Significant biochemical, biophysical and metabolic diversity in circulating human cord blood reticulocytes

Abstract

Background: The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these 'sub-vital' stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies.

Design and methods: Here we use highly purified populations of reticulocytes isolated from cord blood, sorted by flow cytometry into four sequential subpopulations based on transferrin receptor (CD71) expression: CD71high, CD71medium, CD71low and CD71negative. Each of these subgroups was phenotyped in terms of their, morphology, membrane antigens, biomechanical properties and metabolomic profile.

Results: Superficially CD71high and CD71medium reticulocytes share a similar gross morphology (large and multilobular) when compared to the smaller, smooth and increasingly concave reticulocytes as seen in the in the CD71low and CD71negativesamples. However, between each of the four sample sets we observe significant decreases in shear modulus, cytoadhesive capacity, erythroid receptor expression (CD44, CD55, CD147, CD235R, and CD242) and metabolite concentrations. Interestingly increasing amounts of boric acid was found in the mature reticulocytes.

Conclusions: Reticulocyte maturation is a dynamic and continuous process, confounding efforts to rigidly classify them. Certainly this study does not offer an alternative classification strategy; instead we used a nondestructive sampling method to examine key phenotypic changes of in reticulocytes. Our study emphasizes a need to focus greater attention on reticulocyte biology.

Conflict of interest statement

Competing Interests: Bruce Russell, Laurent Rénia, Francois Nosten and Fabio T. M. Costa are PLOS ONE editorial board members. Kayen Low was an employee of JEOL Asia Pte Ltd during her involvement with this manuscript. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Flow cytometry gating strategy and representative morphology of reticulocyte samples.

(A) Flow cytometry dot plot showing CD71 and Hoechst staining using to define strategy gating for different reticulocyte subset sampling: CD71high, CD71medium, CD71low and CD71negative. The mean percentage of each subsets are inserted in brackets (n = 14). Cord blood reticulocyte subsets stained with new Methylene blue (see insets). (B) Morphology of cord blood reticulocytes was observed by scanning electron microscopy. The scale bars represent 1 µm. (C) The blue bordered insets show key nanostructures on the surface of the reticulocytes. The scale bars in these insets represent 100 nm. (D) Intracellular reticulocyte structures were observed by transmission electron microscopy.

Figure 2. Immunophenotyping of reticulocyte samples.

Difference of between mean fluorescence intensity with different erythrocytic antigens and mean fluorescence intensity with secondary antibody on reticulocytes sampled from the four CD71 gates (CD71high, CD71medium, CD71low and CD71negative) (Delta of Mean Fluorescence Intensity (MFI)). The dotted red line represents the threshold of positivity based on the CD15 staining.

Figure 3. Biomechanical properties of reticulocyte samples.

The size and biomechanical characteristics of CD71 sorted cord blood reticulocytes (different color markers represent each of the 4 isolates used). (A) The median diameter (um−/+IQR) of live reticulocytes in physiological media. (B) The median shear flow modulus (−/+IQR) (high shear flow modulus indicates reduced membrane deformability) of the reticulocyte membranes as measured by micropipette aspiration. (C) Representative micropipette aspiration images of CD71high, CD71medium, CD71low and CD71negative (top panel to bottom respectively) measured at an identical pressure. (D) The relative cytoadhesiveness of different reticulocyte samples on CHO cells (two types) and activated platelets.

Figure 4. Metabolomic profile of reticulocyte samples.

Heat map of 14 metabolomic markers (GC-MS) found in differential amounts (dark brown indicating a greater quantity) in CD71 high, medium, low and negative reticulocyte populations. Boric acid was the only marker that increased as the reticulocyte matured from CD71 high to negative. The normalized level of marker scale is shown at the bottom of the heat map.

Figure 5. Summary of the key phenotypic changes in maturing human cord blood samples.

An overview of the key biochemical, biophysical and metabolic changes occurring in the subsets of circulating cord blood reticulocytes relative to RNA content as measured by thiazole orange. Our finding are also put into the context of the ‘R’ reticulocyte characterization (R1 = Immature reticulocytes, R2 = Mature reticulocytes and R3 = Normocytes). .