Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution

Abstract

Delayed myeloid engraftment after cord blood transplantation (CBT) is thought to result from inadequate numbers of progenitor cells in the graft and is associated with increased early transplant-related morbidity and mortality. New culture strategies that increase the number of cord blood progenitors capable of rapid myeloid engraftment after CBT would allow more widespread use of this stem cell source for transplantation. Here we report the development of a clinically relevant Notch-mediated ex vivo expansion system for human CD34(+) cord blood progenitors that results in a marked increase in the absolute number of stem/progenitor cells, including those capable of enhanced repopulation in the marrow of immunodeficient nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Furthermore, when cord blood progenitors expanded ex vivo in the presence of Notch ligand were infused in a clinical setting after a myeloablative preparative regimen for stem cell transplantation, the time to neutrophil recovery was substantially shortened. To our knowledge, this is the first instance of rapid engraftment derived from ex vivo expanded stem/progenitor cells in humans.

Figure 1. Culture of CD34⁺ CB progenitors with Notch ligand results in enhanced and more rapid human engraftment as well as increased SRC frequency

CD34⁺ cord blood progenitors were cultured in the presence of Delta1^ext-IgG or control human IgG. The progeny of 30,000 starting CD34⁺ cells depleted of CD14⁺ cells were infused into individual sublethally irradiated NOD/SCID mice via tail vein injection. The number of cultured cells infused per mouse on average was 2.2 × 10⁷ following culture with Notch ligand and 1.8 ×10⁷ following culture with control human IgG. A third group of mice received 30,000 CD34⁺ non-cultured cells plus 2×10⁵ irradiated CD34^neg carrier cells. \ (a) Bone marrow aspirates were performed at 3 weeks and both femurs and tibiae were harvested at 9 weeks when mice were sacrificed. Y axis represents the percent of human CD45 in the marrow of recipient mice. Results shown are the mean ± sem of mice from 5 independent experiments (total number of mice per group: Delta1^ext-IgG, N=46; Control IgG, N=32; Non-cultured, N=44). Contribution to engraftment from the lymphoid and myeloid compartments are represented by the black (lymphoid, as measured by co-staining of human CD19 and CD45) and gray portions (myeloid, as measured by co-staining of human CD33 and CD45). The white portion are human cells that did not co-stain for CD33 or CD19. (b) Cultured and non-cultured cells were infused into sublethally irradiated mice and human engraftment measured at 10 days, 3 weeks and 9 weeks. The y axis represents the percent of human CD45% in the marrow of recipient mice. Results shown are the mean ± sem of mice from 3 independent experiments.

Figure 2. Clinical grade culture of CB progenitors with Delta1^ext-IgG results in significant in vitro expansion of CD34⁺ cells and more rapid neutrophil recovery in a myeloablative double CBT setting

CD34⁺ cord blood progenitor cells were enriched and placed into culture with Delta1^ext-IgG as described. (a) The mean fold expansion for both total nucleated cells (TNC) and CD34⁺ cells upon harvest is presented. Data shown in the bar graph are the mean ± sem for the ten patients enrolled to date (range in parentheses). (b) The individual and median times (solid line) to absolute neutrophil counts (ANC) of ≥500 μl⁻¹ for patients receiving double unit CB transplants with two non-manipulated units (“conventional”) versus with one ex vivo expanded unit and one non-manipulated unit (“expanded”) is presented. (c) The absolute neutrophil count and specific donor CB unit contribution to myeloid engraftment over time (x axis, days 0 to 80) determined by ampFLP analysis are presented. The bars indicate percent expanded (hatched gray) or non-manipulated (white) unit contributing to myeloid (CD33⁺) engraftment (left axis) weekly through day 28 (day 7, 14, 21 and 28) post transplant and then at day 56 and 80. Eight of ten evaluable patients are presented (see results). The left column represents patients who achieved an ANC of ≥500 μl⁻¹ when the expanded unit was the dominant donor while the right column represents patients who had converted to the non-manipulated donor when the ANC reached ≥500 μl⁻¹. The solid line represents ANC over time (right Y axis). Individual time to ANC ≥500 μl⁻¹ is shown to the left of each graph.

Figure 2. Clinical grade culture of CB progenitors with Delta1^ext-IgG results in significant in vitro expansion of CD34⁺ cells and more rapid neutrophil recovery in a myeloablative double CBT setting

CD34⁺ cord blood progenitor cells were enriched and placed into culture with Delta1^ext-IgG as described. (a) The mean fold expansion for both total nucleated cells (TNC) and CD34⁺ cells upon harvest is presented. Data shown in the bar graph are the mean ± sem for the ten patients enrolled to date (range in parentheses). (b) The individual and median times (solid line) to absolute neutrophil counts (ANC) of ≥500 μl⁻¹ for patients receiving double unit CB transplants with two non-manipulated units (“conventional”) versus with one ex vivo expanded unit and one non-manipulated unit (“expanded”) is presented. (c) The absolute neutrophil count and specific donor CB unit contribution to myeloid engraftment over time (x axis, days 0 to 80) determined by ampFLP analysis are presented. The bars indicate percent expanded (hatched gray) or non-manipulated (white) unit contributing to myeloid (CD33⁺) engraftment (left axis) weekly through day 28 (day 7, 14, 21 and 28) post transplant and then at day 56 and 80. Eight of ten evaluable patients are presented (see results). The left column represents patients who achieved an ANC of ≥500 μl⁻¹ when the expanded unit was the dominant donor while the right column represents patients who had converted to the non-manipulated donor when the ANC reached ≥500 μl⁻¹. The solid line represents ANC over time (right Y axis). Individual time to ANC ≥500 μl⁻¹ is shown to the left of each graph.