Reconstitution of donor-derived immune system after allogeneic hematopoietic stem cell transplantation (HSCT) is essential for recovery and long-term survival. Despite routine use of human umbilical cord blood (hUCB) as a stem cell source for allogeneic HSCT, much remains unknown regarding the kinetics of immune recovery and correlation with different transplant cell dosages. To study the hUCB repopulating potential, different hUCB CD34+ cell dosages were transplanted into immune deficient NSG mice; hematopoietic cells were then collected and engraftment was analyzed.


NOD/SCID/IL-2Rγnull recipient (NSG) mice (Jackson Laboratories, Bar Harbor, ME) were kept in pathogen-free facilities. CD34+ cells were isolated from a pool of six hUCB donors using a CD34+ microbead kit (Miltenyi Biotec). Each sublethal irradiated (220 or 300 cGy) 8 week old female NSG mice received either low dose (15x103, N=15) or high dose (75x103, N=15) CD34+ cells transplanted intravenously via retro-orbital route. Animal experiments were performed in accordance with Institutional Animal Care and Use Committee guidelines. Statistical analysis was performed with Prism software (GraphPad Software, Inc) and Excel. Data are presented as mean ± standard error of the mean (SEM).


To determine the effects of hUCB CD34+ cell dosages on the rate of engraftment, NSG mice were transplanted with low doseor high dose CD34+ cells. The transplanted CD34+ cell dosages were comparable to clinical dosages based on body weight (Mavroudis et al. 1996). The engrafted cells were analyzed for expression of surface markers that define human hematopoietic cells. During the follow up period of up to 18 weeks, the high dose infused group had increased hUCB engraftment compared with the low dose infused group in peripheral blood (Fig 1A), bone marrow (Fig 1B & 1C) and spleen (Fig 1D), which is consistent with reported clinical observations that infused cell dosage is inversely correlated with time to engraftment (Migliaccio et al. 2000 Blood). Interestingly, we observed different lymphoid subset frequencies between low and high dose infused groups at the post-engraftment stage (18 weeks post transplantation) (data not shown).

To investigate different lymphoid subset engraftment frequencies in low and high dose hUCB transplanted recipient mice at early engraftment stage, peripheral blood and hematopoietic organs were collected and analyzed up to 10 weeks post transplantation. The low dose infused group had significantly lower CD3+ (T cells) and CD56+ (NK cells) frequency in peripheral blood at 4 and 8 weeks (Fig 2A & 3A). More importantly, CD3+ (T cells) frequency was close to non-detectable in the bone marrow and spleen in the low dose infused group (Fig 2B & 2C), and CD56 (NK cells) frequency was decreased in the low dose infused group compared with the high dose infused group (Fig 3B & 3C). The absolute CD3+ and CD56+ number, displayed as fold difference, were even more dramatically decreased in the femur (Fig 2D & 3D) and the spleen (Fig 2E & 3E) of low dose infused group.

Because of the substantial difference in T cell subset frequencies between the two dosage groups in bone marrow and spleen, thymuses were collected and analyzed to study T cell development and maturation. Engraftment of hCD45+ cells in the thymuses were observed in 10 out of 15 animals (66.7%) in the low dose infused group and 12 out of 14 animals (85.7%) in the high dose infused group. Interestingly, in animals with high hCD45+ frequency, the total thymocyte CD3+ frequency was lower in the low dose infused group (Fig 4A). Additionally, the low dose infused group had lower CD3+CD4+ T cell frequency (Fig 4B) and higher CD3+CD4+CD8+ T cell frequency (Fig 4C), suggesting low dose infused group had a decreased mature T cell population and increased immature T cell population in the thymus.

In contrast, the low dose hUCB CD34+ cells infused group had increased hCD19 (B cells) frequency in the peripheral blood, bone marrow and spleen (Fig 5A-5C), while the absolute hCD19 (B cells), displayed as fold difference, did not show a statistically significant difference between the two groups (Fig 5D & 5E).


In summary, our findings suggest that (1) transplanted hUCB cell dosage is inversely correlated with time to engraftment (2) low transplanted hUCB cell dosage resulted in skewed immune cell population which may contribute to delayed immune recovery after allogeneic HSCT.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.