Stem cell research is one of the hot topics these days. However, in this filed, purely isolated cells are required; for example, pure hematopoietic progenitor cells are required for hematopoietic stem cell research. To purify hematopoietic progenitor cells, Invitrogen provides the Dynal® CD34 Progenitor Cell Selection System, which is immuno technology combined with magnetic selection system. This system/kit contains Dynabeads® M-450 CD34 (for isolation of hematopoietic progenitor cells), and DETACHaBEAD® CD34 (for detachment of Dynabeads® M-450 CD34). The Dynabeads® M-450 CD34 are uniform, superparamagnetic polymer beads (4.5 um diameter), and the beads are coated with a CD34 primary antibody to specifically select human hematopoietic progenitor cells. CD34 is one of the membrane antigens which is predominantly expressed on human hematopoietic progenitor cells. DETACHaBEAD® CD34 is used to remove the beads and CD34 antibody from purified hematopoietic progenitor cells. Using this kit, CD34 positive hematopoietic progenitor cells can be isolated from human bone marrow, peripheral blood or cord blood, but it is best to use isolated mononuclear cells (MNCs) from bone marrow.
We isolated hematopoietic progenitor cells from human bone marrow. In the first step, MNCs were isolated from bone barrow by using Ficoll (Invitrogen also provides a MNC isolation kit, available seperately). In the second step, CD34+ cells are captured by the Dynabeads and isolated using a Dynal magnet. Cells are then washed 3 times with isolation buffer (phosphate buffered saline (PBS) without Ca2+ and Mg2+, with 2% bovine serum albumin (BSA) or human serum albumin (HSA), 0.6% citrate. 100 IU/ml penicillin-streptomycin solution). These washes remove unbound MNCs which are CD34-, resulting in more pure CD34+ cells. In the final step, beads are removed from the cells by using DETACHaBEAD® CD34; CD34+ cells are then in the supernatant. There are 3 more wash steps with isolation buffer. The collected supernatant is then centrifuged at 400g for 10 min and the pellet is resuspended with 1 mL of isolation buffer. Usually, 1-4% of MNC were isolated as CD34+ hematopoietic progenitor cells from bone marrow tissue. According to the manual from Invitrogen, almost 1% of MNC are isolated as CD34+ hematopoietic progenitor cells from umbilical cord blood; 0.1-0.2% of MNC are isolated as CD34+ hematopoietic progenitor cells from peripheral blood. According to Dynal, at least 95% of isolated cells are CD34+ from bone marrow. Our experiments also showed that 95-99% of isolated cells were CD34+ by using fluorescence activated cell sorting analysis. By using isolated CD34+ cells, we performed in vitro differentiation assays (e.g. chondrogenic, osteogenic, myogenic assays) with the proper differentiation media. We also performed colony forming cell assays to show the activity of hematopoietic progenitor cells. Our isolated hematopoietic progenitor cells could expand with proper growing media to keep self-renewal activity.
Post-doc
Molecular Neurobiology Lab
McLean Hospital/Harvard Medical School