L3. Functional Assay - CD34+ measurement + Bone Marrow transplantation

Question 1

How is HSC’s detected when bone marrow has been donated?

Answer 1

Measuring number of CD34+ by flow cytometry.

1) All cells are measured (SSC/FSC), Lymphocytes are gated
2) CD45+ CD34+ positive –> the hematopoietic stem cells.

Question 2

What is CD45+ used for?

Answer 2

CD45+ is a WBC marker

Question 3

Explain how scientist found out which cells repopulate the bone marrow in a lethally irradiated mouse

Answer 3

1) 2x10^5 bonemarrow from mouse w/ injected HSC’s from donor, reacted with AB’s. Selected undifferentiated cells, injected in mouse –> only 1x10^3 partly enriched cells to repopulate haematopoeisis in the mouse
2) 1x10^3 reacted with FI-ABs against Sca-1. Selected stem cells from progenitor cells via flow cytometry. Resulted that only 30-100 fully enriched cells were needed to restore haematopoesis in the mouse.

Question 4

What is the number of enriched HSCs that reconstruct haematopoiesis in a mouse?

Answer 4

20 HSC –> 4-4 (100% o mice reconstituted
10 HSC –> 10-11 (90,9% of mice reconstituted
5 HSC –> 9-17 (52.9% of mice reconstituted)
2 HSC –> 5-21 (23.8% of mice reconstituted)
1 HSC –> 9 of 41 (21.9% of mice reconstituted).