TY  - JOUR
TI  - Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms
AU  - Thompson-Peach, Chloe A. L.
AU  - Foßelteder, Johannes 
AU  - Reinisch, Andreas 
AU  - Thomas, Daniel 
VL  - 13
IS  - 2
PY  - 2023
DA  - 2023/01/20
SP  - e4592
C1  - Bio-protocol 2023;13:e4592
DO  - 10.21769/BioProtoc.4592
UR  - https://doi.org/10.21769/BioProtoc.4592
AB  - Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient–derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders.Graphical abstractSchematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation
KW  - Hematopoietic stem and progenitor cells
KW  - Megakaryocyte differentiation
KW  - Thrombopoietin
KW  - TPO-independent
KW  - Myeloproliferative neoplasm
KW  - Collagen
KW  - Interleukin-6
KW  - Interleukin-9
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False