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. 2021 Sep 1;96(9):1064-1076.
doi: 10.1002/ajh.26247. Epub 2021 Jun 3.

Comprehensive phenotyping of erythropoiesis in human bone marrow: Evaluation of normal and ineffective erythropoiesis

Hongxia Yan  1   2 , Abdullah Ali  3 , Lionel Blanc  4   5 , Anupama Narla  6 , Joseph M Lane  7   8 , Erjing Gao  1 , Julien Papoin  4 , John Hale  1 , Christopher D Hillyer  1 , Naomi Taylor  2   9 , Patrick G Gallagher  10   11   12 , Azra Raza  3 , Sandrina Kinet  2 , Narla Mohandas  1
Affiliations

Comprehensive phenotyping of erythropoiesis in human bone marrow: Evaluation of normal and ineffective erythropoiesis

Hongxia Yan et al. Am J Hematol. .

Abstract

Identification of stage-specific erythroid cells is critical for studies of normal and disordered human erythropoiesis. While immunophenotypic strategies have previously been developed to identify cells at each stage of terminal erythroid differentiation, erythroid progenitors are currently defined very broadly. Refined strategies to identify and characterize BFU-E and CFU-E subsets are critically needed. To address this unmet need, a flow cytometry-based technique was developed that combines the established surface markers CD34 and CD36 with CD117, CD71, and CD105. This combination allowed for the separation of erythroid progenitor cells into four discrete populations along a continuum of progressive maturation, with increasing cell size and decreasing nuclear/cytoplasmic ratio, proliferative capacity and stem cell factor responsiveness. This strategy was validated in uncultured, primary erythroid cells isolated from bone marrow of healthy individuals. Functional colony assays of these progenitor populations revealed enrichment of BFU-E only in the earliest population, transitioning to cells yielding BFU-E and CFU-E, then CFU-E only. Utilizing CD34/CD105 and GPA/CD105 profiles, all four progenitor stages and all five stages of terminal erythroid differentiation could be identified. Applying this immunophenotyping strategy to primary bone marrow cells from patients with myelodysplastic syndrome, identified defects in erythroid progenitors and in terminal erythroid differentiation. This novel immunophenotyping technique will be a valuable tool for studies of normal and perturbed human erythropoiesis. It will allow for the discovery of stage-specific molecular and functional insights into normal erythropoiesis as well as for identification and characterization of stage-specific defects in inherited and acquired disorders of erythropoiesis.

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Conflict of interest statement

Conflict of interests

None.

Figures

Figure 1.
Figure 1.. Human erythroid progenitor populations can be divided into distinct subsets.
(A) Representative FACS plots illustrating expression of CD71 and CD105 in previously defined erythroid progenitor populations CD34+CD36−, CD34+CD36+ and CD34−CD36+. Based on their expression, six subsets including P1, P2, P3, P4, P5 and P6, were gated and sorted for colony forming assay. (B) Number of colonies generated from 200 FACS-sorted cells of P1-P6, separately, in EPO-alone medium (upper panel) and complete medium (lower panel). Error bars indicate standard deviation (SD) of the mean (n=4). (C) Representative images of colonies generated by P1 to P6 cells, in EPO-alone and complete medium. The photos were taken under an inverted microscope at ×4 magnification, scale bar=500μm. (D) CD117 expression on surface of P1 to P6 cells. Error bars indicate standard deviation (SD) of the mean (n=5).
Figure 2.
Figure 2.. Validation of the continuum of erythroid progenitors
(A) Representative FACS plots for definition of erythroid progenitors from in vitro culture of human CD34+ cells, on day 5 of differentiation. (B) Representative cytospin images of sorted IL3R+CD71−, EP1, EP2, EP3 and EP4 cells from in vitro culture of human CD34+ cells. The cells were sorted on day 5 of differentiation. The images were captured under Leica DM2000 microscope at ×100 magnification, scale bar=10μm. (C) Representative images of colonies generated by sorted IL3R+CD71−, EP1, EP2, EP3 and EP4 cells from in vitro culture of human CD34+ cells, in complete medium. The photos were taken using a Nikon D3500 camera. (D) Representative images of colonies generated by sorted IL3R+CD71−, EP1, EP2, EP3 and EP4 cells from in vitro culture of human CD34+ cells, in complete medium. The photos were taken under an inverted microscope at ×4 magnification, scale bar=1mm. (E) Quantitative analysis of colony forming ability of sorted IL3R+CD71−, EP1, EP2, EP3 and EP4 cells from in vitro culture of human CD34+ cells. The data are from three independent experiments. (F) Representative FACS plots for sorting purity of EP1, EP2, EP3 and EP4 (left panel) and their differentiation progress after two days of culture (right panel). (G) Number of cell divisions of EP1 to EP4 by the end of differentiation. The numbers were calculated based on final erythroid yield of EP1 to EP4 under same culture conditions. The data are from three independent experiments.
Figure 3.
Figure 3.. CD34/CD105 expression profiles distinguish the in vivo continuum of erythroid progenitors
(A) Representative FACS plots illustrating strategy for definition of erythroid progenitors using enriched CD117+ cells from primary bone marrow. The gates of EP1 to EP4 were made based on expression of CD34 and CD105. (B) Representative cytospin images of sorted IL3R+CD71−, EP1, EP2, EP3 and EP4 cells from primary bone marrow. The images were captured under Leica DM2000 microscope at ×100 magnification, scale bar=10μm. (C) Representative images of colonies generated by sorted IL3R+CD71−, EP1, EP2, EP3 and EP4 cells, in complete medium. The photos were taken using a Nikon D3500 camera. (D) Representative images of colonies generated by sorted IL3R+CD71−, EP1, EP2, EP3 and EP4 cells, in complete medium. The photos were taken under an inverted microscope at ×4 magnification, scale bar=1mm. (E) Quantitative analysis of colony forming ability of sorted IL3R+CD71−, EP1, EP2, EP3 and EP4 cells from six independent experiments.
Figure 4.
Figure 4.. Decreases in CD105 expression levels distinguish terminally differentiating erythroblasts in human bone marrow.
(A) Relative mRNA expression of CD105 at distinct stages of erythroid differentiation. The data are from RNA-seq on 3 biological replicates. (B) Representative FACS plots for definition of erythroblasts at distinct stages. The gates were made based on expression of CD105 and GPA. (C) Dot plot overlay of gated erythroblast populations showing their expression of α4-integrin and Band3. The color of each population sources from the gating plot (B). (D) Representative cytospin images of sorted erythroblasts at distinct stages from primary bone marrow. The images were captured under Leica DM2000 microscope at ×100 magnification, scale bar=10μm.
Figure 5.
Figure 5.. An integrated FACS strategy enables stage-wise detection of human adult erythropoiesis.
(A) Representative FACS plots illustrating an integrated gating strategy for human erythroid continuum from BFU-E to reticulocytes from primary human bone marrow. (B) Dot plot overlay of gated erythroid progenitor and erythroblast populations showing their expression of CD105 and GPA. The color of each population sources from the gating plot (A) and the curved arrow indicates the erythroid differentiation trajectory. (C) Representative FACS plots showing sequential maturation of sorted EP1, EP2, EP3 and EP4 in culture along the indicated trajectory in (B). (D) Representative histograms of erythroid-associated genes expression (CD117, CD71, CD36, CD38 and CD45) in primary human bone marrow cells at distinct erythroid differentiation stage.
Figure 6.
Figure 6.. The impaired terminal erythroid differentiation in MDS is associated with defective erythroid progenitor differentiation.
(A) The FACS plots showing erythroid continuum in MDS. The upper panel shows CD105/GPA overlay of erythroid populations, and the lower panel shows further analysis on erythroid progenitor differentiation. (B) Quantification of erythroid progenitors and erythroblasts showing decreased EPs in MDS patients with impaired terminal erythroid differentiation. The Y-axis indicates the cell number of each population in 104 erythroid cells. The black circles are healthy controls and the colored dots represent the different MDS samples. Each color corresponds to a specific MDS sample.
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