Differentiation Of Hemopoietic Stem Cells Research Articles

Roles of Notch Glycoslation in Signaling

Notch receptors are single pass transmembrane glycoproteins with an extracellular domain (ECD) comprised of many epidermal growth factor-like (EGF) repeats. Specific Ser and Thr residues in numerous EGF repeats have an attached O-glycan initiated by fucose, glucose, or N-acetylglucosamine (GlcNAc), and elongated with additional sugars. Notch receptors signal following binding of Notch ligands to the Notch receptor ECD, leading to release of the intracellular domain and activation of many Notch target genes. Notch signaling regulates cell fate decisions in many organs during development and differentiation. Dysregulation of Notch signaling leads to developmental disorders and to cancer. Lunatic (LFNG), Manic (MFNG) and Radical (RFNG) Fringe are GlcNAc transferases that extend Notch EGF repeats which already carry a fucose. EOGT is also a GlcNAc-transferase that transfers O-GlcNAc directly to EGF repeats. We are investigating roles for EOGT and each Fringe in T and B cell development using mice lacking various combinations of the Eogt, Lfng, Mfng and Rfng genes. We are also identifying individual roles for Fng genes using a co-culture assay of bone marrow hemopoietic stem cells (HSC) from compound mutant mice stimulated by OP9 stromal cells expressing one of the four Notch ligands, DLL1, DLL4, JAG1 or JAG2. The combined results show that the O-fucose glycans on NOTCH1 are key regulators of the strength of NOTCH1 signaling which, in turn, determines differentiation of HSC to T cell progenitors.

Correlation of Bone Marrow Morphology and Immunophenotyping in Acute Leukaemia Patients

Background: Acute leukaemia (AL) is a malignant disorder of the blood that is characterized by blocked or impaired differentiation of haemopoietic stem cells, resulting in abnormal accumulation of immature precursors and suppression of growth and maturation of cells in vivo. Objective: To find out correlation between morphological and immunophenotypic study of bone marrow among acute leukaemia patient. Methods/Procedure: This is a comparative cross sectional study of diagnosis of leukaemia by bone marrow study and immunophenotyping from bone marrow sample with bone marrow alone of suspected cases of leukaemia treated in the department of haematology in Dhaka Medical College Hospital (DMCH)from March 2013 to August 2013 .Bone marrow examination and immunophenotyping was done simultaneously but having bone marrow morphology report we have compared with flow report. Results: Out of 50 patients according to Bone marrow study (BMS) 25(50.0%) of the patients had acute myelogenous leukaemia, 24(48.0%) had acute lymphoblastic leukaemia and 1(2.0%) had acute leukaemia. On the other hand, in immunophenotyping 28(56.0%) patients had acute lymphoblastic leukaemia, 20(40.0%) had acute myelogenous leukaemia and 2(4.0%) mixed cell immunophenotyping. Discordance of diagnosis was found in 3(6%) is diagnose as AML which was ALL on flow and one acute leukaemia and one AML was subsequently diagnose as mixed cellular leukaemia. Conclusion: Subjective variation in the accuracy of diagnosis of leukaemia on the basis of bone marrow study alone may occur. Inclusion of immunophenotyping with bone marrow study improves accuracy of leukaemia.

Gadofullerene nanoparticles for robust treatment of aplastic anemia induced by chemotherapy drugs.

Aplastic anemia (AA) is characterized as hypoplasia of bone marrow hematopoietic cells and hematopenia of peripheral blood cells. Though the supplement of exogenous erythropoietin (EPO) has been clinically approved for AA treatment, the side-effects hinder its further application. Here a robust treatment for AA induced by chemotherapy drugs is explored using gadofullerene nanoparticles (GFNPs).Methods: The gadofullerene were modified with hydrogen peroxide under alkaline conditions to become the water-soluble nanoparticles (GFNPs). The physicochemical properties, in vitro chemical construction, stability, hydroxyl radical scavenging ability, in vitro cytotoxicity, antioxidant activity, in vivo treatment efficacy, therapeutic mechanism and biological distribution, metabolism, toxicity of GFNPs were examined.Results: GFNPs with great stability and high-efficiency antioxidant activity could observably increase the number of red blood cells (RBC) in the peripheral blood of AA mice and relieve the abnormal pathological state of bone marrow. The erythropoiesis mainly includes hemopoietic stem cells (HSCs) differentiation, erythrocyte development in bone marrow and erythrocyte maturation in peripheral blood. The positive control-EPO promotes erythropoiesis by regulating HSCs differentiation and erythrocyte development in bone marrow. Different from the anti-AA mechanism of EPO, GFNPs have little impact on both the differentiation of HSCs and the myeloid erythrocyte development, but notably improve the erythrocyte maturation. Besides, GFNPs can notably decrease the excessive reactive oxygen species (ROS) and inhibit apoptosis of hemocytes in blood. In addition, GFNPs are mostly excreted from the living body and cause no serious toxicity.Conclusion: Our work provides an insight into the advanced nanoparticles to powerfully treat AA through ameliorating the erythrocyte maturation during erythropoiesis.

Myeloid Neoplasms with t(12;22)(p13;q12) and MN1 Ectopia Are Refractory to Chemotherapy and Can Benefit from Hematopoietic Stem Cell Transplantation

Translocation of t(12;22)(p13;q12) is a rare but recurrent chromosomal (chr) abnormality in hematologic malignancies involving ETS variant 6 (ETV6) and meningioma 1 (MN1) genes. The pathogenic mechanism of t(12;22)(p13;q12) and the fact that nearly half of the cases lack fusion genes remain mysterious. We focused here on AML cases with t(12;22)(p13;q12) to elucidate their molecular etiology and outcomes of allogeneic hemopoietic stem cell transplantation (allo-HSCT). A total of eight cases with t(12;22)(p13;q12) and additionally one with t(12;17;22)( p13;q21;q13) (six males and three females) were detected in 2782 newly diagnosed AML and MDS patients by G-banding karyotyping (Table 1). The median age of onset was 45 years (range 4-60 years). The cases include three AML-M5, one AML-M2, one AML progressed from MDS (MDS-AML), one MDS with excess blasts 2 (MDS-EB-2), one chronic myelomonocytic leukemia (CMML), and two myeloid/T mixed-phenotype acute leukemia (MPAL) (Table 1). All cases underwent standard or intensive chemotherapy for myeloid malignancies, and six patients underwent allo-HSCT (Fig. 1a). MN1-ETV6, ETV6-MN1, and MN1 expression were analyzed by RT-PCR. Mutations of 58 commonly mutated genes in hematologic malignancies were analyzed by next-generation sequencing in seven cases (Table 1). Fluorescence in situ hybridization (FISH) was performed using the MN1 dual-color break-apart probe on cases P4 and P9, who were negative for MN1-ETV6. 30 × whole-genome sequencing (WGS) was performed on diagnostic bone marrow (BM) samples from the four cases which were negative for MN1-ETV6, and structure variations including translocations (Fig. 1b) were analyzed. 5/9 cases were positive for both MN1-ETV6 and ETV6-MN1fusions (all type I), and the expression of MN1 was significantly up-regulated in 3/4 fusion negative cases (Table 1). FISH analysis showed split signals in both P4 and P9, indicating a fracture within or adjacent to MN1. WGS analysis showed ectopia of intact MN1 in case P1, P3, and P4 (Fig. 1b), including translocated adjacent to the super enhancers (SEs) of the cracked ETV6. A novel MN1-STAT3 fusion was identified and confirmed by RT-PCR in case P9. We observed overexpression of MN1 in MN1-ETV6 negative cases in our study, which strongly indicates that the contribution of the SEs within ETV6 made on the overexpression of MN1 or aberrant activation of the chimeric MN1-ETV6. Both type I/II MN1-ETV6, and the novel MN1-STAT3 in this study retains the main functional domains of MN1 (Fig. 1c). The MN1-STAT3 contains only 69 amino acids of the STAT3 residual without functional domains, and STAT3-MN1 was absent due to the triadic translocation. Thus, the fusion protein will retain the primary function of MN1 but not STAT3 in case P9, which also indicates that it is the functionally retained MN1 within MN1-ETV6 rather than the fusion, that plays the essential role in leukemogenesis. The genomic evidence of ETV6 disruption was observed in 8/9 cases in our study. ETV6 is a tumor suppressor which is essential in hemopoietic stem cell differentiation and multilineage blood cell development. It is conceivable that the MN1 overactivation and the ETV6 disruptive haploinsufficiency synthetically contribute to the leukemogenesis in t(12;22)(p13;q12) malignancies. This scenario is partially similar to the mechanism of GATA2-MECOM in inv(3)(q21q26) AML, within which that the central pathogenesis is the ectopia of the GATA2 SE that activates MECOM expression and confers the GATA2 haploinsufficiency simultaneously. The present study confirmed that myeloid neoplasms with t(12;22)(p13;q12) were rare and responded poorly to conventional chemotherapy, and provide evidence that these patients can benefit from allo-HSCT. Our investigation especially highlights the evidence of a SE-associated orchestrated mechanism of MN1 overexpression and ETV6 haploinsufficiency in t(12;22)(p13;q12) myeloid neoplasms in a spatiotemporally specific manner, rather than the conventional thought of MN1-ETV6/ETV6-MN1 fusion formation. Figure 1 Disclosures No relevant conflicts of interest to declare.

Evolutionary intraembryonic origin of vertebrate hematopoietic stem cells in the elasmobranch spleen.

The electric ray (Torpedo marmorata Risso) provides an animal model for the detection of early intraembryonic hemopoietic stem cells (HSCs) in sea vertebrates. The spleen of this bone-marrowless vertebrate appears to be the major site of HSCs differentiation during development and in adulthood. Splenic development in this species was investigated and hemopoietic stem cells were detected in this organ by immunocytochemistry utilizing CD34 and CD38 antibodies. At stage I (2-cm-long embryos with external gills), the spleen contains only mesenchymal cells. At stage II (3-4 cm-long embryos with a discoidal shape and internal gills), an initial red pulp was observed in the spleen, without immunostained cells. At stage III (10-11- cm-long embryos), the spleen contained well-developed white pulp, red pulp and ellipsoids. Image analysis at stage III showed four cell populations, i.e. CD34+/CD38-, CD34+/CD38+, CD34- /CD38+, and CD34-/CD38- cells. The present findings, obtained from an elasmobranch, indicate that the CD34 and CD38 phenotypes are conserved through vertebrate evolution.

Differentiation of haemopoietic stem cells and progenitor cells from human pluripotent stem cells

BackgroundInduced pluripotent stem cells hold great promise as an unlimited source of clinical grade immunologically matched haemopoietic stem cells (HSCs) for transplantation. However, there are currently no robust, reproducible differentiation protocols for the production of engraftable HSCs. We aimed to characterise the production of haemopoietic progenitor cells (HPCs) and HSCs during differentiation of human embryonic stem cells and induced pluripotent stem cells. MethodsWe used a serum-free, feeder-free, good manufacturing practice-compliant differentiation protocol established by the Novosang Consortium for the production of red blood cells for transfusion. Cell phenotype was assessed by flow cytometry for markers of HPCs and HSCs. Cell function was assessed in vitro by the colony forming units in culture (CFU-C) assay and in vivo with a long-term transplantation assay. FindingsAfter preliminary studies, the work focused on days 6, 8, and 10 of the differentiation protocol. In seven independent experiments, HPCs were reproducibly produced at high levels with over 90% of cells expressing CD43 by day 10. Almost all CD41+ cells coexpressed CD43 at all timepoints (day 10, 65% CD43+CD41+). CFU-Cs emerged in large numbers from day 6 and increased by day 10 (mean 1000 colonies per 105 cells plated [SD 478], six experiments). Cytospin and flow cytometry confirmed production of erythroid, granulocyte and macrophage, macrophage, and mixed colonies. The human embryonic HSC markers—vascular endothelial cadherin and CD45—were coexpressed at low levels (mean 3·6% [SD 2·5], seven experiments). No human engraftment was detected when cells were injected into immune-deficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. InterpretationThis differentiation protocol reproducibly gave a high yield of HPCs. However, there was no evidence of engraftable HSCs being produced. This finding could indicate inadequate extrinsic signalling during differentiation. Further work will therefore focus on co-culturing human pluripotent stem cells with human stromal cells in an attempt to mimic the embryonic HSC niche. FundingWellcome Trust.

IL-27 Abrogates Receptor Activator of NF-κB Ligand-Mediated Osteoclastogenesis of Human Granulocyte-Macrophage Colony-Forming Unit Cells through STAT1-Dependent Inhibition of c-Fos

IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Ralpha (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-kappaB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-kappaB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.

Book reviews

Clinical & Laboratory HaematologyVolume 5, Issue 1 p. 116-120 Book reviews First published: March 1983 https://doi.org/10.1111/j.1365-2257.1983.tb00505.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Book Reviewed in this article: Sickle Cell Disease-A Handbook for the General Clinician Edited by A.F. Fleming. Urokinase: basic and clinical aspects Edited by P.M. Mannucci and A. D'Angelo. Haemutology and Oncology. Butterworth's International Medical Reviews, Pediatrics 1. Edited by M. Willoughby and S.E. Siegel. Fungal Infections in the Compromised Patient Edited by D.W. Warnock and M.D. Richardson. Bleeding Disorders. Investigation and Management G.I.C. Ingram, M. Brozovic and N.G.P. Slater. 2nd edn. The Haemophilias, Methods in Haematology Volume 5 Edited by A.L. Bloom. Cytapheresis and Plasma Exchange, Progress in Clinical and Biological Research, Volume 88 Edited by W.R. Vogler. Topical Reviews in Haematology, Volume Two Edited by S. Roath. Cellular and Molecular Biology of Hemopoietic Stem Cell Differentiation T.W. Mak and E.A. McCulloch. Volume5, Issue1March 1983Pages 116-120 RelatedInformation

Interaction and Regulation of HSC with Osteopontin Is Mediated through α4β1 and α9β1Integrins, Which Are Differentially Expressed in the HSC Pool.

Osteoblasts are a key cellular component of the hemopoietic stem cell (HSC) niche and directly regulate the HSC pool. Molecules synthesised by osteoblasts both promote or inhibit HSC proliferation. Osteopontin (Opn) is an osteoblast produced, RGD containing protein with roles in cell adhesion and migration. Until recently, the role of Opn in hemopoiesis was seen as restricted to the regulation of bone turnover. However, from analysis of hemopoiesis in the Opn null mouse, we have demonstrated that Opn plays a critical role in regulating the HSC pool. Furthermore Opn is critical in trans-marrow migration and lodgement of HSC within the BM after transplantation. When added to in vitro HSC cultures, exogenous thrombin-cleaved Opn also inhibits cell proliferation and potently suppresses HSC differentiation. We have now demonstrated that this interaction occurs in an RGD-independent manner via the cryptic SVVYGLR epitope revealed on the N-terminal fragment of Opn following thrombin cleavage. This epitope has previously been shown to bind to α4β1 and α9β1. HSC are known to express α4β1, but we have now shown that within the HSC pool this occurs in a differential manner, mimicking that of CD38, with more committed CD34+CD38+ cord blood progenitors having the highest levels of expression. In addition, we have shown the previously unrecognised characteristic of human marrow and cord blood HSC, the expression of α9β1, which also occurs in a differential manner, but mimicking CD34. Expression of α9β1 is highest on cord blood CD34+CD38− cells, a population highly enriched for HSC. Using the synthetic SVVYGLR peptide in culture, we re-capitulated the thrombin-cleaved Opn induced suppression of HSC differentiation in a dose dependent manner. Antibody blocking experiments demonstrated that binding to this peptide was occurring through both α4β1 and α9β1. In contrast, suppression of HSC proliferation and differentiation did not occur through the upstream alternate α4β1 binding site. Furthermore, we have now demonstrated endogenous binding of Opn to α4β1 and α9β1 to cord blood HSC in vivo. Together, these data provide strong evidence that Opn is an important component of the HSC niche which acts as a physiological negative regulator. Furthermore, our studies identify the previously unrecognised characteristic of HSC, the expression of α9β1, which together with α4β1 provides two receptors on HSC with differing expression signatures and potentially a mechanism for fine tunning the physiological effects of Opn.

The Reciprocal t(9;22)-Translocation Products ABL/BCR Have Leukemogenic Potential Independently from BCR/ABL.

In 95% of chronic myeloid leukemia (CML) and in 25% of acute lymphatic leukemia (ALL) the t(9;22) translocation fuses the bcr gene on chromosome 22 to the abl gene on chromosome 9 and vice versa. On 22+ the different breakpoints leads to the formation of two different major fusion genes: the major breakpoint (M-bcr) related to CML and the minor (m-bcr) related to ALL. The chimaeric fusion gene on 22+ (Philadelphia-chromosome) encodes for the BCR/ABL protein, the p210(BCR/ABL) in CML and the p185(BCR/ABL) in Ph+ALL. The fusion gene on 9+ encodes for the reciprocal ABL/BCR proteins, the p40(ABL/BCR) in CML and the p96(ABL/BCR) in Ph+ALL. The respective ABL/BCR transcripts are detectable in 65% of CML and 100% of Ph+ ALL patients. The ABL/BCRs are BCR mutants and thus N-terminally truncated Rho-guanine-nucleotide exchange factors (Rho-GEF's). It is known that the N-terminal truncation can confer transformation potential to Rho-GEFs, such as NET-1. In addition, both ABL/BCRs, like wt BCR, contain a C-terminal Rac-GTPase activating protein (GAP)-domain. CML-associated ABL/BCR (p96(ABL/BCR)) differs from the Ph+ ALL-associated p40(ABL/BCR) in that that it misses the ‘dbl homology domain'(DH domains) of potential oncogenic function. Hence it seems that Ph+ALL blasts, in contrast to CML-blasts, express, as a consequence of t(9;22) translocation, two oncogenic fusion proteins, the p185(BCR-ABL) as well as the p40(ABL/BCR) protein. Actually nothing is known about the contribution of the reciprocal t(9;22) translocation products to the CML- and the ALL-phenotype. Thus we studied the phenotype induced by the ABL/BCRs in hemopoietic progenitors.Here we report that both ABL/BCRs i) lost the capacity of wt BCR to suppress the activation of RAC by its Rac-GAP domain, but did not influence the activation status of Rho or cdc42; ii) as a consequence of the deregulation of Rac the cytoskeleton modelling by BCR (Filopodia - cdc42-like phenotype) was altered in p40(ABL/BCR)- and p96(ABL/BCR)-expressing fibroblasts (stress fibers - Rho-like phenotype and “microspikes”, respectively); iii) the increase of migration of BCR-expressing 32D cells into a stroma cell-spheroid model was reverted in p40(ABL/BCR)- and p96(ABL/BCR)-expressing 32D cells; iv) adhesion to TNFalpha activated endothelial cell layer in the “flow chamber” was increased in BCR-positive 32D cells but not in p40(ABL/BCR)- and p96(ABL/BCR)-positive cells. Regarding their leukemogenic potential we showed that i) both ABL/BCRs, in contrast to wt BCR, activated RAS; ii) both ABL/BCRs were unable to transform fibroblasts and to render Ba/F3 cells factor-independent. iii) p96(ABL/BCR) increased the replating efficiency of Sca1+/lin- hemopoietic stem cells (HSC) by selecting a population of immature HSC exclusively expressing c-kit and Sca-1 more strongly than p40(ABL/BCR); iv.) both ABL/BCR blocked the myeloid differentiation of HSC v) the inoculation of p96(ABL/BCR)- or p40(ABL/BCR)-expressing HSC into lethally irradiated recipient mice led in the 40% and 60% of the cases, respectively, to a clinical picture of either acute leukemia or myeloproliferative syndrome within 2–9 months.These data show for the first time that the t(9;22) leads to two leukemogenic fusion proteins - the BCR/ABL and the ABL/BCR - in CML as well as in Ph+ALL, which might represent an additional target for molecular therapy approaches.

Apoptotic mechanisms in the control of erythropoiesis.

Erythropoiesis is a complex multistep process encompassing the differentiation of hemopoietic stem cells to mature erythrocytes. The steps involved in this complex differentiation process are numerous and involve first the differentiation to early erythoid progenitors (burst-forming units-erythroid, BFU-E), then to late erythroid progenitors (colony-forming units-erythroid) and finally to morphologically recognizable erythroid precursors. A key event of late stages of erythropoiesis is nuclear condensation, followed by extrusion of the nucleus to produce enucleated reticulocytes and finally mature erythrocytes. During the differentiation process, the cells became progressively sensitive to erythropoietin that controls both the survival and proliferation of erythroid cells. A normal homeostasis of the erythropoietic system requires an appropriate balance between the rate of erythroid cell production and red blood cell destruction. Growing evidences outlined in the present review indicate that apoptotic mechanism play a relevant role in the control of erythropoiesis under physiologic and pathologic conditions. Withdrawal of erythropoietin or stimulation of death receptors such as Fas or TRAIL-Rs leads to activation of a subset of caspase-3, -7 and -8, which then cleave the transcription factors GATA-1 and TAL-1 and trigger apoptosis. In addition, there is evidence that a number of caspases are physiologically activated during erythroid differentiation and are functionally required for erythroid maturation. Several caspase substrates are cleaved in differentiating cells, including the protein acinus whose activation by cleavage is required for chromatin condensation. The studies on normal erythropoiesis have clearly indicated that immature erythroid precursors are sensitive to apoptotic triggering mediated by activation of the intrinsic and extrinsic apoptotic pathways. These apoptotic mechanisms are frequently exacerbated in some pathologic conditions, associated with the development of anemia (ie, thalassemias, multiple myeloma, myelodysplasia, aplastic anemia). The considerable progress in our understanding of the apoptotic mechanisms underlying normal and pathologic erythropoiesis may offer the way to improve the treatment of several pathologic conditions associated with the development of anemia.

Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34 ++ CD33,38,71 − . Bone marrow and umbilical cord blood samples of CD34 + cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34 ++ CD33,38,71 − phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34 ++ CD33,38,71 − phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34 ++ CD33,38,71 − subset. In comparison to this ‘intrinsic’ potential, the effects of the applied growth stimuli on CD34 ++ CD33,38,71 − cell replication were negligible. In contrast, the overall recovery of the CD34 ++ CD33,38,71 − cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.

Role of cytokines and extracellular matrix in the regulation of haemopoietic stem cells

The understanding of molecular mechanisms regulating the formation, growth and differentiation of haemopoietic stem cells has advanced considerably recently. Particular progress has been made in defining the cytokines, chemokines and extracellular matrix components which retain and maintain primitive haemopoietic cell populations in bone marrow. Furthermore, signal transduction pathways that are critical for haemopoiesis, both in vivo and in vitro, and that are activated by cytokines have also been identified and further characterised. The importance of these processes has, this year, been exemplified by the phenotypes of mice deficient in key signal transduction proteins and the discovery that mutations in the component proteins of some signalling pathways are linked to human diseases. Significant advances in understanding the molecular mechanisms for mobilisation of stem cells from bone marrow have also been made this year; this has potential importance for bone marrow transplantation.

造血幹細胞の増殖と分化の骨髄‐間質細胞による制御

造血幹細胞の増殖と分化の骨髄‐間質細胞による制御

Frequent deletion of chromosome 12p12.3 in children with acute lymphoblastic leukaemia

Cytogenetic deletions of the short arm of chromosome 12 are common recurring alterations found in a wide range of haematological neoplasias, including childhood acute lymphoblastic leukaemia (ALL), the most frequent paediatric malignancy. Such a loss of genetic material suggests the presence of a tumour suppressor gene which plays an important role in growth regulation or in the differentiation of haemopoietic stem cells. To substantiate this hypothesis and to determine more precisely the chromosomal location of this putative gene, we applied a deletion mapping strategy based on the detection of loss of heterozygosity (LOH) at specific genomic loci in tumour cells. 13 polymorphic markers were used to screen DNA samples from 20 children with ALL. LOHs at 12p12.3 were observed in almost 50% of informative B-cell precursor ALL patients analysed. This is one of the most frequent genetic alterations found in this disease. A common region of LOH was delimited by the markers D12S89 (distal) and D12S358 (proximal), separated by a genetic interval of approximately 3 cM. We refined the position of the putative 12p tumour suppressor gene to a physical interval of <1.3 Mb, a crucial step towards the identification of candidate genes. A yeast artificial chromosome clone contig that spans the entire critically deleted region includes two known genes: TEL, a member of the ets family of transcription factors, and p27KIP1, a cyclin-dependent kinase inhibitor.

Identification of a unique membrane-bound molecule on a hemopoietic stem cell line and on multipotent progenitor cells.

Hemopoietic stem cells are a distinct population of cells that can differentiate into multilineages of hemopoietic cells and have long-term repopulation capability. A few membrane-bound molecules have been found to be preferentially, but not uniquely, present on the surface of these primitive cells. We report here the identification of a unique 105-kDa glycoprotein on the surface of hemopoietic stem cell line BL3. This molecule, recognized by the absorbed antiserum, is not present on the surface of myeloid progenitors 32D and FDC-P1 cells, EL4 T cells, and NIH 3T3 fibroblasts. This antiserum can also be used to block the proliferation of BL3 cells even in the presence of mitogen-stimulated spleen cell conditioned medium, which is known to have a stimulating activity on BL3 cells. It can also inhibit development of in vitro, fetal liver cell-derived multilineage colonies, but not other types of colonies, and of in vivo bone marrow cell-derived colony-forming unit spleen foci. These data suggest that gp105 plays an important role in hemopoietic stem cell differentiation.

Cloning of the bovine interleukin-3-encoding cDNA

Interleukin-3 (IL-3) is one of the cytokines that act during the early and late stages of blood cell formation. To enable the study of the role of IL-3 in bovine haemopoietic stem cell differentiation, the polymerase chain reaction was used to amplify an IL-3 cDNA from first-strand cDNAs prepared from RNA isolated from 4- and 5-hour concanavalin-A-stimulated peripheral blood lymphocytes (PBL) from N'Dama cattle. An analysis of the cDNA sequence reveals that it contains a 432-nucleotide (nt) open reading frame which codes for 144 amino acids (aa). Cleavage of the putative signal peptide consisting of the first 17 aa yields the mature form of the protein (14.5 kDa). Comparisons of the bovine IL-3 sequence with the sheep, human and mouse IL-3 sequences show that the bovine sequence shares 90.7, 55.8 and 51.9% nt identity, respectively, in the coding region, and 85.4, 35 and 27.7% aa identity, respectively.

Separate control of the survival, the self-renewal and the differentiation of hemopoietic stem cells.

Hemopoietic stem cells adhere to hemopoietic supportive (MS-5) cells, but not to non-supportive (MS-K) cells. Although a soluble stem cell factor (SCF) was produced by both of these cell lines, little activity was detectable in the supernatant from the cultures of either of these cells, indicating that SCF might be compartmentalized within the extracellular matrix (ECM), and transferred directly to the stem cells via the ECM (44). To probe this possibility, we studied the transfer of SCF from the ECM and the subsequent support of the survival of the hemopoietic stem cells. A stem cell-enriched bone marrow cell fraction was overlaid on SCF-containing ECM. The stem cells survived and proliferated for some days without differentiation under these conditions, whereas stem cells overlaid on ECM without SCF died within a few days. Addition of interleukin-3 (IL-3) to the ECM that contains SCF, induced differentiation of the stem cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced further differentiation of the stem cells, which was accompanied by a decrease in the number of colony-forming unit in spleen (CFU-S). These observations verified the above hypothesis, and indicated that the survival, the self-renewal, and the differentiation of hemopoietic stem cells can be separately controlled at least in vitro.

Influence of growth factors and substrates on differentiation of haemopoietic stem cells

During the last few years there has been major progress in our understanding of the mechanisms underlying the regulation of haemopoietic stem cell proliferation and development. In the past 12 months, advances have been made in identifying how growth factor receptor expression is regulated in primitive haemopoietic cells, in determining the transcription factors that are associated with development, and in recognizing some of the specific molecular interactions that occur between the bone marrow stromal cells and the haemopoietic progenitor cells. These and previous studies clearly demonstrate that the environment can influence the survival, the proliferation and the differentiation of haemopoietic cells at every stage of development.

The renewal and differentiation of hemopoietic stem cells.

Blood-forming tissues are organized in well-defined microenvironments composed of hemopoietic cells and a supportive stroma of connective tissue and endothelium. Hemopoietic cells segregate to various lineages, all derived from a small population of pluripotent stem cells residing in the bone marrow. Regulation of growth and differentiation, particularly under conditions of perturbations, damage, and disease, is mediated by inducer colony-stimulating factors and interleukins counteracted by inhibitory cytokines. Whereas much is known about the mode of induction of differentiation, insufficient information is available to explain the process of stem cell renewal that is crucial for the longevity of the hemopoietic system. It is also only partially known how inhibition of hemopoietic processes occurs, and what molecules in blood-forming tissues signal organization into discrete patterns. This paper reviews recent progress that has opened new avenues to a better understanding of this highly complex issue.