Abstract
Abstract 4742
The regulation of megakaryopoiesis and thrombopoiesis are incompletely understood. Better understanding of the underlying mechanisms would be of biological import, but may also lead to novel approaches for generating of platelets for clinical application. One cell line that undergoes terminal differentiation into megakaryocytes is pre-adipocytes. These cells represent a potential candidate cell for ex vivo generation of megakaryocytes. Here we demonstrate that pre-adipocytes differentiate into MKs and platelets by using an endogenous paracrine loop involving TPO, the primary cytokine involved in megakaryopoiesis, and its receptor c-mpl. We previously reported that pre-adipocytes differentiate into MKs and form platelets when the culture medium is switched from maintenance medium to MK lineage induction (MKLI) medium. Neither media include TPO. Based on these observations, the present study tested the hypothesis that pre-adipocytes might differentiate into MKs and platelets by endogenous TPO and c-mpl expression of sufficient magnitude to drive megakaryopoiesis. We used primary mouse pre-adipocytes (CD45-, CD117-, Sca1+, CD29+, CD73+, CD90+, CD105+, CD106+) from subcutaneous adipose tissues and also the mouse pre-adipocyte line 3T3-L1 cells. The TPO levels, as assessed by ELISA, in the supernatant from 106 pre-adipocytes cultured in 2 ml MKLI medium without exogenous TPO (TPO-) were unmeasurable level on Day 0, 29±14 pg/ml on Day 7 and 8±2 pg/ml on Day 12. Similar results were obtained in the supernatant from 3T3-L1 during MK differentiation. We did not observe measureable TPO in supernatants from pre-adipocytes and 3T3-L1 cells cultured in maintenance medium on Days 0, 7 and 12. We then compared MK differentiation from pre-adipocytes in MKLI media in the absence (TPO-) or addition (final concentration, 50 ng/ml; TPO+) of TPO. The frequency of CD41-postive pre-adipocytes in culture after 6 days was 58±11% for TPO+ and 70±7% for TPO- (p>0.05), consistent with endogenous TPO being sufficient under these circumstances to stimulate MK differentiation of pre-adipocytes. DNA ploidy and c-mpl expression assessed by immunohistochemistry were also similar with or without added exogenous TPO. We next examined the number of CD41-expressing large-sized (>15 μm) cells beginning with 1.2 × 104 preadipocytes in the absence or presence of the c-mpl inhibitor rat anti-mouse c-mpl inhibitor AMM2 (final concentration, 10 μg/ml). In the absence of AMM2, 10-fold more CD41-positive, large cells were seen (6.8±1.7×103) than in its presence (0.6±0.2×103, p<0.05). These observations support that pre-adipocytes differentiate into MKs using endogenous TPO stimulation via c-mpl. We next examined the ability to release platelets from these treated pre-adipocytes in each experimental condition (pre-adipocytes and 3T3-L1 cells, each ± exogenous TPO). Infusion of 2 × 105 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled MKs into irradiated thrombocytopenic mice (7 days post-2.0Gy exposure) led to a time-dependent appearance of CFSE+/CD41+ platelet-sized particles with a peak at 4 hours after the infusion reaching ∼2.5% of the total circulating platelet population (∼30 platelets/infused MK) under all tested experimental conditions. For platelet function, blood samples from each of these mice were perfused over a collagen-coated chip for 10 minutes, and the incorporation of CFSE+ particles into thrombi on the chip was determined (Total Thrombus-formation Analyzing System). A similar degree of platelet incorporation was observed under all experimental conditions and each was with an efficiency similar to that seen when CFSE+ control platelets were infused. These findings demonstrate that pre-adipocytes differentiate into MKs and subsequent platelets by an endogenous TPO release and a paracrine loop involving c-mpl. We propose that such pre-adipocytes could be used as a model of a continuously replicating cell line that upon switching media simultaneous expresses TPO and its receptor c-mpl to establish a paracrine loop leading to terminal differentiation into functional MKs. The basis of why this media switch induces this change needs to be clarified to further develop this pre-adipocyte strategy as a donor-independent source for platelet transfusion as well as for studying mechanism underlying megakaryopoiesis and thrombopoiesis.
Disclosures:
Matsubara: Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.
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