Ex Vivo Expansion and Characterization of Human Umbilical Cord Blood CD34⁺ Stem Cells

Background: Human umbilical cord blood (hUCB) has been considered as an alternative source of hematopoietic stem cells (HSCs) for allogeneic cell transplantation owing to its easy availability and less stringent requirement for HLA matching. However, the absolute number of HSCs in hUCB is much smaller than that in bone marrow or mobilized peripheral blood. Therefore, there is a need to develop a reliable and efficient approach for the expansion of HSCs of hUBC in vitro.

Method: CD34⁺ cells were purified from hUCB using magnetic-activated cell sorting system and cultured in IMDM basal medium supplemented with cytokine cocktails. CD34⁺ cell population was identified by flow cytometry after in vitro expansion. Nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice were randomly allocated into four groups (n=4 for each group). Twenty-four hours after exposure to sub-lethal irradiation of ⁶⁰Co, CD34⁺ cells obtained directly from hUCB or from in vitro expansion were transplanted into NOD/SCID mice. Four groups of mice were injected with saline, 0.4 million unexpanded CD34⁺ cells, 0.4 million in vitro expanded CD34⁺ cells, and 2.9 million in vitro expanded CD34⁺ cells, respectively. Multi-lineage differentiation was assessed using antibodies against a panel of cell surface markers (e.g., CD34, CD45, CD11, CD19, CD15, CD71, CD66, CD41a, and CD42b) at 1, 3, and 8 weeks post transplantation. Circulation peripheral blood was also collected for various analyses.

Result: Human CD34⁺ cells were increased about 7.3 folds after 4 days’ culture in vitro. For in vivo mouse study, no cells expressing human cell surface marker were detected in mice injected with saline (negative control). One week after injection with CD34⁺ cells, human surface markers including CD34, CD45, and CD15 were easily detectable in all three groups. Three weeks after cell injection, CD34⁺ cells were decreased below 1% in group 2 and 3 but not in group 4. On the other hand, CD45⁺ and CD15⁺ populations were increased in groups 2, 3, and 4. There was a strong correlation between the numbers of injected CD34⁺ cells and differentiated cell populations. This trend was also observed with cells expressing megakaryocytic marker CD41 and erythroid progenitor cell marker CD71 in mouse groups 2 to 4. Eight weeks post transplantation, human CD34⁺ cells were hardly detected in any groups. However, peripheral blood and bone marrow cells expressing human cell surface markers of various hematopoietic lineages were detectable in groups 2-4. These results indicated that CD34⁺ cells from hUCB either before or after expansion in vitro are capable of differentiation into hematopoietic cells of various lineages in vivo. Our study also indicates that in vitro expanded CD34⁺ cells are successfully engrafted to bone marrow of NOD/SCID mice.

Conclusion: An efficient experimental method has been developed for the expansion of hUBC CD34⁺ cells in vitro and these cells can be potentially explored for various applications in the clinic.