Thrombopoietin and its receptor Wendling, F; Vainchenker, W
European cytokine network (Montrouge),
09/1998, Letnik:
9, Številka:
3
Journal Article
Recenzirano
Thrombopoietin (TPO), the primary physiological regulator of platelet production, was initially thought to be a lineage-specific factor acting predominantly on megakaryocytopoiesis. Detailed studies ...establish that this cytokine mediates biological effects on a broad spectrum of hematopoietic progenitor cells, including stem cells. TPO is a hormone constitutively produced mainly by the liver and kidney. Plasma TPO levels are regulated by the platelet and megakaryocyte mass through Mpl receptor binding, internalization and degradation. The Mpl receptor is a member of the hematopoietin receptor superfamily lacking intrinsic kinase activity. Upon ligand-induced Mpl homodimerization, the major signaling events for proliferation are mediated through the JAK2/STAT5 pathway, while differentiation might occur through a prolonged activation of the MAPK pathway. Preclinical and clinical studies demonstrate the potential use of TPO in a variety of contexts, but it is too early to evaluate its benefit in reducing platelet transfusion.
Ex-vivo expansion of mature hematopoietic cells and progenitors is a major cell therapy technology aiming to shorten chemotherapy-induced cytopenias. The goal of amplifying the most primitive ...hematopoietic stem cell populations in clinically acceptable conditions is currently difficult, due to the absence of clinical grade early acting-cytokines and lack of adequate stem cell assays. Stem cell factor (SCF) and granulocyte colony stimulating factor (G-CSF) are both clinically applicable and they have been shown to induce a dose-response effect with regard to the expansion of mature cell populations when used in combination with early- acting cytokines. The combined effects of high concentrations of G-CSF and SCF on hematopoietic cells and progenitors and the effects of this procedure on the more primitive stem cell subsets have not been studied. We have developed a preclinical protocol to evaluate the feasibility of expansion of granulomonocytic cells using SCF and G-CSF with the goal of translating this protocol to the clinical application. In preliminary assays, the effects of low and high concentrations of both cytokines on total nucleated cell numbers and progenitors from purifed CD34+ cells (50 104cells/ml) have been tested. As compared to low concentrations (G-CSF 10 ng/ml and SCF 50 ng/ml) high concentrations of G-CSF and SCF (100 ng/ml and 300 ng/ml respectively) induced a two-fold increase of the nucleated cells and progenitors after 9 days of culture (n=7 experiments). High concentrations were therefore chosen for further experiments. CD34+ cells purified from mobilized peripheral blood grafts (purity 99%) have been cultured in a clinically acceptable medium containing clinical grade fetal calf serum ( 10 %), G-CSF (100 ng/ml) and SCF (300 ng/ml) for 9 days. Cells were analyzed using apoptosis tests, immunophenotyping and clonogenic assays at day 0 and day+9. In several experiments ( n= 9) a mean 21-fold expansion of total viable cell numbers was obtained, with a mean 4-fold expansion of clonogenic progenitors. Expanded cells had consistently CD11b+, CD13+, CD15+ phenotype and 2–4% of them remained CD34+. In a scale-up experiment started with 23.106 CD34+ cells, the total cell numbers expanded 16-fold at day+9, with generation of high numbers of CD11b + (18%), CD13+(98%) and CD15+ cells (91%) demonstrating the feasibility of the protocol in clinical scale. To determine if ex-vivo expansion would lead to a significant exhaustion of more primitive stem cells, we have evaluated the NOD/SCID-repopulating cell (RC) contents of the cultures before (day 0) and after ex-vivo expansion (day 9), in mice transplanted with day 0 and day+9 cells. In two experiments, the numbers of NOD/SCID mice engrafted with CD45+ human cells have been found increase from 30% ( 3/10) at day 0 to 100 % ( 10/10 ) at day 9 (Exp1 ) and from 50 % (3/6) at day 0 to 90 % ( 7/8 ) at day9 (Exp 2), demonstrating the persistence of NOD/SCID-RC potential after ex-vivo expansion. Overall the large-scale granulomonocytic cell production protocol that we developed could be of major interest in order to maintain the dose-intensity in high dose chemotherapy regimens and to shorten neutropenia after autologous PBSC transplantation while maintaing the stem cell potential of the graft.
In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from ...5-FU-treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R-infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R-transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R- or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R-infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R- or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R-infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.
The ability of ex vivo expansion to increase the long‐term repopulating capacity of a graft is still unknown. One problem is the most reliable way to quantify transplantable cells. We addressed this ...point in a baboon model based on autologous transplantation of serial limiting doses of non‐manipulated or ex vivo‐expanded mobilized CD34+ cells and determined the threshold doses of non‐manipulated and expanded cells which supported long‐term multilineage engraftment. In the expansion group, CD34+ cells were cultured for 6 d with a combination of early acting cytokines (Flt3‐ligand, stem cell factor, thrombopoietin and interleukin 3). Grafted cells were characterized by their surface antigens and biological properties semisolid assays, long‐term culture‐initiating cells (LTC‐IC) and non‐obese diabetic severe combined immunodeficient reconstituting cells (SRC). Animals were followed for at least 12 months post transplantation. The expansion protocol yielded 12·3‐fold, 16·9‐fold, 3·7‐fold, 3·5‐fold and 2·2‐fold increases in CD34+ cells, granulocyte‐‐macrophage colony‐forming units (CFU‐GM), megakaryocyte CFU (CFU‐MK), LTC‐IC and SRC respectively. It induced a modest increase in the long term reconstitutive ability of the graft; the threshold value for long‐term engraftment was 0·5 × 106/kg CD34+ cells in the control group and 0·3 × 106/kg CD34+ cells in the expansion group, although one animal in this latter group remained hypoplastic. Frequencies of SRC had a high predictive value of long‐term engraftment (r > 0·80). The main advantage of the protocol was the acceleration of granulocyte recovery, achieved at the different doses tested. In conclusion, these experiments suggest that this ex vivo expansion protocol marginally amplifies long‐term reconstituting cells.
Thrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production through binding to its receptor, Mpl, encoded by the c-mpl proto-oncogene. ...Circulating levels of TPO are regulated by receptor-mediated uptake and degradation. To better understand this mode of TPO regulation, we examined whether expression of Mpl was regulated by its ligand. Using RNase protection analysis, we found no differences in the levels ofc-mpl transcripts in megakaryocytes (MKs) produced in vitro either in the presence or absence of TPO and in platelets (PLTs) obtained from mice hyperstimulated in vivo by ectopic secretion of TPO. Similarly, Western blot analysis of MKs produced in the presence or absence of TPO showed no difference in Mpl levels. Levels of Mpl, GpIIb, or P-selectin were virtually identical in platelet lysates obtained from normal, TPO knockout and mildly TPO-stimulated mice. In contrast, the expression of Mpl was significantly reduced in PLTs from severely thrombocythemic mice. These results show that TPO does not have a major effect on the transcription or translation of Mpl. However, they do suggest that an excess of circulating TPO can lead to the disappearance of Mpl from PLTs via catabolism.
Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis ...from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups, making these studies difficult. Here, we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion, the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs, especially for their homing potential.
To evaluate the functional conservation of signal transduction mechanisms between haematopoietic receptors and to characterize the molecules activated in this phenomenon, we introduced granulocyte ...colony‐stimulating factor receptor (G‐CSFR) cDNA into mouse fetal liver cells using a retroviral vector. In semi‐solid medium assays, G‐CSFR‐infected cells gave rise to all types of colonies granulocyte‐macrophage (GM), megakaryocyte (MK) and mixed lineage (GEMM) colony‐forming units (CFU) and erythroid burst‐forming units (BFU‐E) in the presence of G‐CSF alone. The direct effect of G‐CSF on erythroid differentiation of G‐CSFR‐transduced erythroid progenitors was demonstrated by the development of erythroid colonies using G‐CSFR‐expressing Lin− cells cloned at one cell per well in liquid culture in the presence of G‐CSF. Interestingly, while Stat5, but not Stat3, was activated in erythroid cells in response to erythropoietin (EPO), both were activated in erythroid and granulocytic cells stimulated by G‐CSF. Furthermore, G‐CSF induced the growth of erythroid colonies from G‐CSFR‐expressing fetal liver cells from EPO receptor−/− (EPO‐R−/−) or Stat5a−/− Stat5b−/− mice, demonstrating that erythroid differentiation can occur in the absence of EPO‐R or Stat5. These data show that forced expression of G‐CSFR allows G‐CSF‐dependent multilineage proliferation and differentiation of haematopoietic progenitors and rescues EPO‐R−/− erythroid cells. While G‐CSF induces Stat5 activation in G‐CSFR‐expressing erythroid cells, this activation is not necessary for the terminal erythroid differentiation induced by G‐CSF.
The mechanisms of the chronic myeloid leukemia (CML) clones proliferative advantage over normal clones are currently unknown. They may involve an insensitivity to a negative regulation or a growth ...factor-independent proliferation. Clonogenic progenitors from CML patient blood or marrow in chronic phase were grown either in the presence or absence of recombinant growth factors. No erythroid colonies were observed in the absence of any cytokine. In contrast, erythroid colonies composed of fully mature hemoglobinized erythroblasts (day 12 burst-forming units-erythroid) were obtained in the presence of Steel factor (SF) alone. Addition of erythropoietin (Epo) to SF either had no effect on the cloning efficiency or increased up to 50% the number of erythroid colonies. No erythroid growth was observed when cultures were stimulated by interleukin-3 or granulocyte-macrophage colony-stimulating factor alone. Similar erythroid growth in the presence of SF but without Epo was obtained in “serum-free” cultures when purified blood CML CD34+ cells were grown. This growth of erythroid colonies in the absence of Epo was not accounted for by an autocrine stimulation loop by Epo, because neutralizing antibodies against Epo did not inhibit it. This abnormal response to growth factor was specifically observed in the CML clone, as shown by the presence of the BCR-ABL transcript in all of these erythroid colonies. The direct implication of BCR-ABL was further documented (1) by studies of α-interferon–treated patients with a chimerism in which the abnormal growth correlates with the presence of the malignant clone and (2) by the use of antisense oligonucleotide against BCR-ABL transcript, which abrogated this abnormal growth. Finally, erythroid growth in the SF presence was greatly diminished by herbimycin A, whereas, at the same concentration, this tyrosine kinase inhibitor had no marked effect on erythroid colony formation in the presence of SF plus Epo on CML or normal marrow cells. This result suggests that the BCR-ABL kinase activity leads directly to this Epo-independent terminal differentiation requiring, however, the presence of SF.
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