Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL
Received 13 July 2009; received in revised form 31 August 2009; accepted 2 September 2009. published online 09 September 2009.
Objective
The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs.
Materials and Methods
hCB CD34+ or CD34+CD38– hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency–repopulating cell assays.
Results
During a 7-day culture of CD34+ or CD34+CD38– hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency–repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34+ cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1α protein and enhanced activation of its downstream target genes.
Conclusion
This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.
aResearch Promotion and Coordination Department, Nissan Chemical Industries, Tokyo, Japan
bSynthesis Research Department, Chemical Research Laboratories, Nissan Chemical Industries, Chiba, Japan
cPharmaceutical Research Department, Biological Research Laboratories, Nissan Chemical Industries, Saitama, Japan
eDepartment of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan
fCenter for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Japan
Offprint requests to: Atsushi Iwama, M.D., Ph.D., Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670 Japan
ABSTRACT