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Hematopoietic progenitor cell lines with erythroid and megakaryocyte potential

ABSTRACT: Red blood cells (RBC) and platelets are generated from a common, bi-potent hematopoietic progenitor cell in the bone marrow. Important events orchestrating the RBC- and platelet cell lineages have been defined, however, more detailed investigation is limited by shortcomings of suitable experimental systems. Here we show that retroviral delivery of an estrogen-regulated form of Hoxa7 into unseparated bone morrow cells along with thrombopoietin (TPO) can be used to immortalize conditionally bi-potent hematopoietic progenitor cells (Hoxa7-TPO). Hoxa7-TPO cells have lost self-renewal capacity and the potential to differentiate into leukocytes, but retain the potential to differentiate into red blood cells and platelets. Consistent with this lineage potential, gene expression and phenotype analysis of non-differentiated Hoxa7-TPO cells show a close relationship to primary bi-potent megakaryocyte erythrocyte progenitors (MEP). Mature Hoxa7-TPO-derived RBC and platelets are phenotypically and functionally indistinguishable from their primary counterparts. Consistent with this close relationship to primary cells, CRISPR/ Cas9-mediated deletion of known differentiation factors, such as Klf1, Nfe2 and Gp1ba mediates the expected impact on RBC and platelet generation in vivo, with GP1ba-deletion recapitulating the human Bernard-Soulier syndrome. The simplicity of this system, including its amenability to genetic manipulation, along with the unlimited proliferative capacity of Hoxa7-TPO cells will facilitate studies related to cell differentiation and function of the RBC- and megakaryocyte lineages.

ORGANISM(S): Mus musculus

PROVIDER: GSE244976 | GEO | 2025/06/24

REPOSITORIES: GEO

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Project description:The haematopoietic cytokine thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for maintenance of the haematopoetic stem cell compartment. Tpo is a heavily glycosylated, hepatocyte-derived cytokine which functions by binding to its receptor (TpoR) on target cells and thereby activating intracellular signalling cascades that induce their proliferation and/or differentiation. In addition to its role in signal propagation, TpoR is expressed on the surface of platelets, where it contributes to regulation of Tpo levels by sequestering circulating cytokine. TpoR belongs to the homodimeric Class I cytokine receptor family but is unusual due to a duplication of the Cytokine binding Homology Region (CHR). Almost thirty years after initial discovery of TpoR, the structure of the human Tpo:TpoR interaction was recently reported. Here we determine the structure of extracellular portion of the murine Tpo:TpoR signalling complex using single particle cryo-EM. The structure reveals that Tpo:TpoR forms a largely symmetrical 1:2 complex. The cytokine cross-links the same site on the membrane-distal CHR of both receptor chains using opposing surfaces and with significantly different affinities. This orients the two membrane-proximal CHRs such that they contact one another adjacent to the plasma membrane. The potential cytokine-binding site in CHR2 is glycosylated and does not interact with Tpo. A large insertion in CHR1 that is unique to Tpo forms a partially structured loop that is disulphide bonded to CHR2 and, in one receptor chain, contacts cytokine. Biochemical analyses indicate that the glycosylated C-terminal domain of Tpo does not influence receptor binding. We demonstrate that the therapeutic TpoR agonist Romiplostim binds to the same site on the receptor as does cytokine. Our study characterises the Tpo/TpoR interaction structurally and biochemically to allow for the future development of potent TpoR agonists for therapeutic use.

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Hematopoietic progenitor cell lines with erythroid and megakaryocyte potential

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