PDPN/CLEC-2 axis modulates megakaryocyte subtypes in a hematopoietic stem cell-regulating megakaryocyte-dominant manner

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 16N∼32N and 8N∼16N, respectively. The number of platelets decreased in the co-culture with the PDPN WT feeder compared to those in the co-culture with the PDPN KO feeder. Megakaryocyte subtypes were immunocytochemically detected in in vitro differentiated CD41-positive megakaryocytes. 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. Conclusion The PDPN/CLEC-2 axis modulates megakaryocyte subtype differentiation, with a predominance of HSC-regulating megakaryocytes.

This study aimed to characterize the megakaryocytes that develop in response to the PDPN/CLEC-2 axis.To our knowledge, this is the first report to demonstrate that bone marrow microenvironmental factors such as PDPN modulate megakaryocyte subtype differentiation.The present findings improve our understanding of how megakaryocytes interact with their environment in the bone marrow.

Mice
C57BL/6NcrSlc mice were purchased from CLEA Japan, Inc. (Tokyo, Japan).They were bred and maintained under standard conditions [12 h light/dark cycle with stable temperature (25 °C) and humidity (60%)].This study was approved by the Animal Care and Use Committee of the Hokkaido University (22-0087).
Establishment of immortalized bone marrow podoplanin (PDPN)-expressing stromal cell-line and its PDPN KO-line Bone marrow stromal cells were isolated as previously described [16].Briefly, bone marrow cells were harvested from the femurs and tibias of young mice (7-12 weeks old).To use each cell line as a feeder, the cells were treated with culture medium containing 10 mg/mL mitomycin C (Nacalai Tesque) for 2 h and collected after washing with phosphate-buffered saline (PBS).The mitomycin C-treated feeders were re-seeded at a density of 0.65 × 10 5 /cm 2 (2.5 × 10 5 cells/well in 12-well plates).
In vitro megakaryocyte differentiation and co-culture with feeder cells Bone marrow hematopoietic progenitors were cultured in IMDM supplemented with 10% FBS, antibiotics, and 50 ng/mL thrombopoietin (PeproTech) for 5 days to induce megakaryocyte differentiation.On culture day 3, GM6001 (50 mM final concentration; Millipore), a broad-range matrix metalloproteinase inhibitor, was added to the culture medium to suppress the shedding of surface proteins from megakaryocytes.For the coculturing experiments, hematopoietic progenitors (1.0 × 10 5 cells per well) were cultured on a feeder cell layer in 12-well plates.

Immunocytochemistry
The cells were plated and cultured on 15 mm growth cover glasses (Thermo Fisher Scientific) in 12-well plates.The cells were fixed with 4% paraformaldehyde.For the coculture of megakaryocytes and feeder cells, we carefully pipetted drops of the fixative into the wells and fixed the cells for 1 h to preserve cell-cell contact.The cells were then permeabilized, if necessary, with 0.5% Triton X-100 in PBS for 10 min.

Statistics
Quantitative data are depicted as mean ± standard distribution of the mean.
Representative data from at least three independent experiments are shown.
Comparisons between two groups were performed using the unpaired t-test.Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software).

Results and Discussion
PDPN/CLEC-2 axis promotes megakaryocyte proliferation and polyploidization, but decreases the resultant platelet generation.
To investigate megakaryocyte subtype differentiation via the PDPN/CLEC-2 axis, we first established an immortalized bone marrow PDPN-expressing stromal cell line and a PDPN knockout line (PDPN WT and KO feeder cells, respectively, Fig. 1).
Bone marrow hematopoietic progenitors were committed to megakaryocytes in coculture with PDPN WT or KO feeder cells, and the number and ploidy of megakaryocytes were evaluated (Fig. 2A).In addition, the number of resultant platelets in culture with PDPN WT or KO feeder cells were also evaluated.2F and G).The PDPN/CLEC-2 axis induces megakaryocyte proliferation and alters the ploidy pattern of megakaryocytes, but decreases the resultant platelet generation.
Megakaryocyte subtypes are distinguished by specific marker proteins: LSP1 in immune-skewed, MYLK4 in HSC-regulating, and BMAL1 in platelet-producing megakaryocytes [4].These subtype marker proteins were immunocytochemically detected in in-vitro differentiated CD41-positive megakaryocytes (Fig. 3A).The size of each megakaryocyte subtype was assessed by measuring the major axis diameter (Fig. 3B).The diameter of the LSP1-positive, the MYLK4-positive, and the BMAL1-positive megakaryocytes were 44.80 ± 7.01 m, 49.29 ± 20.87 m, and 51.65 ± 24.59 m, respectively.Immune-skewed LSP1-positive megakaryocytes have a small cytomorphology [4], and megakaryocytes with high ploidy (>16N) and a large cytoplasm exhibit HSC regulation and platelet production functions [18].In the present study, LSP1-positive megakaryocytes tended to be smaller than the other megakaryocyte subtypes, but the difference was not statistically significant among the megakaryocyte subtypes due to the wide size distribution of MYLK4-positive and BMAL1-positive megakaryocytes.Wang et al. reported that human megakaryocyte subtypes are produced via distinct routes [6].We consider that the smaller MYLK4-and BMAL1positive megakaryocytes are cells in the early stages of their differential route.We evaluated the ability of each megakaryocyte subtype to form platelets (Fig. 3C).In the in vitro differentiated megakaryocytes, BMAL1 was detected in 83% of the proplateletforming megakaryocytes, whereas LSP1 and MYLK4 were not detected in the proplatelet-forming megakaryocytes (Fig. 3D).Based on these observations, we confirmed that the immunocytochemical approach for detecting LSP1, MYLK4, and BMAL1 represented their corresponding megakaryocyte subtypes.We evaluated the percentage of each megakaryocyte subtype in co-cultures with PDPN WT or KO feeder cells (Fig. 4).The percentage of LSP1-positive megakaryocytes did not differ between the co-culture with PDPN WT and KO feeders (7.89±0.96% in PDPN WT vs 12.72±2.45% in PDPN KO, Fig. 2B).The percentage of MYLK4-positive megakaryocytes was significantly increased in the co-culture with PDPN WT feeder as compared with that with PDPN KO feeder (67.27±0.32% in PDPN WT vs 57.47±3.09% in PDPN KO, Fig. 2C).In contrast, the percentage of BMAL1-positive megakaryocytes was significantly decreased in the co-culture with PDPN WT feeder as compared with that with PDPN KO feeder (39.99±3.32% in PDPN WT vs 54.90±3.58% in PDPN KO, Fig. 2D).These observations suggest that the PDPN/CLEC-2 axis induces a state of HSC-regulating megakaryocyte predominance in this population.

Flushed
bone marrow cells were suspended in cold Iscove's modified Dulbecco's medium (IMDM; Wako) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and penicillin-streptomycin (Wako) and seeded into 12-well plates or cell-culture dishes.The culture medium was changed daily for 5-7 d to remove non-adhesive cells until proliferation of adhesive bone marrow stromal cells.To immortalize these stromal cells, the human telomerase reverse transcriptase gene (hTERT) was introduced via retroviral infection.The recombinant virus was prepared by transfecting the pBABE-Puro-hTERT retroviral vector (Cell Biolabs Inc.) into Plat-E retroviral packaging cells (Cell Biolabs Inc.) using Lipofectamine 3000 (Thermo Fisher Scientific).After infecting these stromal cells with the resultant virus-containing culture medium, the immortalized stromal cells were cloned by puromycin selection.Podoplanin expression in cloned stromal cells was evaluated by immunocytochemistry and flow cytometry, as described in the following section.The cloned PDPN-expressing stromal cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Wako) containing 10% FBS and penicillin-streptomycin.The PDPN KO-line was generated by genome editing using the Guide-it CRISPR/Cas9 System (#632601; TaKaRa Bio).The genomic target loci (Pdpn exon 1) and guide sequence alignments are shown in Fig 1A.Plasmids were constructed according to the manufacturer's instructions.The constructed plasmid was transfected into immortalized PDPN-expressing stromal cells by electroporation using Gene Pulser Xcell (Bio-Rad).Transfectants were isolated by limiting the dilution.Genomic DNA extracted from the cloned transfectant was subjected to PCR using KOD FX (Toyobo) and the .The PCR amplicons were analyzed by direct Sanger sequencing using the BigDye Terminator v1.1 Cycle Sequencing Kit and an ABI Prism 310 Genetic Analyzer (Thermo Fisher Scientific).

Figure 1 .
Figure 1.Establishment of immortalized PDPN KO feeder cell line.A. Information on genomic target loci and guide sequence alinement in the Guide-it CRISPR/Cas9 System.B. Representative immunocytochemistry images of PDPN WT and KO feeders.Blue, DAPI; Green, PDPN; Red, CD44.Scale bar, 100 m.C. Flowcytometry histogram of PDPN expression in PDPN WT and KO feeder cells.

Figure 2 .
Figure 2. Cellular characteristics of megakaryocytes and their resultant platelets in coculture with PDPN WT or KO feeder cells.A. Graphical illustration of co-culture in bone marrow hematopoietic progenitors with PDPN WT or KO feeder cells.B and C. The number of CD41-positive megakaryocytes on PDPN WT or KO feeder cells.Representative immunocytochemical images (B) and their quantitative data (C).Scale bar, 100 m.D and E. Representative ploidy histograms of megakaryocytes co-cultured with PDPN WT or PDPN KO feeder (D and their quantitative data (E).****p < 0.0001.n = 3 per group.F and G.The number of CD41/CD42b-positive platelets on PDPN WT or KO feeder cells.Representative flowcytometric scatter-grams (F) and their quantitative data (G).*p < 0.05.**p < 0.01.TPO, thrombopoietin; MK, megakaryocyte; PDPN, podoplanin.