Safety and efficacy of ex vivo expanded CD34+ stem cells in murine and primate models

Abstract

Background: Hematopoietic stem cell (HSC) transplantation has been widely applied to the treatment of malignant blood diseases. However, limited number of functional HSCs hinders successful transplantation. The purpose of our current study is to develop a new and cost-efficient medium formulation that could greatly enhance the expansion of HSCs while retaining their long-term repopulation and hematopoietic properties for effective clinical transplantation.

Methods: Enriched human CD34⁺ cells and mobilized nonhuman primate peripheral blood CD34⁺ cells were expanded with a new, cost-efficient expansion medium formulation, named hematopoietic expansion medium (HEM), consisting of various cytokines and nutritional supplements. The long-term repopulation potential and hematologic-lineage differentiation ability of expanded human cells were studied in the non-obese diabetic/severe combined immunodeficiency mouse model. Furthermore, the efficacy and safety studies were performed by autologous transplantation of expanded primate cells in the nonhuman primate model.

Results: HEM could effectively expand human CD34⁺ cells by up to 129 fold within 9 days. Expanded HSCs retained long-term repopulation potential and hematologic-lineage differentiation ability, as indicated by (1) maintenance (over unexpanded HSCs) of immunophenotypes of CD38^-CD90⁺CD45RA^-CD49f⁺ in CD34⁺ cells after expansion; (2) significant presence of multiple human hematopoietic lineages in mouse peripheral blood and bone marrow following primary transplantation; (3) enrichment (over unexpanded HSCs) in SCID-repopulating cell frequency measured by limiting dilution analysis; and (4) preservation of both myeloid and lymphoid potential among human leukocytes from mouse bone marrow in week 24 after primary transplantation or secondary transplantation. Moreover, the results of autologous transplantation in nonhuman primates demonstrated that HEM-expanded CD34⁺ cells could enhance hematological recovery after myelo-suppression. All primates transplanted with the expanded autologous CD34⁺ cells survived for over 18 months without any noticeable abnormalities.

Conclusions: Together, these findings demonstrate promising potential for the utility of HEM to improve expansion of HSCs for clinical application.

Keywords: Ex vivo expansion; Human cord blood; Long-term HSC; NOD/SCID mice; Nonhuman primates; Transplantation.

Fig. 1

Effect of different medium conditions on HSC expansion ex vivo. a CD34⁺ cell expansion with TPO concentrations ranging from 0 to 100 ng/mL in SGF2 medium (IMDM supplemented with 200 ng/mL SCF, 200 ng/mL Flt-3 L, and nutrition supplements). b CD34⁺ cell expansion with IL-3 concentrations ranging from 0 to 100 ng/mL in SGF3 medium (SGF2 with 20 ng/mL TPO). c CD34⁺ cell expansion with G-CSF concentrations ranging from 0 to 100 ng/mL in SGF4 medium (SGF3 with 15 ng/mL IL-3). d CD34⁺ cell expansion with GM-CSF concentrations ranging from 0 to 100 ng/mL in SGF5 medium (SGF4 with 10 ng/mL G-CSF). e CD34⁺ cell expansion with SR1 concentrations ranging from 0 to 2 μM in SGF6 medium (SGF5 with 10 ng/mL GM-CSF). f A summary of CD34⁺ cell expansion in media with different growth factor combinations. SGF7 is SGF6 supplemented with 1 μM SR1. Expansion was calculated as fold increase (after/before expansion) in cell counts on each day (days 0, 4, and 9). Data are shown as mean ± SD, n = 6. **p < 0.01, ***p < 0.001; one-way ANOVA followed by Dunnett’s multiple comparison test

Fig. 2

Effects of HEM on the ex vivo expansion of human HSCs from different sources. Human CD34⁺ cells purified from fresh UCB, cryopreserved CB, and mPB were cultured with HEM to day 4 or day 9. Total cell (a) and CD34⁺ cell (b) expansion was calculated as fold increase (after/before expansion) in cell counts on each day (days 0, 4, and 9). Data are shown as mean ± SD, n = 10. c Types of CFU colonies formed from the hematopoietic stem/progenitor cells before/after expansion. Equal numbers of unexpanded human CD34⁺ cells and 9-day HEM-expanded CD34⁺ cells (1 × 10⁴ per dish) were assayed as described in Methods. Data are shown as mean ± SD, n = 4. Pairwise comparisons (unexpanded vs expanded) were performed for each type of colony using Student’s t test (p > 0.05 for all pairs). d–f Representative flow analysis for multi-immunophenotyping of the expanded cells on day 0, day 4, and day9. Gates of CD34⁺ CD38⁻, CD90⁺, CD45RA⁻, and CD49f⁺ cell populations were set based on the fluorescence minus one (FMO) of each cell surface marker antibody. CD38⁻, CD90⁺, CD45RA⁻, and CD49f⁺ were analyzed in CD34⁺ cell population

Fig. 3

Functional assessment of human cells in PB and BM of NOD/SCID mice transplanted with various types of human HSCs. Three weeks (a) and eight weeks (b) after intravenous human CD34⁺ transplantation in mice, the presence of human cells was analyzed in the PB of mice transplanted with unexpanded human HSCs, low-dose expanded HSCs, or high-dose expanded HSCs. Normal saline (NS) was injected as the vehicle control. Data are shown as mean ± SD, n = 16. c At week 8 post-transplantation, engrafted human cells were detected in the BM by flow cytometry. The percentages of cells expressing human hematologic-lineage markers shown are calculated on the total (mouse plus human) cell population from mouse PB or BM. Data are shown as mean ± SD, * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA followed by Dunnett’s multiple comparison test

Fig. 4

Long-term engraftment of human cells in NOD/SCID mice. a Extent of human cell engraftment in the BM of NOD/SCID mice transplanted with normal saline (NS; blue dots), unexpanded HSCs (red dots), low-dose expanded HSCs (green dots), and high-dose expanded HSCs (purple dots) at 24 weeks post-transplantation. b, c Proportion of CD45⁺CD15⁺ and CD45⁺CD19⁺ cells in the three HSC transplantation groups at week 24 post-transplantation. d Representative flow cytometry of human CD45⁺CD15⁺ (myeloid) lineage and CD45⁺CD19⁺ (lymphoid) lineage in mouse BM at 24 weeks post-transplantation. e Extent of human cell engraftment in mouse BM at 8 weeks following secondary transplantation in NOD/SCID mice. Data points are color-coded for each group as a. f, g Proportion of CD45⁺CD15⁺ and CD45⁺CD19⁺ cells in the three HSC transplantation groups at week 8 after secondary transplantation. Data are shown as mean ± SD, n = 8 in each group. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA followed by Dunnett’s multiple comparison test. h Comparison of the frequency of SRC in unexpanded CD34⁺ cells and HEM-expanded CD34⁺ cells. The human CD45⁺ cell proportion in the mouse BMs was analyzed by flow cytometry 8 weeks after transplantation

Fig. 5

Autologus transplantation in a nonhuman primate model. a Scheme for autologus transplantation in a nonhuman primate model. CTX treatment was administered on days − 4 and − 3, and transplantation was performed on day 0. b Types of CFU colonies formed from fresh isolated CD34⁺ cells (pre-expansion) and 9-day HEM-expanded cells (Post-expansion). Data are shown as mean ± SD, n = 4. c Percentage of GFP⁺ cells in PB nucleated cells (CD45⁺ population) at various time points during the first month following autologous transplantation. d Percentage of GFP⁺ cells in primate PB CD45⁺, CD14⁺, and CD20⁺ cell population at 1 month post-transplantation. e GFP⁺ cells in the primate BM CD45⁺ population at 1 month after transplantation. PB and BM samples were from experimental group (E-1, E-2, E-3, E-4, and E-5). BM was harvested from primate femora. f Recovery time of white blood cell (WBC), neutrophil (NEU), platelet (PLT), and lymphocyte (LYM) were determined by comparing the baseline before/after CD34⁺ cell transplantation. Blank control, n = 2; negative control, n = 2; experiment group, n = 5. The lines of each group indicate the median in statistical analysis