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

Abstract

Limited CD34+ cell number in human umbilical cord blood (hUCB) samples stimulated the development of in vitro HSC expansion. hUCB CD34+ cells cultured with cytokine cocktails were increased about 7.3 folds after 4 days' culture in vitro. For in vivo study, CD34+ cells obtained directly from hUCB or from in vitro expansion were transplanted into NOD/SCID mice (n=4 for each group). 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. Peripheral blood (PB) was collected and multi‐lineage differentiation was assessed at 1, 3, and 8 weeks post transplantation. CD34+ cells were decreased below 1% in group 2 and 3 but not in group 4 after 3 weeks. On the other hand, CD45+, CD41+, CD71+and CD15+ populations were increased in groups 2‐4 compared with those of 1 week. 8 weeks post transplantation, human CD34+ cells were hardly detected in PB from any groups. However, PB and bone marrow cells expressing human cell surface markers of various hematopoietic lineages were detectable in groups 2‐4 [Figure 1]. 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 long term engrafted to bone marrow of mice [Figure 2]. An efficient experimental method has been developed for the expansion of hUCB CD34+ cells in vitro and these cells can be potentially explored for various applications in the clinic.