Embryonic stem cells are isolated from the inner cell mass of blastocysts at app

Question

Embryonic stem cells are isolated from the inner cell mass of blastocysts at approximately 4 days post- fertilization. These cells ultimately give rise to all of the cell types found in the body. I wanted to study the ability of embryonic stem cells to function as hematopoietic (bone marrow) stem cells. I made a mistake and isolated the cells from a mouse blastocyst at 6 days post-fertilization. I transduced these cells with a plasmid that expresses GFP so that I could trace them in vivo. I then used these GFP labeled cells in a bone marrow transplant into mice. Blood was isolated from these mice 2 months after bone marrow transplantation of labeled embryonic stem cells and was analyzed by FACS to see how well the embryonic stem cells were able to function as bone marrow stem cells. Markers of blood cells: CD45= all blood cells; B220= B cells; Mac-1= monocytes

A. Did the embryonic stem cells give rise to any blood in this animal and if so how much (4 pts)? __________________ B. Did the animals own bone marrow give rise to any of the blood in this animal and if so how much (4 pts)? _________ C. Explain the results seen with the B220 antibody (4 pts).
D. Explain the results seen with the Mac-1 antibody (4 pts).

Explanation / Answer

We first need to look at the FACS quadrant.

The lower left square is for cells which are negative for both X and Y axis marker (negative).

The upper right corner square is for cells which are positive for both X and Y axis marker (double positive).

The upper left square is for cells which are only positive for Y axis marker ( single positive).

The lower right square is for cells which are only positive for X axis marker (single positive).

A. If we look at the FACS quadrant for CD45 antibody, in the upper right square there is 70% cells which will be then double positive, meaning 70% of the cells are showing both GFP and CD45 marker. Thus embryonic stem cells give rise to blood cell. It is 70% of the blood cells.

B- The animals own bone marrow also gives rise to blood cell. According to the CD45 FACS result, the lower right suare represents 30% cells which are positive for CD45 cell. But at the same time these 30% cells are negative for GFP. Thus this 30% cells are coming from the animals own bone marrow.

C. B220 antibody tagged the B cells. According to the FACS results, 30% of the cells are negative for both B220 and GFP ( lower left square). 35% of the cells are positive for GFP ( upper left sqaure). These 35% cells are also present in upper right square. But there is no cells present in the lower right squere, Thus the cells which are positive for B220 antibody are all GFP labelled cells which are originaed from embryonic stem cell.

D. Mac-1 antibody labelled the monocytes. According to the results, 30% of the cells are only positive for Mac-1 , whereas 70% of the GFP labelled cells are possitive for mac-1 antibody. So we can say that 70% of the monocytes in the blood has been originated from embryonic stem cells and the own bone marrow of the animal cell give rise to 30% monocyte.

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