Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis

Abstract

Summary. Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum-free cell-culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum-free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) þIL-3 was TPO able to stimulate a small increase (ϳ25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34 þ cells were cultured for 7 d in serum-free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre-cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit-erythroid (BFU-E) colonies as cells which were pre-cultured in the absence of TPO. Further, when primitive CD34 þ , Kit þ MNC were cultured for 3-7 d under serum-free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.

The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions – comparison to other cytokines and growth factors

“…This effect was not previously observed in our serum supplemented cloning system, 12 which was probably 'contaminated' by INS present in the serum. 8,38 We also found, that when the IGF-1R expression was downregulated using an antisense strategy, the effect of INS on the late development of BFU-E colonies was still preserved. This suggests that INS acted through its own INS-R and not through the structurally similar IGF-1R.…”

Polycythemia vera: myths, mechanisms, and management

“…61 Thrombopoietin in conjunction with SCF promotes the production of neutrophils from CD34 ϩ cells 62 and in conjunction with erythropoietin, the production of erythroid progenitor cells, 63 an effect that may be due to abrogation of apoptosis. 64 Thrombopoietin overexpression in mice caused granulocytosis, thrombocytosis, osteomyelofibrosis with extramedullary hematopoiesis, and death, 65 while ectopic expression of Mpl caused fatal erythroblastosis. 66 Importantly, the retrovirus, MPLV, which encodes an Mpl gene truncated in its extracellular domain and fused with a viral envelope protein gene, induced a syndrome in mice that mimicked polycythemia vera, 67 while hematopoietic progenitor cells infected with MPLV were growth factor-independent in vitro and capable of terminal differentiation in the absence of growth factors.…”

Numerous growth factors, cytokines, and chemokines are secreted by human CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner

“…Apoptosis was detected in these cells by means of the in situ apoptosis detection kit Apoptag (Oncor, Gaithersburg, MD) according to the manufacturer's protocol as described. 20,25 In this assay, terminal deoxynucleotide transferase was used to label the fragmented ends of DNA that have undergone apoptosis with digoxigenin-deoxyuridine triphosphate. The digoxigenin label was subsequently labeled with anti-digoxygenin antibody, which allowed simple, direct enumeration of apoptotic cells with a fluorescence microscope.…”