Effect of anti-CD52 antibody alemtuzumab on ex-vivo culture of umbilical cord blood stem cells

Abstract

Background: Excessive maturation of hematopoietic cells leads to a reduction of long-term proliferative capability during cord blood (CB) expansion. In this study, we report the effects of anit-CD52 (Alemtuzumab, Campath) on both short- and long-term ex vivo expansion of CB hematopoietic stem cells (HSC) by evaluating the potential role of Alemtuzumab in preserving the repopulating capability in CB HSC and nonlymphoid progenitors.

Methods: Ex vivo expansion experiments were carried out using freshly purified CB CD34(+)cells in StemSpantrade mark SFEM medium in the presence of stem cell factor, Flt3-Ligand and thrombopoietin at 50 ng/ml. Alemtuzumab (10 microg/ml) was used to deplete CD52(+) cells during the cultures. Flow cytometry was used to monitor CB HSC and their differentiation. Colony forming unit (CFU) assays and long term culture-initiating cell (LTC-IC) assays were performed on cells obtained from day 0 (before culture) and day 14 after cultures. Secondary cultures was performed using CD34(+) cells isolated at 35 days from primary cultures and further cultured in StemSpantrade mark SFEM medium for another 14 days to confirm the long term effect of alemtuzumab in liquid cultures.

Results: Compared to cytokines alone, addition of alemtuzumab resulted in a significant increase in total nucleated cells, absolute CD34(+) cells, myeloid and megakaryocytic progenitors, multi-lineage and myeloid CFU and LTC-IC.

Conclusion: The results from current study suggested that the use of alemtuzumab for ex vivo expansion of CBHSC maybe advantageous. Our findings may improve current technologies for CBHSC expansion and increase the availability of CB units for transplantation. However, in vivo studies using animal models are likely needed in further studies to test the hematopoietic effects using such expanded CB products.

Figure 1

CD34 selection efficiency by aldefluor assay and flow-cytometry assay. After CD34 selection, cells were stained with CD34-PE and Aldefluor. CD34+ stem cells were selected according to forward scatter (FSC) and side scatter (SSC) properties using the gated region R1 to remove cell debris, residual platelet and red cell contamination. Figures were representative of 6 separate experiments. A: Phenotype of day 0 CD34⁺ selected cells that initiate the culture. The percentages of cells positive for the CD34-PE and Bodipy-aminoacetate (BAAA) were obtained from gates R1. B: Isotype expression pattern for IgG-PE and BAAA. C: Expression profile of stem cells co-staining with CD34-PE and BAAA. As shown in the Fig, based on R1 gating, 81.05% cells were both CD34 positive and BAAA brightly stained, 12.65% cells were CD34 positive but BAAA negative. 86.50% of the CD34+ cells were brightly stained with BAAA.

Figure 2

Effects of alemtuzumab on total nucleated cells and CD34⁺ cells. 100,000 CD34+ cells were cultured in 2 ml StemSpan™ SFEM Medium supplemented with 10% of healthy human serum, 50 ng/ml of stem cell factor, TPO and Flt3 Ligand. 10 μg/ml of alemtuzumab was added to test the alemtuzumab's effects on HSC expansion. Cultures were maintained at 37°C with humidified air containing 5% CO₂ for 5 weeks. A small fraction of the cultures were harvested at day 7, 14 to test absolute total nucleated cell expansion. (A), CD34⁺ cell percentages (B), Absolute CD34⁺ cell numbers in control and alemtuzumab treatment groups. Results are representative of 6 separate experiments from different fresh cord blood samples.

Figure 3

Effect of alemtuzumab on megakaryocytes. Cells were cultured in the same condition as described in Fig 2 and harvested at Day 7 and Day 14 for analysis. A: Absolute CD41⁺ cell numbers. B: Relative CD41⁺ cell expansion with Alemtuzumab versus control. (N = 6, p < 0.05).

Figure 4

CFU and LTC- IC expansion fold under different culture conditions. A: CFU expansion fold under different culture conditions: After 14 days culture, cells were harvested from different culture conditions and put to Methocult H4435 medium for another 2 weeks. CFU-GM, BFU-E/CFU-E and CFU-GEMM were counted according to their morphologic characters. Comparison of absolute CFU (total CFU together with three different types of CFU) expansion fold after 14 days culture with or without alemtuzumab treatment is shown. The results of three separate experiments performed in duplicate are expressed in term of the CFU expansion fold on day 14 compared to day 0 initiating cells. B: LTC-IC expansion fold under different culture conditions: LTC-IC was performed on cells before and after 14 days of culture and, in the absence or presence of alemtuzumab. Results are from 3 separate experiments from performed in triplicate on separate fresh cord blood samples.

Figure 5

Alemtuzumab long-term efforts on CD34⁺ cells secondary culture. CD34⁺ cells were isolated from day 35 cultures and put into secondary culture with the original cytokine combination (50 ug/ml SCF, TPO and Flt-3 Ligand) and 10% human serum, with or without 10 μg/ml alemtuzumab (control group). After a further 7 to 14 days of culture, cells were harvested to analyze CD34 and CD52 expression profiles. A: Expression profiles of CD34 and CD52 at Day 7 and Day 14 of secondary cultures. B: Absolute expansion fold of CD34+ cells of secondary cultures. The absolute expansion fold is calculated by the absolute CD34+ cells number as compare to secondary culture initiate CD34+ numbers. The results are from three separate experiments.