Hypersensitivity of megakaryocyte progenitors to thrombopoietin in essential thrombocythemia.

Essential thrombocythemia (ET) is a clonal myeloproliferative disorder characterized by an elevation of platelets in peripheral blood and excessive proliferation of megakaryocytes in bone marrow. The pathological mechanisms for the elevation of megakaryocytes and platelets in ET remain unclear. To study the hypersensitivity of megakaryocyte progenitors to thrombopoietin (TPO), a 9-year-old girl, diagnosed with ET, underwent dose-response experiments with recombinant human TPO, using her bone marrow non-adherent mononuclear cells and CD34 positive cells. Spontaneous colony-forming unit-megakaryocytes (CFU-Meg) were observed in serum-deprived cultures of non-adherent mononuclear cells, whereas they disappeared in cultures of CD34 positive cells. The patient's CFU-Meg showed maximal growth at concentrations of TPO lower than those for normal children. Dose-response curves demonstrated a 50-80 fold increase in sensitivity of the patient's CFU-Meg to TPO. We observed hypersensitivity of megakaryocyte progenitors to TPO in a child with ET. Our results suggest that spontaneous CFU-Meg formation in patients with ET may be due to hypersensitivity to TPO.     

Release from quiescence of CD34+ CD38- human umbilical cord blood cells reveals their potentiality to engraft adults.

Using optimal culture conditions in which the transforming growth factor beta 1 (TGF-beta 1) inhibitory loop has been interrupted by antisense TGF-beta 1 oligonucleotides or anti-TGF-beta serum, we have compared the proliferative capacities and the abilities of the CD34+ CD38- cell populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38- fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of colony-forming units (CFU)-granulocyte/erythrocyte/macrophage/megakaryocyte, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult transplantation. In addition, the colonies resulting from the umbilical cord blood samples were significantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparations, as estimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is partially compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbilical cord blood is a suitable source of cells for adult transplantation.     

Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation

Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here, we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, angiopoietin-like 5, and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation.     

Thrombopoietin: biology and clinical potentials

Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. In vitro, TPO induces the growth of colony-forming units-megakaryocyte (CFU-MK) and the generation of mature polyploid megakaryocytes, which subsequently form extended cytoplasmic processes, termed proplatelets. On more differentiated CFU-MK, but not on megakaryocytes, TPO is critical for enhancing proplatelet formation. TPO has multilineage effects in hematopoiesis, not only stimulating megakaryocytopoiesis but also acting in synergy with other cytokines to enhance proliferation and survival of committed erythroid progenitors and primitive hematopoietic stem cells. Surface c-MPL, the receptor for TPO, defines a phenotype of hematopoietic stem cells with long-term repopulating ability. Treatment with various cytokine combinations, including TPO, results in an extensive ex vivo expansion of hematopoietic stem cells and blood cell precursors. In normal animals, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or glycosylated TPO increases the number of bone marrow megakaryocytes and their progenitors and greatly enhance the production of morphologically and functionally normal platelets. In contrast, they have only minimal effects on peripheral white blood cell and red blood cell counts. PEG-rHuMGDF used alone markedly expands circulating levels of multiple types of hematopoietic progenitors, and its effect is enhanced in combination with granulocyte colony-stimulating factor (G-CSF). Although PEG-rHuMGDF augments platelet aggregation induced by agonists in vitro, it has no influence in an animal model of thrombus formation. PEG-rHuMGDF or glycosylated TPO has a profound effect in a variety of animal models of thrombocytopenia, including myelosuppressive therapy. PEG-rHuMGDF treatment accelerates multilineage hematopoietic recovery, effectively improving thrombocytopenia, and, in most models, neutropenia and anemia. The concurrent administration of PEG-rHuMGDF and G-CSF does not interfere with the in vivo activity of cytokines but rather has synergistic effects. To further accelerate hematopoietic recovery, PEG-rHuMGDF administration should start at the earliest time following myelosuppressive treatment; this time sensitivity may result from the presence of a greater number of residual hematopoietic progenitors in the bone marrow soon after treatment. Moreover, if a relatively large dose of PEG-rHuMGDF is administered, a single intravenous injection is fully effective in improving impaired hematopoiesis. This effectiveness appears to be related to the persistence of PEG-rHuMGDF in the circulation. The safety and efficacy of two forms of the recombinant hormone, PEG-rHuMDGF and glycosylated human full-length TPO produced in mammalian cells, are currently under clinical investigation.     

Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation

Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2.4 times higher in CB than in PB cells. Importantly, the TPO-induced ploidy level was notably different: whereas 82.7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.     

5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies

Background and Objectives   Mesenchymal stem/progenitor cells (MSCs) are multipotent progenitors that differentiate into such lineages as bone, fat, cartilage and stromal cells that support haemopoiesis. Bone marrow MSCs can also contribute to cardiac repair, although the mechanism for this is unclear. Here, we examine the potential of MSCs from different sources to generate cardiomyocytes in vitro , as a means for predicting their therapeutic potential after myocardial infarction.
Materials and Methods   Mesenchymal stem/progenitor cells were isolated from the perivascular tissue and Wharton's jelly of the umbilical cord and from cord blood. Their immunophenotype and differentiation potential to generate osteoblasts, chondrocytes, adipocytes and cardiomyoxcytes in vitro was compared with those of bone marrow MSCs.
Results   Mesenchymal stem/progenitor cells isolated from umbilical cord and cord blood were phenotypically similar to bone marrow MSCs, the exception being in the expression of CD106, which was absent on umbilical cord MSCs, and CD146 that was highly expressed in cord blood MSCs. They have variable abilities to give rise to osteoblasts, chondrocytes and adipocytes, with bone marrow MSCs being the most robust. While a small proportion (~0·07%) of bone marrow MSCs could generate cardiomyocyte-like cells in vitro, those from umbilical cord and cord blood did not express cardiac markers either spontaneously or after treatment with 5-azacytidine.
Conclusion   Although MSCs may be useful for such clinical applications as bone or cartilage repair, the results presented here indicate that such cells do not generate cardiomyocytes frequently enough for cardiac repair. Their efficacy in heart repair is likely to be due to paracrine mechanisms.     

Effects of recombinant human thrombopoietin on megakaryocyte colony formation and megakaryocyte ploidy by human CD34+ cells in a serum-free system

In serum-free cultures of human CD34 cells, recombinant human thrombopoietin (TPO) induced megakaryocyte colony formation in a dose-dependent fashion that was further enhanced by the presence of interleukin-3 (IL-3) and stem cell factor (SCF), but not by IL-6, IL-11 or erythropoietin. TPO gave rise to much smaller colonies and at an earlier time than IL-3, indicating that TPO affects predominantly more mature megakaryocytic progenitors. In liquid cultures, TPO increased the percentage and the absolute number of ≥8N megakaryocytes, but it did not shift their modal ploidy from 2N. TPO-induced endomitosis was totally inhibited by the presence of, or previous exposure of cells to, IL-3 and/or SCF. The mechanism by which TPO overcomes in vivo the negative effects of IL-3 and SCF on megakaryocyte ploidy remains unknown.     

Differential effects of human granulocyte colony-stimulating factor (hG-CSF) and thrombopoietin on megakaryopoiesis and platelet function in hG-CSF receptor-transgenic mice.

Granulocyte-colony stimulating factor (G-CSF) has been found to act on the neutrophilic lineage. We recently showed that human G-CSF (hG-CSF) has effects similar to early-acting cytokines such as interleukin-3 (IL-3) in the development of multipotential hematopoietic progenitors in transgenic (Tg) mice expressing receptors (R) for hG-CSF. In the present study, we examined the effects of hG-CSF on more mature hematopoietic cells committed to megakaryocytic lineage in these Tg mice. The administration of hG-CSF to the Tg mice increased the numbers of both platelets in peripheral blood and megakaryocytes in the spleen, indicating that hG-CSF stimulates megakaryopoiesis in the Tg mice in vivo. The stimulatory effect of hG-CSF was also supported by the results of studies in vitro. hG-CSF supported megakaryocyte colony formation in a dose-dependent fashion in clonal cultures of bone marrow cells derived from the Tg mice. Direct effects of hG-CSF on megakaryocytic progenitors in the Tg mice were confirmed by culture of single-cell sorted from bone marrow cells. hG-CSF showed a stronger effect on maturation of megakaryocytes in the Tg mice than that of IL-3 alone, but weaker than that of TPO alone. In addition, hG-CSF induced phosphorylation of STAT3 but not Jak2 or STAT5, while TPO induced phosphorylation of both. In contrast to TPO, hG-CSF did not enhance ADP-induced aggregation. Thus, hG-CSF has a wide variety of functions in megakaryopoiesis of hG-CSFR-Tg mice, as compared with other megakaryopoietic cytokines, but the activity of hG-CSF in megakaryocytes and platelets does not stand up to a comparison with that of TPO. Specific signals may be required for the full maturation and activation of platelets.     

Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34⁺ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34⁺ cell fraction, whereas in the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.     

Role of interleukin-2 (IL-2), IL-7, and IL-15 in natural killer cell differentiation from cord blood hematopoietic progenitor cells and from gamma c transduced severe combined immunodeficiency X1 bone marrow cells

Natural killer (NK) cells are characterized by their ability to mediate spontaneous cytotoxicity against susceptible tumor cells and infected cells. They differentiate from hematopoietic progenitor cells. Patients with X-linked severe combined immunodeficiency (SCID X1) carry mutations in the gamma c cytokine receptor gene that result in lack of both T and NK cells. To assess the role of interleukin-2 (IL-2), IL-7, and IL-15 cytokines, which share gamma c receptor subunit, in NK cell differentiation, we have studied NK cell differentiation from cord blood CD34 (+) cells in the presence of either stem cell factor (SCF), IL-2, and IL-7 or SCF and IL-15. The former cytokine combination efficiently induced CD34 (+) CD7 (+) cord blood cells to proliferate and mature into NK cells, while the latter was also able to induce NK cell differentiation from more immature CD34 (+) CD7 (-) cord blood cells. NK cells expressed CD56 and efficiently killed K562 target cells. These results show that IL-15 could play an important role in the maturation of NK cell from cord blood progenitors. Following retroviral-mediated gene transfer of gamma c into SCID X1 bone marrow progenitors, it was possible to reproduce a similar pattern of NK cell differentiation in two SCID-X1 patients with SCF + IL-2 + IL-7 and more efficiently in one of them with SCF + IL-15. These results strongly suggest that the gamma c chain transduces major signal(s) involved in NK cell differentiation from hematopoietic progenitor cells and that IL- 15 interaction with gamma c is involved in this process at an earlier step than IL-2/IL-7 interactions of gamma c are. It also shows that gene transfer into hematopoietic progenitor cells could potentially restore NK cell differentiation in SCID X1 patients.     

Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures

In contrast to myeloid and B-lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+ CD38+ and CD34+ CD38(low) cells from both cord blood and adult bone marrow reproducibly develop into CD4+ CD8+ T cells when introduced into NOD-SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T-cell potential of human primitive progenitor cells. The frequency of T-cell progenitors among cord-blood-derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD-SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD41(low) CD8- sCD3- TCR alphabeta- CD5+ CD1a+ T cells were mixed with CD4+ CD8+ cells and more mature CD4+ CD8- TCR alphabeta+ cells. However, in FTOC initiated with bone marrow T progenitors, <10% double-positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T-cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine-stimulated CD34+ CD38(low) cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.     

Differential maturation of megakaryocyte progenitor cells from cord blood and mobilized peripheral blood

OBJECTIVE: In comparison with stem cell transplantation using bone marrow or cytokine-mobilized peripheral blood, cord blood transplantation is characterized by delayed engraftment, in particular platelet recovery. The differences in the kinetics of engraftment may be related to quantitative differences in the numbers of stem cells and megakaryocyte progenitor cells and/or to qualitative differences between megakaryocyte progenitor cells in these grafts. We compared the hematopoietic composition of these grafts and determined the distribution of mature and immature megakaryocyte progenitor cells in cord blood and mobilized peripheral blood and their in vitro kinetic behavior. METHODS: Megakaryocyte progenitor cell subpopulations from cord blood (CB) and mobilized peripheral blood (PBSC) were expanded in vitro in the presence of mpl-ligand. The developmental differences during expansion of megakaryocyte progenitors were analyzed by flow cytometry and progenitor assays. RESULTS: We found that the immature (CD34(+)/CD41(-)) subpopulation from CB contains more than 98% of all megakaryocyte progenitor cells, responsible for 99% of all megakaryocytic cells cultured during 2 weeks. The CB CD34(+)/CD41(+) subpopulation shows no contribution to megakaryocytic cell formation. In contrast, in PBSC the mature (CD34(+)/CD41(+)) subpopulation contains 7% of all megakaryocyte progenitor cells. Moreover, CD34(+) cells from CB and PBSC also showed distinct phenotypic differences during maturation in vitro. PBSC megakaryocyte progenitor cells transiently express both CD34 and CD41 during maturation in vitro, whereas CB progenitor cells transiently lack expression of both markers before differention into (CD34(-)/CD41(+)) megakaryocytic cells. CONCLUSION: The in vitro data indicate the presence of different developmental stages of megakaryocyte progenitor cells in CB as compared to PBSC. These differences in composition and maturation between CB and PBSC may be related to the different kinetics of engraftment following transplantation of these stem cell sources.     

The effect of thrombopoietin on erythroid progenitors in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red blood cell aplasia. Usually, erythropoiesis in vitro is defective, and decreased numbers of erythroid progenitors are found in colony assays performed with bone marrow cells. Recently, some investigators showed that thrombopoietin (TPO) also works on common multipotent progenitor cells and stimulates erythropoiesis. We examined the effect of TPO together with erythropoietin (EPO), stem cell factor (SCF), and/or interleukin-3 (IL-3) on erythroid burst-forming units (BFU-E) of bone marrow nonadherent mononuclear cells from 2 patients with DBA using a serum-free culture system. Very few BFU-E appeared in cultures containing EPO alone. Adding IL-3, SCF, or both to cultures containing EPO induced an increase in the number of BFU-E. When TPO was added to cultures containing EPO and IL-3, SCF, or both, the number of BFU-E further increased. Especially in patient 2, BFU-E formation was grossly equal to that in normal controls. We conclude that TPO enhances bone marrow erythropoiesis in patients with DBA in the presence of EPO and SCF and/or IL-3. The data raise the possibility of the combination of TPO and SCF as a therapeutic agent in DBA.     

Ex vivo expansion of human adult stem cells capable of primary and secondary hemopoietic reconstitution

OBJECTIVE: Ex vivo expansion of human hemopoietic stem cells (HSC) is an important issue in transplantation and gene therapy. Encouraging results have been obtained with cord blood, where extensive amplification of primitive progenitors was observed. So far, this goal has been elusive with adult cells, in which amplification of committed and mature cells, but not of long-term repopulating cells, has been described. METHODS: Adult normal bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells were cultured in a stroma-free liquid culture in the presence of Flt-3 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6 (IL-6), or interleukin-3 (IL-3). Suitable aliquots of cells were used to monitor cell production, clonogenic activity, LTC-IC output, and in vivo repopulating capacity. RESULTS: Here we report that BM and MPB HSC can be cultured in the presence of FL, TPO, SCF, and IL-6 for up to 10 weeks, during which time they proliferate and produce large numbers of committed progenitors (up to 3000-fold). Primitive NOD/SCID mouse repopulating stem cells (SRC) are expanded sixfold after 3 weeks (by limiting dilution studies) and retain the ability to repopulate secondary NOD/SCID mice after serial transplants. Substitution of IL-6 with IL-3 leads to a similarly high production of committed and differentiated cells but only to a transient (1 week) expansion of SRC(s), which do not possess secondary repopulation capacity. CONCLUSION: We report evidence to show that under appropriate culture conditions, adult human SRC can also be induced to expand with limited differentiation.     

Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK)

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that acts as a stimulator of pre-B lymphocyte cell growth and as a chemoattractant for T cells, monocytes, and hematopoietic stem cells. More recent studies also suggest that megakaryocytes migrate in response to SDF-1. Because genetic elimination of SDF-1 or its receptor lead to marrow aplasia, we investigated the effect of SDF-1 on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-MK]). We report that SDF-1 augments the growth of CFU-MK from whole murine bone marrow cells when combined with thrombopoietin (TPO). The addition of SDF-1 to interleukin-3 (IL-3) or stem cell factor (SCF) had no effect. Specific antagonists for CXCR4 (the sole receptor for SDF-1), T22, and 1-9 (P2G) SDF-1 reduced megakaryocyte colony growth induced by TPO alone, suggesting that many culture systems contain endogenous levels of the chemokine that contributes to the TPO effect. To examine whether SDF-1 has direct effects on CFU-MK, we developed a new protocol to purify megakaryocyte progenitors. CFU-MK were highly enriched in CD41(high) c-kit(high) cells generated from lineage-depleted TPO-primed marrow cells. Because the growth-promoting effects of SDF-1 were also observed when highly purified populations of CFU-MK were tested in serum-free cultures, these results suggest that SDF-1 directly promotes the proliferation of megakaryocytic progenitors in the presence of TPO, and in this way contributes to the favorable effects of the bone marrow microenvironment on megakaryocyte development.