Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Aishwarya Swaminathan; Yung Hwang; Ashley Winward; Merav Socolovsky

doi:10.3791/64373

Method Article

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

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DOI:

10.3791/64373

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November 4th, 2022

Aishwarya Swaminathan¹ , Yung Hwang¹ , Ashley Winward¹ , Merav Socolovsky¹

¹Department of Molecular, Cell and Cancer Biology, UMASS Chan Medical School

Summary

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Abstract

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming "units" known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit⁺ CD55⁺ CD49f^med/lowCD105^med/highCD71^med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71^medCD150^high) and P1-hi (CD71^highCD150^low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit⁺ CD55⁺ CD49f^med/lowCD105^med/high CD71^lowCD150^high) contains all of the BFU-e progenitors; Population P3 (P3, Kit⁺ CD55⁺ CD49f^med/highCD105^med/lowCD150^low CD41^low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit⁺ CD55⁺ CD49f^med/highCD105^med/lowCD150^highCD41⁺) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit⁺ CD55⁺ CD49f^med/highCD105^med/lowCD150^highCD41^-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Introduction

Erythropoiesis may be divided into two principal phases: early erythropoiesis and erythroid terminal differentiation (Figure 1)¹^,²^,³. In early erythropoiesis, hematopoietic stem cells commit to the erythroid lineage and give rise to early erythroid progenitors, which were first identified in the 1970s based on their colony-forming potential in semi-solid medium⁴^,⁵^,⁶^,⁷^,⁸^,