PDPN/CLEC-2 axis modulates megakaryocyte subtypes in a hematopoietic stem cell-regulating megakaryocyte-dominant manner
Rikuto Nara a), Hinako Notoh a), Tomoyuki Sasaki b), Nagaharu Tsukiji b), Toshiaki Shirai b), Ayuka Kamata a), Nobuaki Suzuki c), Atsuo Suzuki d), Shuichi Okamoto e), Takeshi Kanematsu f), Naruko Suzuki f), Akira Katsumi g), Tetsuhito Kojima e), h), Katsue Suzuki-Inoue b), Tadashi Matsushita c), f), Shogo Tamura c), i)
a) Graduate School of Health Sciences, Hokkaido University, Japan
b) Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, Chuo, Yamanashi, Japan
c) Department of Transfusion Medicine, Nagoya University Hospital, Nagoya, Japan
d) Department of Medical Technique, Nagoya University Hospital, Japan
e) Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan
f) Department of Clinical Laboratory, Nagoya University Hospital, Nagoya, Japan
g) Department of Hematology, National Center for Geriatrics and Gerontology, Obu City, Japan
h) Aichi Health Promotion Foundation, Nagoya, Japan
i) Department of Clinical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan
Abstract
Introduction
Megakaryocytes are classified into several subtypes including LSP1-positive immune-skewed, MYLK4-positive hematopoietic stem cell (HSC)-regulating, and BMAL1-positive platelet-producing megakaryocytes. Podoplanin (PDPN)-expressing stromal cells generate a microenvironment that promotes megakaryopoiesis in the bone marrow. In this context, PDPN interacts with C-type lectin-like receptor-2 (CLEC-2) on megakaryocyte progenitors, which induces megakaryocyte proliferation. However, the megakaryocyte subtypes developed by the regulation of the PDPN/CLEC-2 axis have not yet been elucidated.
Materials and methods
We established an immortalized bone marrow PDPN-expressing stromal cell line and a PDPN-knockout line (PDPN WT and KO feeder cells, respectively). Bone marrow hematopoietic progenitors were committed to megakaryocytes in co-culture with PDPN WT or KO feeder cells. The number and ploidy of megakaryocytes, resultant platelets, and the polarization of megakaryocyte subtypes were investigated.
Results
The number of megakaryocytes was significantly increased in the co-culture with PDPN WT feeder cells compared to that with PDPN KO feeder cells. The megakaryocytes on the PDPN WT and KO feeders showed their main ploidy at 16 N∼32 N and 8 N∼16 N, respectively. The number of platelets was decreased in the co-culture with the PDPN WT feeder compared to that in the co-culture with the PDPN KO feeder. For each megakaryocyte subtype, the percentage of MYLK4-positive megakaryocytes significantly increased and the percentage of BMAL1-positive megakaryocytes significantly decreased when co-cultured with the PDPN WT feeder. These results were also confirmed in the co-culture of CLEC-2 conditional KO megakaryocytes with PDPN WT feeder cells.
Conclusion
The PDPN/CLEC-2 axis modulates megakaryocyte subtype differentiation, with a predominance of HSC-regulating megakaryocytes.