Hematopoietic Stem Cells, but Not Progenitors, Are the Exclusive Target for STAT5-Induced Long-Term Self-Renewal.
Authors: Fatrai Szabolcs , Wierenga Albertus T.J. , Vellenga Edo , Daenen Simon M. G. J. , Schuringa Jan J. 
Published: April 2020

Constitutive activation of STAT5 has been associated with leukemic transformation. Previously, we demonstrated that overexpression of activated STAT5 in human cord blood (CB) cells results in increased stem cell self-renewal and long-term expansion which coincided with the induction of erythropoiesis. In the present study we investigated whether STAT5-imposed long-term self-renewal is exclusively restricted to HSCs, or whether long-term self-renewal can also be imposed on progenitor cells. We generated retroviral expression systems where STAT5 is fused to the estrogen receptor ligand binding domain (ER) allowing the induction of STAT5 activity by treatment of cells with 4-hydroxytamoxifen (4-OHT). Human CB cells were transduced with control and STAT5-ER retroviral vectors followed by MoFlo sorting into four populations: hematopoietic stem cells (HSC, defined as CD34+CD38low), common myeloid progenitors (CMP, CD34+CD38+CD123+CD45RA−), granulocyte-macrophage progenitors (GMP, CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors (MEP, CD34+CD38+CD123−CD45RA−). Sorted populations were plated on MS5 bone marrow stromal cocultures and STAT5 activity was induced by 4-OHT treatment. HSC cells expressing activated STAT5 displayed a long-term proliferative advantage as well as a significant increase in cobblestone formation. This coincided with elevated levels of Colony Forming Cells (CFCs) that were maintained over 5 weeks. In contrast, STAT5 was unable to induce cobblestone formation in progenitor cocultures and only a transient STAT5-induced increase in cell numbers was observed in cocultures initiated with CMPs and MEPs. Also, CFC numbers dropped significantly after 2 weeks and neither of the cultures could be maintained longer than 3 weeks regardless of STAT5 activity. FACS measurements and cytospins showed a block in myelopoiesis and an increase in erythroid differentiation in STAT5-ER-transduced HSC, CMP, and MEP populations, while the differentiation potential of the GMP remained unaffected. Next, we aimed to identify STAT5 target genes which were upregulated in the STAT5 HSC population that were not responsible for erythroid differentiation and played a role in STAT5-induced self-renewal and long-term expansion. GATA1 was down-modulated in STAT5-transduced CB cells by a lentiviral RNAi approach, which completely abrogated erythropoiesis but maintained enhanced HSC self-renewal. Microarray was performed on both GATA1 downmodulated STAT5-transduced CB cells as well as on STAT5-transduced HSC and progenitor populations. Micoarray data from the two experiments were compared and 39 GATA1- independent STAT5 target genes were identified in the STAT5 HSC population. Many of these genes encoded for membrane proteins or proteins involved in adhesion, migration and signal transduction, and these are currently under investigation. In summary, our data show that hematopoietic stem cells, but not progenitors are the exclusive target for STAT5- induced long-term self-renewal. We identified a set of genes that is upregulated in the STAT5-transduced HSC population in a GATA1-independent manner which is potentially responsible for STAT5-induced self-renewal and long-term expansion.