32D TPO, a TPO-Dependent Subclone of the 32d Cell Line, Expresses CXCR4 and Proliferates in Response to SDF-1.

Both Thrombopoietin (TPO) and Stromal Derived Factor 1 (SDF-1) have been reported to regulate megakaryocyte differentiation in vivo and in vitro. Using growth factors [G-CSF, M-CSF, GM-CSF, erythropoietin (EPO) or thrombopoietin (TPO)], as selective pressure, factor-dependent clones were consistently isolated from the murine multipotential IL-3-dependent cell line 32D. The different clones acquired a phenotype consistent with the growth factor for which they were dependent. In particular, the EPO-dependent clones showed erythroblast morphology and expressed high levels of CD71 and TER119, on the other hand, the TPO-dependent clones were megakaryocytes in morphology and expressed CD41 and CD61 (see Figure). The TPO-dependent subclones retain the capacity to grow in IL-3, but respond to neither EPO, G-CSF or GM-CSF. Here it is shown that the TPO-dependent clone, but not its EPO-dependent sister (see figure) or parental 32D cell lines, express robust level of CXCR4 on their surface. 32D TPO cells are also capable to respond to SDF-1 (the CXCR4 ligand). In fact, these cells die in 3 days in the absence of growth factors but proliferate equally well (from 5x10⁴ to 6–9x10⁵ cells/mL) in the presence of TPO (100 ng/mL) and/or SDF-1 (100 ng/mL), retaining a robust megakaryocytic phenotype in both culture conditions. By expression profile 32D TPO cells growing in TPO express the same level of Gata1 (both ~2^−ΔCt = 3.6 ± 0.5x10⁻² ) and Platelet Factor-4 (both 2^−ΔCt = 3.5±1x10⁻¹) more Gata2 (2^−ΔCt = 2.3±0.1x10⁻¹ vs 1.3±0.2x10^−1, respectively), and Fog1 (2^−ΔCt = 4.5±1x10⁻² vs 3.1±0.5x10^−2, respectively), and less Acetylcholinesterase (2^−ΔCt = 4.96±1x10⁻⁵ vs 6.68±2x10^−5, respectively), and Von Willebrand Factor (2^−ΔCt = 2.7±0.3x10⁻² vs 3.4±0.5x10^−2, respectively), than those growing in SDF-1. These data indicate that a TPO-dependent cell line acquires the ability to respond to SDF-1, supporting the notion that megakaryocytic differentiation can be sustained equally well by TPO or SDF-1. To our knowledge, the 32D TPO cell line is the first megakaryocytic cell line that is dependent for growth on the presence of SDF-1. As such, it might represent a suitable model to compare TPO and SDF-1 signalling in megakaryocytes.

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