Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL.
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.


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-1alpha protein and enhanced activation of its downstream target genes.


CONCLUSIONS
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.
Nishino
Taito
T
Research Promotion and Coordination Department, Nissan Chemical Industries, Tokyo, Japan.
Miyaji
Katsuaki
K
Ishiwata
Norihisa
N
Arai
Kazutaka
K
Yui
Makiko
M
Asai
Yasuyuki
Y
Nakauchi
Hiromitsu
H
Iwama
Atsushi
A
D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor / Q000166:cytology / Q000187:drug effects; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000637:transplantation; D036101:Cord Blood Stem Cell Transplantation; D053648:Cytokine Receptor Common beta Subunit / Q000235:genetics / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D005312:Fetal Blood / Q000166:cytology; D005786:Gene Expression Regulation / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics; D053650:Interleukin-3 Receptor alpha Subunit / Q000235:genetics / Q000502:physiology; D007951:Leukemia, Myeloid / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011828:Radiation Chimera; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000502:physiology; D053628:Receptors, Thrombopoietin / Q000819:agonists* / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D034061:Thrombopoiesis / Q000187:drug effects*; D013926:Thrombopoietin / Q000494:pharmacology
C115876:CSF2RB protein, human; D053648:Cytokine Receptor Common beta Subunit; D018076:DNA, Complementary; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C115022:IL3RA protein, human; D053650:Interleukin-3 Receptor alpha Subunit; D017467:Receptors, Erythropoietin; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; C082218:MPL protein, human; D013926:Thrombopoietin