Hematopoietic stem cells (HSC) are cells that are capable of giving rise to progenitors leading to both cells of lymphoid origin such as T cells and B cells, as well as cells of myeloid origins such as macrophages, monocytes, neutrophils and others. HSC are known to be present in adult bone marrow, placenta, umbilical cord blood, mobilized peripheral blood, liver, spleen and kidney tissues. CD34 expression has been used as a marker for identification of HSC in humans.
CD34 is a cell surface glycoprotein present in umbilical cord and bone marrow cells. Although the exact function of CD34 is unknown, it is known to act as an adhesion factor in cell-cell interactions. It belongs to a family of single pass transmembrane sialomucin proteins. Cells that are CD34+ and CD38- are found to be undifferentiated and so, the expression of CD34 can be used as a marker for isolation of undifferentiated primitive cells.
StemCell technologies have developed an EasySep® human CD34+ selection kit for isolation CD34+ cells from a variety of tissues including umbilical cord blood, peripheral mononuclear cells and bone marrow. The kit contains the EasySep CD34+ selection cocktail and EasySep nanoparticles that label CD34 cells for magnetic separation. The procedure is simple and easy to follow. A single cell suspension of mononuclear cells is prepared from cord blood or bone marrow by Ficoll gradient separation. The cells are then mixed with the separation cocktail and incubated at room temperature for 15 mins. After mixing the cells with antibody-labeled nanoparticles for 10 mins, the volume of the suspension is brought up to 2.5 ml with medium. The tube is then placed in the EasySep magnet. The magnet is picked up with the tube in it and inverted to remove the supernatant. More media is added to the tube and the procedure repeated to remove all the cells that are not bound to the nanoparticles and therefore are CD34-. After repeating the procedure 5 times, the tube is removed from the magnet and CD34+ cells are eluted with additional media.
The EasySep CD34 Kit has been used in our laboratory for over 4 years, for isolation of hematopoietic stem cells from fetal human liver, cord blood and mobilized peripheral blood. The attractive feature of this kit is the relative ease with which a reasonably pure population of CD 34 expressing cells can be isolated from a heterogeneous cell suspension.Since there is no trauma of flushing out the CD34+ cells, cell viability is not compromised. However, to use this kit effectively, it is necessary to purchase the EasySep magnet which is sold by StemCell technologies. The company also offers a PE conjugated CD34 antibody for flow cytometric or fluorescence microscopic verification of the isolated cells. We have been able to achieve over 95% purity of isolated cells using this kit.
In summary, the EasySep CD34 Kit from StemCell technologies is an excellent tool for isolation of CD34 expressing stem cells from various tissues. An initial purchase of the EasySep magnet is well worth the expense.
Scientist
Children’s Research Center of Michigan
Children’s Hospital of Michigan