid and myeloid cells of human origin indicating derivation from a human in vivo repopulating cell with lymph myeloid potential
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