Presence Of Thrombopoietin Research Articles

Fetal Stromal Niches Enhance Human Embryonic Stem Cell–Derived Hematopoietic Differentiation and Globin Switch

Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

Generation of HLA‐deficient platelets from hematopoietic progenitor cells

Exposure to allogeneic blood products often leads to the development of human leukocyte antigen (HLA) antibodies. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA Class I antigens constitutes a significant clinical problem. We developed an RNA interference (RNAi)-based approach to silence the expression of HLA Class I molecules on PLTs derived from CD34+ progenitor cells. A lentiviral-based system was used to express short-hairpin RNA (shRNA) targeting β2-microglobulin (β2m) transcripts in CD34+ progenitor cells. Differentiation to PLTs was performed by incubating progenitor cells in the presence of thrombopoietin and interleukin-3. The transduction of RNAi cassettes containing the sequences for shRNAs targeting β2m caused up to 85% reduction of progenitor cells HLA Class I antigen expression, which was maintained in the culture-derived PLTs. The HLA-deficient PLTs derived from HLA-silenced CD34+ cells proved to be fully functional in in vitro tests when compared to peripheral blood-derived PLTs. Our data show that in vitro generating HLA Class I-deficient PLTs from hematopoietic progenitor cells prove to be feasible. As malignancy risks associated with insertional mutagenesis are not to be expected in anucleated PLTs, provision of HLA-deficient PLTs from large-scale production units may become reality in the management of patients suffering from PLT transfusion refractoriness.

CD61/ Integrin β3 Ligation Contributes to the Thrombopoietin-Mediated Niche Function of Mouse Hematopoietic Stem Cells.

Abstract 383Hematopoietic stem cells (HSCs) reside in a specialized microenvironment, bone marrow (BM) ‘niche' for maintaining self-renewal and multi-lineage differentiation capacities through the life. We recently reported that murine HSCs (CD34- c-Kit+ Sca-1+Lin-: CD34-KSL) but not CD34+KSL hematopoietic progenitors highly express CD61 (integrin β3), correlating well with long-term repopulating activity (LTRA) of HSCs (Umemoto et al., J. Immunol, 2006). However, the roles of CD61 on HSC regulation and underlying molecular mechanisms in the BM niche are to date unknown. We herein demonstrate a novel role of CD61 using CD61-deficient (CD61-/-) and mutant CD61 mice that exhibit 747 tyrosine residue substituted with alanine in the cytoplasmic tail of β3 integrin subunit (CD61Y747A KI) whereby the integrin inside-out and outside-in signaling are impaired. We found that the CD61-deficiency influences the sensitivity to 5-fluorouracil (5-FU) administration (0.25 mg/g i.v. one shot), increasing the mortality of CD61-/- mice (CD61-/- mice died within 14 days, n=20, vs. WT mice survive over 90 days, n=20, P<0.0001). HSCs per se appeared to be responsible for these results because CD61-/- CD34-KSL cells but not CD61-/- CD34+KSL cells showed accelerated cell-cycle progression compared to those of WT, suggesting that CD61 might contribute to maintenance of quiescent state of HSCs within the BM. It is known that thrombopoietin (TPO) acts on not only potent hematopoietic differentiation from HSCs but also for maintaining quiescent state of HSCs. Accordingly, we examined the relationship between TPO effect and integrin signaling through CD61 in HSCs. The in vitro single cell culture system showed the decreased cell proliferation and survival in CD61-/- CD34-KSL cells that were cultured in the presence of TPO (50 ng/mL), compared to the WT CD34-KSL cells (P<0.01). In contrast, CD61-deficency did not influence the phenotype of cultured CD34-KSL cells in the presence of stem cell factor (SCF, 50 ng/ml). Interestingly, these results were mirrored by CD61Y747A KI CD34-KSL cells and their littermate control CD34-KSL cells. Furthermore, when 40 CD34-KSL cells after 5 days culture in the presence of TPO and they were then applied into long-term competitive repopulation assay, CD61Y747A KI CD34-KSL cells showed less LTRA than that of WT (P<0.01). These results indicated that tyrosine at 747 of the β3 integrin subunit was required for the TPO-dependent expansion/maintenance of HSC in vitro. Whereas, to initiate integrin outside-in signaling in the presence of TPO, CD34-KSL cells were cultured on a well of 96-well plates pre-coated with various CD61 ligands (either vitronectin, 5 μg/ml or osteopontin, 5 μg/mL), resulting in the enhancement of their LTRA, compared to without these ligands. Since these ligands could interact with integrin receptors besides CD61, we utilized the antibody to CD61, 2C9.G2 that is capable of phosphorylating CD61 to mimic the actions of outside-in signaling. Using CD34-KSL cells were cultured with 2C9.G2, we further confirmed that CD61 signaling allowed CD34-KSL cells to maintain undivided state in vitro and increase LTRA in vivo, which were dependent upon the presence of TPO but neither SCF nor other growth factors including insulin growth factor-2 and fibroblast growth factor-1. More interestingly, both stimulation with CD61 ligation and TPO influenced ex vivo expansion of HSCs when started at 40 CD34-KSL cells in a 96-well culture well. Collectively, our results indicate that CD61/β3 integrin signaling plays important roles in TPO-mediated expansion/maintenance of HSC within the BM niche, which are possibly mediated through the tyrosine phosphorylation at 747 tyrosine residue of the β3 integrin cytoplasmic domain. Disclosures:No relevant conflicts of interest to declare.

Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL

The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.

Thrombopoietin controls proliferation of embryonic multipotent hematopoietic progenitors

Thrombopoietin (TPO) stimulation was reported to increase the number of multipotent hematopoietic progenitor (MPP) colonies in a methylcellose colony assay using cells from mouse embryos. Here, we investigated the expression of Mpl, the TPO receptor, in the cells from the yolk sac (YS) and the embryo proper (EP). MPPs in c-Kit(+) population in the mouse embryo expressed Mpl. Using liquid cultures, we found that MPPs from YS and EP proliferated in the presence of TPO and stem cell factor (SCF), whereas their numbers were maintained by TPO alone. In contrast, proliferation induced by TPO and SCF was not observed in MPPs of the bone marrow. Interestingly, examination of MPPs from the fetal liver indicated that their proliferative activity was intermediate between that of early embryonic and adult MPPs. These data suggest that early embryonic MPPs switch to adult MPPs in the embryo. Furthermore, the proliferation of early embryonic MPPs was suppressed by AG490, a Janus kinase2 (JAK2) inhibitor; and TPO could be replaced by constitutively active signal transducer and activator of transcription 5 (STAT5) for the proliferation. Thus, JAK2 and STAT5 mediate at least a part of the proliferative signal in early embryonic MPPs.

Role for MKL1 in megakaryocytic maturation

AbstractMegakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34+ cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood, and reduced ploidy in bone marrow megakaryocytes. In conclusion, MKL1 promotes physiologic maturation of human and murine megakaryocytes.

Roles for the AML1/RUNX1 Point Mutation in the Pathogenesis of MDS/AML.

Myelodysplastic syndermes (MDS) are clonal hematological disorders of hematopoietic stem cells (HSC), which are characterized by ineffective hematopoiesis, dysplastic morphology of blood cells, and high possibility to transit to acute myeloid leukemias (AML). Until now, a number of genetic or epigenetic alterations involved in the pathogenesis of MDS have been identified. Among them, point mutations of AML1/RUNX1 were found in 15 to 17% of MDS/AML (high risk MDS and AML following MDS) patients. Although it has been reported that loss-of-function mutations of AML1 inhibited hematopoietic differentiation and they led to MDS/AML, the direct role for AML1 mutations in the onset of MDS/AML has not been elucidated. To clarify the influence of mutated AML1 on the biology of HSCs, we transduced C-terminus-truncated form of AML1 (AML1dC: dominant-negative form of wild-type AML1), which were derived from high-risk MDS patient, into murine Lineage-Sca1+c-Kit+(LSK) cells. In lethally irradiated murine bone marrow transplantation (BMT) experiment, AML1dC-transduced LSK cells showed higher peripheral blood contribution than mock-transduced cells at 4 weeks after BMT. In mice that received transplants of AML1dC-transduced cell, the ratio of donor-derived hematopoietic cells gradually increased over several months after the transplantation, but not in mice that received transplants of mock-transduced cells. AML1dC-transduced cells more strongly contributed to all hematopietic lineages than mock-transduced cells, especially granulo-monocytic lineage. However, AML1dC-transduced LSK cells did not develop MDS/AML in transplanted mice during over 7 months of observation. Methylcellose colony assay revealed that AML1dC-transduced cells formed CFU-Mix more efficiently than mock-transduced cells in the presence of thrombopoietin (TPO). To further explore the effect of AML1dC on the HSCs differentiation into myeloid cells, we utilized the Tet-off system in the OP9 system. In this system, after deprivation of LIF from the culture medium, Flk-1+ hemangioblasts that have both potential to develop into hematopoietic cells and endothelial cells develop from ES cells after 4.5 days and definitive hematopoietic stem/progenitor cells appear after 8.5 days. After collection of hemangioblasts on day 4.5, we inducibly expressed AML1dC by depriving Tet from the culture medium, cultured for 7 days, and performed FACS analysis on day 11.5. After the culture with Tet, 37.8% of AML1dC-transduced ES cells were positive for Mac-1 in the GFP-negative (AML1dC-negative) fraction. In contrast, after the culture without Tet, 53.3 % of AML1dC-transduced ES cells were positive for Mac-1 in the GFP-positive (AML1dC-expressed) fraction. Because in vivo and in vitro assays suggested that mutated AML1 had some role in the proliferation of MDS clone, we analyzed the activation of signal transduction molecules in AML1dC-transduced LSK cells. As the results, TPO-induced STAT5 phosphorylation was enhanced in AML1dC-transduced LSK cells compared to mock-transduced cells. Moreover, TPO-induced STAT3 activation was detected in AML1dC-transduced LSK cells, but not in mock-transduced LSK cells. There was no significant difference in the phosphorylation of ERK1/2 between AML1dC- and mock-transduced LSK cells. In the realtime PCR analysis, the mRNA expression of CIS, which is one of the STAT5-target genes, was increased 2 to 4-fold in AML1dC-transduced LSK cells compared to mock-transduced cells. Next, to elucidate the effect of mutated AML1 on the determination of HSC fate, we performed colony assay using AML1dC-transduced common lymphoid progenitor (CLP) cells. As the results, lymphoid colony-forming ability was attenuated in AML1dC-transduced CLPs compared to mock-transduced CLPs and only AML1dC-transduced CLPs could form CFU-Mix in the methylcellose culture medium including stem cell factor, IL-3, IL-6, erythropoietin and TPO. These results suggest that AML1dC transduction reprograms CLP to multipotent progenitor or converts CLP to common myeloid progenitor. Together, we show that C-terminus-deletion mutant of AML1 enhances hematopietic stem cell activity and promotes HSC commitment into myeloid progenitors. These results indicate that some types of mutated AML1 give rise to the proliferation of malignant clone via cytokine-induced STATs activation.

In Vitro Myelo-Lymphoid Colony Formation by Mouse Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) clonally differentiate into all myeloid, B-lymphoid, and T-lymphoid lineages. Mouse HSCs are known to form in vitro colonies comprised of morphologically identifiable myeloid cells such as neutrophils, macrophages, erythroblasts, and megakaryocytes. Whether HSCs are able to differentiate along B-and T-lymphoid lineages in such colonies remains obscure. The co-culture systems with stromal cells such as S17, OP9, OP9/Delta cells have been shown to support B- and T-cell development. These systems have been used to identify subclasses of progenitors with lymphoid potentials. However, neither B cells nor T cells have been successfully generated from HSCs in vitro. This is most likely due to the lack of culture conditions which support HSCs to differentiate into a certain stage of lymphoid progenitors. In this study, we attempted to use serum-free single-cell culture to identify cytokines which fill the developmental gap between HSCs and lymphoid progenitors. Here we show that myelo-lymphoid colonies are formed by HSCs in the presence of thrombopoietin (TPO), interleukin (IL)-11, or IL-12 together with stem cell factor (SCF). CD34-negative/low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) bone marrow cells were individually cultured with a combination of cytokines for 7 days. All cells in each colony were transplanted into each from a group of lethally irradiated mice, along with compromised bone marrow cells. The recipient mice were periodically analyzed after transplantation to detect transient myeloid and lymphoid reconstitution. All myeloid, B-, and T-lymphoid progenitor activities were detected in single colonies formed in the presence of SCF+TPO, SCF+IL-11, SCF+IL-12. Only myeloid progenitor activity was predominantly detected in single colonies formed in the presence of SCF+IL-3, consistent with previous observations in blast colony assays. All these combinations of cytokines support self-renewal in HSCs to varying degrees. We conclude that TPO, IL-11, and IL-12 directly act on HSCs and support them to differentiate into progenitors with lymphoid differentiation potential. Early differentiation pathways in HSCs are likely to be used in common by myeloid and lymphoid lineages and be supported in common by multiple cytokines.

In vitro clinical‐grade generation of red blood cells from human umbilical cord blood CD34+ cells

There is no appropriate alternative source of red blood cells (RBCs) to relieve the worsening shortage of blood available for transfusion. Therefore, in vitro generation of clinically available RBCs from hematopoietic stem cells could be a promising new source to supplement the blood supply. However, there have been few studies about the generation of clinical-grade RBCs by coculture on human mesenchymal stem cells (MSCs) and various cytokine supplements, even though the production of pure RBCs requires coculture on stromal cells and proper cytokine supplements. Umbilical cord blood (CB) CD34+ cells were cultured in serum-free medium supplemented with two cytokine sets of stem cell factor (SCF) plus interleukin-3 (IL-3) plus erythropoietin (EPO) and SCF plus IL-3 plus EPO plus thrombopoietin (TPO) plus Flt-3 for 1 week, followed by coculture upon MSCs derived from bone marrow (BM) or CB for 2 weeks. Almost pure clinical-grade RBCs could be generated by coculturing with CB-MSCs but not BM-MSCs. Expansion fold and enucleation rate were significantly higher in coculture with CB-MSCs than BM-MSCs. Despite a 2.5-fold expansion of erythroblasts in the presence of TPO and Flt-3 for 8 days, the final RBC count was higher without TPO and Flt-3. This study is the first report on generating clinical-grade RBCs by in vitro culture with human MSCs and compared effectiveness of several cytokines for RBC production. This provides a useful basis for future production of clinically available RBCs and a model of erythropoiesis that is analogous to the in vivo system.

Inhibition of GSK-3β promotes survival and proliferation of megakaryocytic cells through a β-catenin-independent pathway

The interaction of thrombopoietin (TPO) with its receptor c-Mpl initiates intracellular signals that are critical for megakaryopoiesis. Previously we and others have shown that TPO activates PI3K and Akt and that this pathway is important for megakaryocyte growth. Here, we investigate the importance of the Akt substrate glycogen synthase kinase (GSK)-3β in TPO signaling. GSK-3β is phosphorylated and inhibited by Akt as part of the PI3K pathway. GSK-3β can also be inhibited by Wnt signaling through a distinct mechanism, leading to reduced phosphorylation and accumulation of the transcription factor β-catenin. Therefore, we asked if TPO and Wnt3a can both inhibit GSK-3β in megakaryocytic cells, and if they can act synergistically to promote cell growth. Although both TPO and specific chemical inhibitors of GSK-3β result in increased survival and proliferation in a megakaryocytic cell line model, treatment with Wnt3a failed to increase cell growth either in the absence or presence of TPO, despite inducing high levels of β-catenin. Similarly, expression of a constitutively active version of β-catenin did not increase cell growth either in the absence or presence of TPO, suggesting that the effects of GSK-3β inhibition downstream of TPO signaling are distinct from those induced by Wnt3a and independent of β-catenin. The growth promoting effects of TPO are not mediated by either of the two known GSK-3β targets, cyclin D or HIF-1α. We conclude that GSK-3β is phosphorylated and inhibited by TPO-induced Akt, promoting survival and proliferation in megakaryocytic cells through a pathway that does not involve β-catenin.

Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors

AbstractHuman embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell–derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin αIIbβ3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

Downregulation of Signal Transducer and Activator of Transcription 5 (STAT5) in CD34+ Cells Promotes Megakaryocytic Development, Whereas Activation of STAT5 Drives Erythropoiesis

Although it has been proposed that the common myeloid progenitor gives rise to granulocyte/monocyte progenitors and megakaryocyte/erythroid progenitors (MEP), little is known about molecular switches that determine whether MEPs develop into either erythrocytes or megakaryocytes. We used the thrombopoietin receptor c-Mpl, as well as the megakaryocytic marker CD41, to optimize progenitor sorting procedures to further subfractionate the MEP (CD34(+)CD110(+)CD45RA(-)) into erythroid progenitors (CD34(+)CD110(+)CD45RA(-)CD41(-)) and megakaryocytic progenitors (CD34(+)CD110(+)CD45RA(-)CD41(+)) from peripheral blood. We have identified signal transducer and activator of transcription 5 (STAT5) as a critical denominator that determined lineage commitment between erythroid and megakaryocytic cell fates. Depletion of STAT5 from CD34(+) cells by a lentiviral RNAi approach in the presence of thrombopoietin and stem cell factor resulted in an increase in megakaryocytic progenitors (CFU-Mk), whereas erythroid progenitors (BFU-E) were decreased. Furthermore, an increase in cells expressing megakaryocytic markers CD41 and CD42b was observed in STAT5 RNAi cells, as was an increase in the percentage of polyploid cells. Reversely, overexpression of activated STAT5A(1*6) mutants severely impaired megakaryocyte development and induced a robust erythroid differentiation. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed changes in expression of a number of genes, including GATA1, which was downmodulated by STAT5 RNAi and upregulated by activated STAT5.

Plant Sterols Cause Macrothrombocytopenia in a Mouse Model of Sitosterolemia

Mutations in either ABCG5 or ABCG8 cause sitosterolemia, an inborn error of metabolism characterized by high plasma plant sterol concentrations. Recently, macrothrombocytopenia was described in a number of sitosterolemia patients, linking hematological dysfunction to disturbed sterol metabolism. Here, we demonstrate that macrothrombocytopenia is an intrinsic feature of murine sitosterolemia. Abcg5-deficient (Abcg5(-/-)) mice showed a 68% reduction in platelet count, and platelets were enlarged compared with wild-type controls. Macrothrombocytopenia was not due to decreased numbers of megakaryocytes or their progenitors, but defective megakaryocyte development with deterioration of the demarcation membrane system was evident. Lethally irradiated wild-type mice transplanted with bone marrow from Abcg5(-/-) mice displayed normal platelets, whereas Abcg5(-/-) mice transplanted with wild-type bone marrow still showed macrothrombocytopenia. Treatment with the sterol absorption inhibitor ezetimibe rapidly reversed macrothrombocytopenia in Abcg5(-/-) mice concomitant with a strong decrease in plasma plant sterols. Thus, accumulation of plant sterols is responsible for development of macrothrombocytopenia in sitosterolemia, and blocking intestinal plant sterol absorption provides an effective means of treatment.

New developments with umbilical cord blood

In this issue of Cytotherapy, three papers present, between them, the derivation of mesenchymal stromal cells (MSC) and colony-forming unit megakaryocytes (CFU-Meg) from human umbilical cord blood (UCB) cells and their immuno-equivalence to adult bone marrow (BM)-derived MSC. Briefly, Choi et al. [1] describe the derivation of viable MSC from human UCB and use both phenotypic/ enzymatic expression and reverse transcriptase-polymerase chain reaction (RT-PCR) to demonstrate their adipoand osteogenic lineages. They use atelocollagen matrix and type II collagen synthesis to demonstrate the chondrogenic potential of these UCB-derived MSC. In another paper, Shirvaikar et al. [2] describe the generation of CFU-Meg from UCB in the presence of thrombopoietin (TPO) and interleukin-3 (IL-3) and demonstrate synthesis of both matrix metalloproteinase-9 (MMP) and the membrane-type (MT) variety, MT1-MMP. They also demonstrate expression of CXCR4, the unique receptor for the chemokine stromal cell-derived factor-1 (SDF-1), a chemokine important for both progenitor cell homing and up-regulation of MMP-9 and MT1-MMP. Lastly, Ennis et al. [3] explore the immunogenicity of human UCB perivascular cells (HUCPVC) and demonstrate immuno-equivalence to BM-derived MSC using standard lymphoproliferative assays and T-cell CD25 and CD45 cell-surface receptor expression. What is novel about marrow vs. UCB-derived MSC and SDF-1? MSC found in human BM are multipotent progenitors of mesenchymal tissues and have novel regenerative and immune properties [4]. SDF-1 exerts its effect via its unique receptor, CXCR4. SDF-1 is a potent chemoattractant for all cells expressing CXCR4, including T cells and hematopoietic stem cells. Although low levels of CXCR4 expression are found in minor subpopulations of cultured MSC, the majority of marrow and UCBderived MSC lack CXCR4 and, hence, do not migrate toward an SDF-1 gradient. In addition, SDF-1 stimulates pre-B-lymphocyte cell growth, is important in hematopoetic stem cell survival, and increases survival of human CD34 cells [5]. SDF-1 is important in stem cell migration and CXCR4 mediates its effects. SDF-1-induced migration appears to be dependent on CXCR4 expression. SDF-1 activates adhesion molecules on progenitor cells, and monoclonal antibodies (MAb) against SDF-1 inhibit transendothelial migration of hematopoetic progenitor cells [6]. SDF-1 activates CXCR4 CD34 cells and leads to their adhesion and transendothelial migration. In rhabdomyosarcoma cells that are CXCR4 , SDF-1 increases MMP-2 and decreases tissue inhibitors of MMP (TIMP) secretion [7]. Hence, SDF-1 may facilitate, through its actions on CXCR4, stem cell migration to injured tissue and, through its actions on MMP-2 and TIMP, stem cell-mediated repair of injured tissues. Taken together, observations by Shirvaikar et al. on this issue of up-regulation of metalloproteinases by SDF-1 emphasizes the critical role SDF-1 plays in UCB-derived regenerative cellular medicine. We have demonstrated previously that transduction of human MSC with a retroviral vector expressing CXCR4 improved in vitro migration to SDF-1 [8]. However, given safety and ethical concerns over retroviral transduction of human progenitor cells to be administered clinically, identification of a UCB source of progenitor cells with naturally sufficient CXCR4 expression to facilitate SDF-1mediated homing remains an important discovery and has strong potential in cell therapy strategies, both from a safety as well as a feasibility standpoint.

Serum thrombopoietin compared with ZAP-70 and immunoglobulin heavy-chain gene mutation status as a predictor of time to first treatment in early chronic lymphocytic leukemia

In an effort to confirm previous reports we analyzed clinico-biological implications of increased serum levels of thrombopoietin (TPO) in a series of 71 previously untreated Binet stage A B-cell chronic lymphocytic leukemia (CLL) patients. Serum levels of TPO did not correlate with peripheral blood lymphocytosis (p = 0.928), Rai substages (p = 0.516), platelet count (p = 0.572), hemoglobin level (p = 0.228), LDH (p = 0.144) and β2-microglobulin (p = 0.520). The same applied when correlation with ZAP-70 (p = 0.562), CD38 (p = 0.258) or mutational status of IgVH (p = 0.0794) were sought. The risk of disease-progression according to known and putative prognostic parameters was also evaluated as time to first treatment (TFT). The univariate Cox proportional hazard model demonstrated that the absence of IgVH mutational status (p = 0.0005) and ZAP-70-positivity (p = 0.02) were associated with a shorter TFT. In contrast, Kaplan-Meier estimates of TFT, plotted after setting as cut-off the median value for TPO (i.e., 46 pg/mL), failed to demonstrate any statistical difference between two groups (p = 0.342). Looking for cellular source of TPO we investigated the presence of TPO at gene expression level in 60 B-CLL patients belonging to an independent series. Here we provide evidence for the presence of a low TPO gene expression transcript in B-CLL cells. In conclusion, our results indicate that in early B-cell CLL circulating level of TPO does not provide a useful insight into the complex interrelationship of prognostic variables.

Caspase-3 Activation Is Involved in the Early Megakaryocyte Differentiation, but Not in Platelet Production.

Megakaryocytes (Mgk) differentiate from hematopoietic stem cells through the complex process of endomitosis and cytoplasmic development, and finally form proplatelets in the terminal stage of the differentiation. To elucidate the role of a proapoptotic effecter protein, caspase-3, in megakaryopoiesis and thrombopiesis, we analyzed the phenotype of Mgk in vav-Bcl-2 transgenic (Tg) mice, which overexpress Bcl-2 exclusively in hematopoietic cells by the regulation of vav promotor. As was expected, Bcl-2-overexressing Mgk were remarkably resistant to caspase-3 activation induced by the depletion of thrombopoietin (TPO) from the culture. Percentages of proplatelet formation (PPF) assayed by culturing primary Mgk were slightly increased in the Tg mice. Platelet production from cultured Mgk, which was assayed by flowcytometry, was also slightly increased, indicating normal ability for Plt production from Bcl-2-overexpressing mature Mgk. To further analyze the ability for thrombopoiesis, we assayed the trends of Plt recovery following experimental thrombocytopenia. As Bcl-2-overexpressing mice had giant splenomegaly, we used splenectomized mice, which showed normal Plt counts. After the injection of 5-FU, we observed significant delay of the Plt recovery in the Tg mice, as well as the lack of the overshoot of Plt counts during the recovery phase. To gain insights about the mechanism underlying the delayed Plt recovery, we analyzeded the ploidy of DNA content. In the steady state, peak ploidy of Mgk obtained from the bone marrow of the Tg mice shifted to 2–4N. In a sharp contrast, in Plt recovery phase after 5-FU injection, peak ploidy shifted to 16–32N, and the percentages of Mgk possessing less than 16N ploidy were dramatically decreased. Consistent with this finding, we observed the delay of PPF in Bcl-2-overexpressing cells when Mgk were derived from c-kit+/lineage- progenitor cells in the presence of TPO. These data indicate that differentiation of immature Mgk into mature Plt-producing stage is significantly delayed in the Tg mice, but once the maturation proceeds, Mgk normally produce Plt. Taken together, we conclude that caspase-3 activation is involved in the early stage of the differentiation of Mgk, but not in Plt production.

Interrelation between polyploidization and megakaryocyte differentiation: a gene profiling approach

AbstractPolyploidization is a part of the normal developmental process leading to platelet production during megakaryocyte (MK) differentiation. Ploidization is mainly involved in cell enlargement, but it is not clear whether gene expression is modified during MK ploidization. In this study, human MKs were grown from CD34+ cells in the presence of thrombopoietin and sorted according to their ploidy level. A pangenomic microarray technique was applied to compare gene expression in 2N-, 4N-, 8N-, and 16N-sorted MKs. Using hierarchical clustering, we demonstrated that 2N and 4N MKs or 8N and 16N MKs are 2 different close populations with 105 discriminating genes. In the second approach, we determined the profile of genes that were continuously down- and up-regulated during polyploidization. Among the 100 down-regulated genes, 24 corresponded to genes involved in DNA replication and repair. The great majority of up-regulated genes corresponded to genes directly involved in platelet functions, such as genes encoding specific platelet glycoproteins and α-granule proteins, actin and microtubule cytoskeleton, factors involved in signaling, and transport proteins. Together, these results suggest that MK polyploidization per se does not regulate gene expression but is intrinsically included in the differentiation process.

Continuous expression of Bcl-xL protein during megakaryopoiesis is post-translationally regulated by thrombopoietin-mediated Akt activation, which prevents the cleavage of Bcl-xL.

One of the important biological activities of thrombopoietin (TPO) is to prevent the apoptosis of megakaryocytes. As the antiapoptotic protein Bcl-xL, which has been proven to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role in megakaryopoiesis. We investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early and mid-stages of the differentiation. Both in vitro in cell culture and in vivo in an animal model, expression of Bcl-xL protein was maintained until the platelet-producing stage. TPO depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both megakaryocytes and TPO-dependent megakaryocytic UT-7/TPO cells, suggesting that Bcl-xL protein level in TPO-dependent cells is post-translationally regulated. In agreement with this finding, we recognized the appearance of a 12-kD fragment of Bcl-xL upon TPO depletion. This cleavage of Bcl-xL was inhibited by a caspase-3-specific inhibitor. Furthermore, pretreatment of UT-7/TPO with a phosphatidylinositol 3-kinase (PI3 K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3 K inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this, we prepared UT-7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage. Bcl-xL protein is expressed throughout megakaryopoiesis until platelets are produced, and its expression level is at least in part post-translationally regulated through TPO-mediated Akt activation.

Lnk negatively regulates self-renewal of hematopoietic stem cells by modifying thrombopoietin-mediated signal transduction

One of the central tasks of stem cell biology is to understand the molecular mechanisms that control self-renewal in stem cells. Several cytokines are implicated as crucial regulators of hematopoietic stem cells (HSCs), but little is known about intracellular signaling for HSC self-renewal. To address this issue, we attempted to clarify how self-renewal potential is enhanced in HSCs without the adaptor molecule Lnk, as in Lnk-deficient mice HSCs are expanded in number >10-fold because of their increased self-renewal potential. We show that Lnk negatively regulates self-renewal of HSCs by modifying thrombopoietin (TPO)-mediated signal transduction. Single-cell cultures showed that Lnk-deficient HSCs are hypersensitive to TPO. Competitive repopulation revealed that long-term repopulating activity increases in Lnk-deficient HSCs, but not in WT HSCs, when these cells are cultured in the presence of TPO with or without stem cell factor. Single-cell transplantation of each of the paired daughter cells indicated that a combination of stem cell factor and TPO efficiently induces symmetrical self-renewal division in Lnk-deficient HSCs but not in WT HSCs. Newly developed single-cell immunostaining demonstrated significant enhancement of both p38 MAPK inactivation and STAT5 and Akt activation in Lnk-deficient HSCs after stimulation with TPO. Our results suggest that a balance in positive and negative signals downstream from the TPO signal plays a role in the regulation of the probability of self-renewal in HSCs. In general, likewise, the fate of stem cells may be determined by combinational changes in multiple signal transduction pathways.

Co-culture of cord blood CD34+ cells with human BM mesenchymal stromal cells enhances short-term engraftment of cord blood cells in NOD/SCID mice

The major challenge for cord blood transplantation (CBT) is higher rates of delayed and failed engraftment. In an attempt to broaden the application of CBT to more candidates, ex vivo expansion of hematopoietic stem/progenitor cells in CB is a major area of investigation. The purpose of this study was to employ human BM mesenchymal stromal cells (hBM-MSC) as the feeding-layer to expand CB cells ex vivo. In this study, hBM-MSC were isolated and characterized by morphologic, mmunophenotypic and RT-PCR analysis. The hBM-MSC at passage 3 were employed as the feeding-layer to expand CB CD34(+) cells in vivo in the presence of thrombopoietin, flt3/flk2 ligand, stem cell factor and G-CSF. The repopulating capacity of the ex vivo-expanded CB cells was also evaluated in a NOD/SCID mice transplant experiment. After 1 or 2 weeks of in vitro expansion, hBM-MSC supported more increasing folds of CB in total nucleated cells, CD34(+) cells and colony-forming units (CFU) compared with CB without hBM-MSC. Furthermore, although NOD/SCID mice transplanted with CB cells expanded only in the presence of cytokines showed a higher percentage of human cell engraftment in BM than those with unexpanded CB CD34(+) cells, expanded CB cells co-cultured with hBM-MSC were revealed to enhance short-term engraftment further in recipient mice. Our study suggests that hBM-MSC enhance in vitro expansion of CB CD34(+) cells and short-term engraftment of expanded CB cells in NOD/SCID mice, which may be valuable in a clinical setting.