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

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, Sandrina Kinet and Narla Mohandas are co-senior authors.

Molecular and cellular mechanisms that regulate human erythropoiesis

“…It is possible to enrich for different subsets of erythroid progenitors based on CD34/CD36/CD71 and CD105 sequential expression in CD123 (IL-3 receptor) negative cells. 8 Combining expression of GYPA (CD235A) and CD105, or alternatively band 3/SLC4A1 acquisition (CD233) and CD49d loss, can allow tracking of erythroid maturation ( Figure 1 ). 9 This cellular tracking can be helpful to elucidate the mechanisms of ineffective erythropoiesis.…”

“… 9 This cellular tracking can be helpful to elucidate the mechanisms of ineffective erythropoiesis. 8,10 Importantly, although these surface markers enable enrichment of distinct stages of this differentiation process, sorted populations are still heterogeneous, and some subsets with intermediate antigen expression can be challenging to definitively classify. 10 Conversely, emerging single-cell genomic studies suggest a more continuous view of this process, with a continuum of cell states that vary by global gene expression.…”

Epo-IGF1R crosstalk expands stress-specific progenitors in regenerative erythropoiesis and myeloproliferative neoplasm

“…In humans, intermediate populations between BFU-E and CFU-E have been observed, 32-34 and IRS2 expression increases upon erythroid differentiation in CD34+ cell cultures. 35 We therefore tried to correlate our findings in the human setting.…”

Erythroid Cell Research: 3D Chromatin, Transcription Factors and Beyond

“…Erythroid research remains at the forefront of many research fields. The new molecular markers [ 110 , 157 , 158 ] and complex (epi-)genetic [ 96 , 98 , 159 ] and metabolic networks [ 160 , 161 , 162 ] that are continuously unraveled pave the way towards an in-depth understanding of erythropoiesis. Furthermore, the recent discovery of the molecular bases of new blood group antigens keep providing key fundamental and clinical insights into erythropoiesis and open up new avenues for the management of transfusion issues associated with rare blood group-bearing patients [ 163 , 164 , 165 ].…”