A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis

Platelet bioreactor-on-a-chip

“…on April 2, 2019. by guest www.bloodjournal.org From has been that in vitro cultures extend proPLTs at a significantly slower rate than what has been observed in vivo. Application of physiological shear stress in our microfluidic bioreactor increased the proPLT extension rate by an order of magnitude above static culture controls 41 (supplemental Figure 6I) to ;30 mm/min (Figure 3E), which agree with physiological estimates of proPLT extension rates from intravital microscopy studies in living mice 11 and support increased PLT production in vitro.…”

“…Increasing shear stress within the physiological range did not affect the median proPLT extension rate or the distribution of proPLT extension rates in primary MK culture ( Figure 4F), and proPLT projections in primary mouse MKs retrovirally transduced to express green flourescent protein-b1 tubulin were comprised of peripheral MTs that formed coils at the PLT-sized ends ( Figure 4G and supplemental Movie 8). ProPLTs reached lengths exceeding 5 mm (supplemental Figure 4A) and resisted shear stress up to 1200 mPa in vitro; recapitulating physiological examples of proPLT production from intravital microscopy 11 and demonstrating that abscission events were not caused by membrane tethering. To confirm that shear stress-induced proPLT extension was cytoskeletal-driven, primary mouse MKs were incubated with 5 mM Jasplakinolide ([Jas] actin stabilizer) or 1 mM erythro-9-(3-[2-hydroxynonyl]) (EHNA, cytoplasmic dynein inhibitor) prior to infusion into the microfluidic bioreactor.…”

“…Primary mouse MKs cultured under physiological shear stress produced fewer, longer proPLTs that were less highly branched relative to static cultures (supplemental Movies 1-2). ProPLTs in shear cultures were uniformly extended into the lower channel and aligned in the direction of flow against the vascular channel wall, recapitulating physiological proPLT production 11,30,32 (supplemental Movie 2). The percent of proPLT-producing primary mouse MKs under physiological shear stress were doubled over static cultures 26 to ;90% ( Figure 3D).…”

“…By blocking the upper right channel output to direct flow across the bioreactor, primary mouse MKs infused along the top channel become sequentially trapped between the columns and extend proPLTs into the lower channel ( Figure 2B), recapitulating physiological proPLT extension (white arrow). 11,[30][31][32] To model threedimensional ECM organization and physiological BM stiffness (250 Pa) 33 (supplemental Figure 1A-D), primary mouse MKs were infused in a 1% alginate or 3 mg/mL growth factor reduced Matrigel solution. Alginates are naturally derived polysaccharides that are cell-inert and can be modified with RGD-containing cell adhesion ligands to specifically reproduce MK-matrix interactions in the BM, such as glycoprotein (GP)IIb/IIIa receptor binding to fibrinogen.…”

“…[11][12][13][14][15] Nearly 100% of human adult MKs must produce ;10 3 PLTs each to account for circulating PLT counts. 16 Although functional human PLTs were first grown in vitro in 1995, 17 to date only ;10% of human MKs initiate proPLT production in culture.…”