Figure 1.
Figure 1. STAT5 and Bcl-xL can induce erythroid differentiation. (A) A constitutively active form of STAT5 induced erythroid differentiation in UT7 cells. UT7 cells were transduced either with the empty retrovirus MIGR or with the retrovirus coding for a constitutively phosphorylated STAT5 (STAT5CA). After cell sorting according to GFP expression, cells were cultured in the presence of GM-CSF, without Epo. GpA expression was monitored by flow cytometry at day 4 after retrovirus infection. In the cells expressing STAT5CA, the percentage of GpA+ cells was much greater than in UT7 transduced with the empty vector (MIGR: 9% ± 1%; STAT5CA: 30% ± 3%; n = 3; Student t test: P = .004). (B) STAT5CA expression in human primary progenitors induced Epo-independent terminal erythroid differentiation. CD34+ cells from PB were cultured in the presence of Epo, SCF, IL-3, and DXM and then transduced twice with the different retroviral vectors (MIGR or STAT5CA). Transduced CD36+/GpA- cells were sorted at day 6 and cultured in the presence of SCF without Epo. Flow cytometry analysis showed 48 hours later that the percentage of GpA+ cells was higher with STAT5CA (48% ± 6%) than with the empty vector MIGR (19% ± 2%; n = 3; Student t test: P = .04). Determination of GpA expression was done using a more sensitive cell analyzer than the one used for cell sorting, and this explains why around 20% of the MIGR-transduced cells were found GpA positive. Moreover, cells transduced with the empty vector died after 48 hours of Epo removal, whereas STAT5CA-expressing cells further survived and proliferated (data not shown). (C) Bcl-xL overexpression in human primary progenitors induced GpA expression despite the absence of Epo. Primary cells were transduced either with the empty vector MIGR or with a vector coding for the Bcl-xL cDNA. Two days after cell sorting and Epo removal, 38% ± 4% of Bcl-xL-overexpressing cells were GpA positive, whereas only 18% ± 4% of cells infected with the empty vector MIGR were GpA positive (n = 3; Student t test: P = .04). Data are represented as mean ± SEM. As observed in STAT5CA-expressing cells, Bcl-xL-overexpressing cells could further proliferate and differentiate. When transduced with the empty vector, all cells died 48 hours after EPO removal.

STAT5 and Bcl-xL can induce erythroid differentiation. (A) A constitutively active form of STAT5 induced erythroid differentiation in UT7 cells. UT7 cells were transduced either with the empty retrovirus MIGR or with the retrovirus coding for a constitutively phosphorylated STAT5 (STAT5CA). After cell sorting according to GFP expression, cells were cultured in the presence of GM-CSF, without Epo. GpA expression was monitored by flow cytometry at day 4 after retrovirus infection. In the cells expressing STAT5CA, the percentage of GpA+ cells was much greater than in UT7 transduced with the empty vector (MIGR: 9% ± 1%; STAT5CA: 30% ± 3%; n = 3; Student t test: P = .004). (B) STAT5CA expression in human primary progenitors induced Epo-independent terminal erythroid differentiation. CD34+ cells from PB were cultured in the presence of Epo, SCF, IL-3, and DXM and then transduced twice with the different retroviral vectors (MIGR or STAT5CA). Transduced CD36+/GpA- cells were sorted at day 6 and cultured in the presence of SCF without Epo. Flow cytometry analysis showed 48 hours later that the percentage of GpA+ cells was higher with STAT5CA (48% ± 6%) than with the empty vector MIGR (19% ± 2%; n = 3; Student t test: P = .04). Determination of GpA expression was done using a more sensitive cell analyzer than the one used for cell sorting, and this explains why around 20% of the MIGR-transduced cells were found GpA positive. Moreover, cells transduced with the empty vector died after 48 hours of Epo removal, whereas STAT5CA-expressing cells further survived and proliferated (data not shown). (C) Bcl-xL overexpression in human primary progenitors induced GpA expression despite the absence of Epo. Primary cells were transduced either with the empty vector MIGR or with a vector coding for the Bcl-xL cDNA. Two days after cell sorting and Epo removal, 38% ± 4% of Bcl-xL-overexpressing cells were GpA positive, whereas only 18% ± 4% of cells infected with the empty vector MIGR were GpA positive (n = 3; Student t test: P = .04). Data are represented as mean ± SEM. As observed in STAT5CA-expressing cells, Bcl-xL-overexpressing cells could further proliferate and differentiate. When transduced with the empty vector, all cells died 48 hours after EPO removal.

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