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Thrombopoietin maintains megakaryopoietic HSPC
antibodies and propidium iodide was added prior to analysis to identify live cells.
mRNA expression
Cells were sorted into Trizol (Thermo Fisher Scientific) and RNA was isolated according to the manufacturer’s protocol. Complementary DNA was reverse transcribed by a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) and mRNA expression analyzed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) in a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). Expression levels were calcu- lated relative to the expression of Gapdh.
In vitro culture
BMMNC were isolated as above and cKit enrichment was per-
formed using magnetic activated cell sorting (MACS)-conjugated cKit antibodies (Miltenyi Biotec) and an AutoMACS cell sorter according to the manufacturer’s protocol. Enriched cells were stained with antibodies and sorted to StemSpanII (StemCell Technologies) containing stem cell factor (SCF, 20 ng/mL, Peprotech) and THPO (20 ng/mL, Peprotech), or SCF, THPO, interleukin-3 (IL3, 20 ng/mL, Peprotech), interleukin-6 (IL6, 20 ng/mL, Peprotech) and erythropoietin (EPO, 10 ng/mL, R&D Systems). On day 10 of culture colonies were stained with CD41, CD16/32, CD71 (C2, BD Biosciences), Ter119, CD11b,
A B C D
EFGH
IK
JL trol mice (A), as well as Thpo-/- and ThpoWT/WT mice (B). (C, D) Thymus CD4+ and CD8+ T- cell numbers were analyzed in the two mod- els. (E-H) Peripheral blood white blood cells (E, F) and red blood cells (G, H) were quan- tified. (I-L) Lymphoid progenitors (I, J) and myeloid progenitors (K, L) were quantified In the bone marrow of the two murine models. (n=6) PB: peripheral blood; WBC: white blood cells; RBC: red blood cells; BMMNC: bone marrow mononuclear cells; CLP: com- mon lymphoid progenitors; GMP: granulo- cyte/ macrophage progenitors; MegE: megakaryocute/erythroid progenitors; CFU-
Figure 2. Thrombopoietin maintains hematopoietic stem and progenitor cells with megakaryopoietic potential. (A, B) Mature B-cell counts were analyzed in the bone marrow of ThpoΔ/Δ Alb-Cre and Thpofl/fl con-
E: colony-forming unit erythroid.
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