Superior survival of ex vivo cultured human reticulocytes following transfusion into mice

Abstract

The generation of cultured red blood cells from stem cell sources may fill an unmet clinical need for transfusion-dependent patients, particularly in countries that lack a sufficient and safe blood supply. Cultured red blood cells were generated from human CD34⁺ cells from adult peripheral blood or cord blood by ex vivo expansion, and a comprehensive in vivo survival comparison with standard red cell concentrates was undertaken. Significant amplification (>10⁵-fold) was achieved using CD34⁺ cells from both cord blood and peripheral blood, generating high yields of enucleated cultured red blood cells. Following transfusion, higher levels of cultured red cells could be detected in the murine circulation compared to standard adult red cells. The proportions of cultured blood cells from cord or peripheral blood sources remained high 24 hours post-transfusion (82±5% and 78±9%, respectively), while standard adult blood cells declined rapidly to only 49±9% by this time. In addition, the survival time of cultured blood cells in mice was longer than that of standard adult red cells. A paired comparison of cultured blood cells and standard adult red blood cells from the same donor confirmed the enhanced in vivo survival capacity of the cultured cells. The study herein represents the first demonstration that ex vivo generated cultured red blood cells survive longer than donor red cells using an in vivo model that more closely mimics clinical transfusion. Cultured red blood cells may offer advantages for transfusion-dependent patients by reducing the number of transfusions required.

Figure 1.

Cytospin and confocal analyses of day 21 cultured adult reticulocytes. Cultured reticulocytes were processed for morphological analyses by cytospin and stained with Leishman’s solution before (A) and after leucofiltration (B). Leucofiltration resulted in >99% pure population of cultured reticulocytes. (C) Leucofiltered cultured reticulocytes were also processed for live cell confocal imaging using anti-CD235a-FITC antibody.

Figure 2.

Validation of macrophage depletion for transfusion model. Murine peripheral blood samples were collected at designated time points and cells were labelled with PE-conjugated anti-mouse F4/80 (A) or FITC-conjugated anti-human CD235a (B & C) and analyzed by flow cytometry. (A) Circulating levels of murine macrophages measured in liposome treated (n=8) and untreated NSG mice (n=10) over a 10 day period. (B & C) NSG mice that had either been treated with clodronate liposomes to remove macrophages at day -3 and day -1 (n=3) or left untreated (n=2) were inoculated with RBCs from a single donor on day 0. (B) Percentage of human RBCs in the mouse circulation. (C) Clearance rates of human cells in untreated and macrophage depleted mice. Human cells were normalized to 100% at 10 minutes after injection. Data shown as mean±SE. ***P<0.00003. RBCs: red blood cells; PB: peripheral blood.

Figure 3.

Live images of mouse blood samples containing GPA-positive human cRBCs and adult RBCs. Macrophage depleted mice were injected with 2×10⁸ cord blood cultured reticulocytes (half-life = 54.8 hours) (A), adult cultured reticulocytes (half-life = 48.2 hours) (B) or with donor RBCs (half-life = 29.5 hours) (C). Live confocal imaging of peripheral blood aspirates taken at 10 minutes, 8, 24, 48 and 72 hours postinjection was performed on cells labelled with anti-CD235a-FITC antibody. RBCs: red blood cells; cRBCs: cultured red blood cells; CB: cord blood.

Figure 4.

Phenotypic maturation of transfused cells. (A) The diameters of anti-CD235a-FITC labelled cord and adult cRBCs and human donor RBCs were measured before injection into mice (0 minutes) and at various time points following transfusion. Diameters of murine RBCs were also determined. Data represent mean±SE measurements of cRBCs from 1 CB sample (half-life = 54.8 hours) and 2 adult donors (half-life = 48.2 hours) and RBCs from 1 donor (half-life = 29.5 hours). (B) Confocal images of representative CD235a labelled adult cRBCs at 0 minutes and 48 hours (half-life = 48.2 hours) with corresponding bright field images. (C) Proportion of CD71-positive cells in the GPA-positive human cell population. Numbers in key represent the number of mice inoculated with cells from a single donor of each cell source. Data represent mean±SE. GPA: glycophorin A. CB: cord blood: RBCs: red blood cells; cRBCs: cultured red blood cells.

Figure 5.

Detection and survival of human cRBCs and adult RBCs in transfused NSG mice. cRBCs or adult blood cells were inoculated into the lateral tail vein of macrophage depleted NSG mice. Human cells were detected by measuring expression of CD235a in murine blood by flow cytometry. (A) Proportion of human cells surviving in mouse circulation. Data represent mean±SE of 8 independent experiments for the adult cultured reticulocytes, 5 independent experiments for standard donor cells and 4 independent experiments for cord blood cultured reticulocytes. The mice in each independent experiment were injected with cells from the same donor. The proportion of human cells were normalized, with the levels detected 10 minutes after inoculation set to 100%. Survival of both adult and cord cRBCs in NSG mice was significantly greater than adult red cells at all measured time points over the entire time course of the experiment (P≤0.02). (B) Half-life of human cells in transfused NSG mice, by experiment and by source. Data from mice where the levels of human cells decreased to ≤50% by the end of the experiment were used to calculate the half-life. Each point represents an individual mouse, results from mice inoculated with cells from the same donor are stacked vertically, lines represent mean±SE. GPA: glycophorin A; CB: cord blood: RBCs: red blood cells; cRBCs: cultured red blood cells.

Figure 6.

cRBCs demonstrate better survival than RBCs from the same donor. (A) Direct paired comparison of cRBCs and RBCs from the same donor. Five day old adult red cells were transfused into NSG mice (n=5). Twenty-one days later cRBCs generated from this sample were transfused into a separate group of mice (n=6). At the same time a third group of mice was transfused with unmodified red cells from the same donor that were now 26 days old (n=4). ANOVA showed that overall survival of cRBCs was significantly better than day 5 and day 26 adult red cells (P<0.0001). (B) Half-life of human cells in transfused NSG mice, by source. Each point represents an individual mouse, mean±SE are shown. RBCs: red blood cells; cRBCs: cultured red blood cells; GPA: glycophorin A.