Expansion Of Hematopoietic Cells Research Articles

Perspektiven der Forschung bei kutanen Lymphomen

Primary cutaneous lymphomas are characterized by an expansion of hematopoietic cells in the special microenvironment of the skin. They represent a special challenge both for researches and for clinicians who treat patients with these disorders. New research data concerning the biology of lymphocytes and the cutaneous microenvironment have increased our knowledge of these diseases in the last decades. The new WHO/EORTC classification definitely will facilitate a more detailed investigation of the various subtypes.

BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis

The bone morphogenetic protein (BMP) signaling pathway is essential during gastrulation for the generation of ventral mesoderm, which makes it a challenge to define functions for this pathway at later stages of development. We have established an approach to disrupt BMP signaling specifically in lateral mesoderm during somitogenesis, by targeting a dominant-negative BMP receptor to Lmo2+ cells in developing zebrafish embryos. This results in expansion of hematopoietic and endothelial cells, while restricting the expression domain of the pronephric marker pax2.1. Expression of a constitutively active receptor and transplantation experiments were used to confirm that BMP signaling in lateral mesoderm restricts subsequent hemato-vascular development. The results show that the BMP signaling pathway continues to function after cells are committed to a lateral mesoderm fate, and influences subsequent lineage decisions by restricting hemato-vascular fate in favor of pronephric development.

Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34(+) cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34(+) cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34(+) cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34(+) cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.

Expansion ex vivo des cellules souches hématopoïétiques humaines par la transduction passive de l'homéoprotéine HOXB4

Expansion of human hematopoietic stem cells (HSCs) is a challenge for cellular therapy. It currently relies on either the use of recombinant cytokines or transfer of transcription factor genes. Among these, the HOXB4 homeoprotein is of particular interest since it promotes the expansion of mouse HSCs without inducing leukemia. To prevent potential deleterious side effects associated with stable HOXB4 gene transfer into the cells, we took advantage of the ability of homeoproteins to passively pass through cell membranes. We have shown that, when co-cultured with stromal cells engineered to secrete HOXB4, human stem cells and immature progenitors clearly were expanded. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. The role that HOXB4 plays on stem cell expansion has also been tested on human lymphoid progenitors. We found that our model of protein transfer was also able to induce the expansion of the immature lympho-myeloid and pro-T/NK progenitors as well as of more mature NK progenitors. We then looked for synergistic activities between HOXB4 and other homeoproteins such as HOXC4. We found that HOXC4 was able to promote the expansion of human hematopoietic cells in vitro roughly as HOXB4 did and that the presence of both HOXB4 and HOXC4 molecules induced even higher expansion levels of these cells. Our method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

Exogenous gene expression and growth regulation of hematopoietic cells via a novel human artificial chromosome

A number of gene delivery systems are currently being developed for potential use in gene therapy. Here, we demonstrate the feasibility of 21deltaqHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. We first introduced a 21deltaqHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21deltaqHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21deltaqHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21deltaqHAC could still be detected at two weeks post-transplantation in immunodeficient mice. We also showed effective expansion of hematopoietic cells by introducing the 21deltaqHAC containing ScFvg, a gp130-based chimeric receptor that transmits growth signals in response to specific-antigen of this receptor. All of these results demonstrate the usefulness of HAC in gene therapy.

Cocultures of Umbilical Cord Blood Hematopoietic Progenitor Cells and Umbilical Cord Endothelium.

Purpose. The expansion of hematopoietic progenitor cells (HPC) from cord blood is limited so far. For HPC expansion and differentiation we evaluated a coculture model of cord blood-derived HPC and endothelial cells (EC) isolated from the umbilical cord.Methods. CD34(+) HPC were cultured directly in contact with the endothelial monolayer or indirectly using a 0.4μm microporous transmembrane. EC were stimulated by various cytokines including TPO, interleukin (IL) 1β, IL4, and IL6. After 1 and 2 weeks, the HPC were counted and analyzed by flow cytometry. Their plating efficiencies were determined by methylcellulose assays, and their reconstitutive potential was analyzed by cobblestone assays. DNA microarrays on the expanding cells as well as on cytokine-stimulated EC were also performed.Results. EC stimulation with thrombopoietin and IL4 had no significant effect on mononuclear cell expansion. In contrast, IL1β stimulation resulted in a 10 and 100fold increase of the cell number after one and two weeks, respectively. More than 90% of these cells were CD33(+) and had a plating efficiency equivalent to that of HPC post isolation (7.3 ±1.2 versus 8.3±0.68%). The number of 5-wk cobblestone-area forming cells among these expanded cells was diminished, whereas the number of 2-wk cobblestone areas did not change (10.6 versus 8.5). The expanded cells could further mature into functional granulocytes. Time-course DNA microarrays of IL1β-stimulated EC demonstrated an increased expression of genes encoding tumor necrosis factor-induced proteins, interleukins, and endothelial adhesion molecules. When IL1β was replaced by IL6, a 7-fold expansion of morphologically progenitor-like cells took place. 10–13% of these cells were positive for CD133, had a plating efficiency of 10.2±1.7%, and showed on average 14.5 five-week cobblestone areas.Conclusion. Cocultures of cord blood-derived HPC with umbilical cord-derived EC offer alternative methods for the expansion and differentiation of hematopoietic cells.

SPRY1 Is a Negative Regulator of Long-Term In Vivo Engraftment and Ex Vivo Expansion of Primitive Human Umbilical Cord Blood Cells.

Hematopoietic stem cells (HSCs) are functionally defined by their capacity to home to the bone marrow microenvironment, proliferate and differentiate to restore normal hematopoiesis in a myeloablated recipient; however, the molecular determinants of these processes are not well understood. By comparing the gene expression profiles of highly purified human HSC-enriched and HSC-depleted cell populations, and subsequently validating the hematopoietic function of a subset of these differentially expressed genes using zebrafish (Danio rerio), we previously identified human Sprouty 1 (SPRY1), an evolutionarily conserved antagonist of RTK signaling, as a potential regulator of mammalian HSC development and/or cell-fate decisions. To directly assess the role of SPRY1 in mammalian HSC and hematopoietic progenitor cell (HPC) function, we constructed a dual-promoter lentiviral vector to co-express SPRY1 and green fluorescent protein (GFP) (SPRY1-LV) and a control lentiviral vector to express GFP alone (GFP-LV) in the CD34+ fraction of human umbilical cord blood (UCB). While the enforced expression of SPRY1 in CD34+ UCB cells had no effect on the frequency or morphology of colonies generated in short-term in vitro colony-forming cell (CFC) assays (SPRY1-LV = 27.6 ± 12.3% and GFP-LV = 28.4 ± 20.0%; n = 3), it profoundly inhibited the capacity of UCB CD34+ cells to engraft in the bone marrow NOD-SCID mice in vivo. The estimated frequency of week 11 SCID-repopulating cells (SRC) (± 1 standard error) for SPRY1-LV (n = 13 mice) and GFP-LV (n = 15 mice) cells was 1 in 12,678 cells (6,167 – 26,064) and 1 in 3,412 cells (2,272 – 5,124), respectively, as determined using limiting dilution conditions and Poisson statistics. Furthermore, in 14 day cultures designed for the ex vivo expansion and/or maintenance of primitive hematopoietic cells, ectopic expression of SPRY1 in CD34+ UCB cells dramatically inhibited the expansion of total nucleated cells (SPRY1-LV = 37.9 ± 10.1 fold; GFP-LV = 71.1 ± 5.8 fold; n = 3; p<0.05) and CFCs (SPRY1-LV = 5.7 ± 1.2 fold; GFP-LV = 20.8 ± 14.9 fold; n = 3), although it had no effect on expansion of CD34+CD38− cells. We are currently investigating potential mechanisms for the observed affects of SPRY1 on primitive hematopoietic cells, paying particular attention to the possible effects of SPRY1 expression on “early-acting” hematopoietic cytokines and growth factors that activate RTKs - including FGF1, FGF2, VEGF, SCF, FLT3L, and ANGPT1. In conclusion, enforced expression of SPRY1 negatively regulates primitive hematopoietic cell engraftment in vivo and expansion in vitro, thereby presenting the first example of a role for a Sprouty family member, SPRY1, in primitive human hematopoietic cell function. Moreover, this data further validates the use of model organisms, such as zebrafish, for evaluating the functional roles of transcripts identified in large-scale gene expression profiling experiments in mice and humans.

PEGylated murine Granulocyte–macrophage colony-stimulating factor: Production, purification, and characterization

Granulocyte–macrophage colony-stimulating factor (GM-CSF) regulates proliferation, differentiation, and function of hematopoietic progenitor cells. Aside from expansion of hematopoietic cells, GM-CSF has shown efficacy in other diseases, including Crohn’s disease. While GM-CSF being clinically used in humans, the ability to perform mechanistic studies in murine models is difficult due to the limited availability and rapid clearance of murine GM-CSF in the peripheral blood. To address these issues, we efficiently expressed murine GM-CSF under the control of the AOX1 gene promoter in Pichia pastoris using the Mut S strain KM71H. We describe the unique conditions that are required for efficient production by high-density fermentation and purification of mGM-CSF protein. Recombinant mGM-CSF protein was purified by tangential flow ultrafiltration and preparative reverse phase chromatography. To address limited half life or rapid clearance in mice, recombinant murine GM-CSF was modified by lysine-directed polyethylene glycol conjugation (PEGylation). PEG-modified and unmodified proteins were characterized by amino terminus sequence analysis and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Under the mild reaction conditions, the recombinant protein is efficiently modified by PEGylation on an average of 2–3 sites per molecule. In vivo treatment of mice with PEGylated mGM-CSF, but not the unmodified recombinant mGM-CSF, reproduces the potent colony stimulating effects of human GM-CSF in patients on myeloid progenitor populations, as assessed by FACs analysis. This simplified approach for the expression, purification, and modification of a biologically potent form of murine GM-CSF should facilitate the study of central mechanisms of action in murine disease models.

Acute promyelocytic leukemia: A model of molecular target based therapy

Leukemia, a group of hematological malignancies characterized by clonal expansion of hematopoietic cells with uncontrolled proliferation, decreased apoptosis and blocked differentiation, is one of the most notorious enemies of mankind which accounts for some 300,000 new cases and 222,000 deaths each year worldwide 1234. Leukemia can be divided into acute or chronic, lymphoid or myeloid types, based on the disease progression and hematopoietic lineages involved 5. The responses of leukemia to therapies differ from one type or subtype to another. Hence, to improve the clinical outcome, the therapeutic strategies should be disease pathogenesis-based and individualized. The close collaboration between bench and bedside may not only shed new lights on leukemogenesis, gain insights into therapeutic mechanisms, but also provide opportunities for designing more rational therapies. The development of curative approaches for acute promyelocytic leukemia (APL) may serve as a paradigm 6.

A Gain-of-Function Mutation ofJAK2in Myeloproliferative Disorders

Polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis are clonal myeloproliferative disorders arising from a multipotent progenitor. The loss of heterozygosity (LOH) on the short arm of chromosome 9 (9pLOH) in myeloproliferative disorders suggests that 9p harbors a mutation that contributes to the cause of clonal expansion of hematopoietic cells in these diseases. We performed microsatellite mapping of the 9pLOH region and DNA sequencing in 244 patients with myeloproliferative disorders (128 with polycythemia vera, 93 with essential thrombocythemia, and 23 with idiopathic myelofibrosis). Microsatellite mapping identified a 9pLOH region that included the Janus kinase 2 (JAK2) gene. In patients with 9pLOH, JAK2 had a homozygous G-->T transversion, causing phenylalanine to be substituted for valine at position 617 of JAK2 (V617F). All 51 patients with 9pLOH had the V617F mutation. Of 193 patients without 9pLOH, 66 were heterozygous for V617F and 127 did not have the mutation. The frequency of V617F was 65 percent among patients with polycythemia vera (83 of 128), 57 percent among patients with idiopathic myelofibrosis (13 of 23), and 23 percent among patients with essential thrombocythemia (21 of 93). V617F is a somatic mutation present in hematopoietic cells. Mitotic recombination probably causes both 9pLOH and the transition from heterozygosity to homozygosity for V617F. Genetic evidence and in vitro functional studies indicate that V617F gives hematopoietic precursors proliferative and survival advantages. Patients with the V617F mutation had a significantly longer duration of disease and a higher rate of complications (fibrosis, hemorrhage, and thrombosis) and treatment with cytoreductive therapy than patients with wild-type JAK2. A high proportion of patients with myeloproliferative disorders carry a dominant gain-of-function mutation of JAK2.

Cytokine production and hematopoiesis supporting activity of cord blood–derived unrestricted somatic stem cells

Cytokine production and hematopoiesis-supporting stromal activity of cord blood (CB)-derived unrestricted somatic stem cells (USSC) in comparison to bone marrow mesenchymal stem cells (BMMSC) and hematopoietic progenitor expansion solely driven by recombinant cytokines were assessed. USSC generation was initiated from fresh and cryopreserved CB. Cytokine production by USSC and BMMSC was determined qualitatively by cytokine mRNA expression array analyses or quantitatively by Multiplex or ELISA analyses. To evaluate hematopoiesis-supporting activity, CB CD34+ cells were expanded in cocultures with USSC and BMMSC or in the presence of Flt3-L, SCF, and TPO. Expansion of CD34+ cells, total cells, colony-forming cells (CFC), and LTC-IC were determined after 1, 2, 3, and 4 weeks of culture. USSC constitutively produced SCF, LIF, TGF-1beta, M-CSF, GM-CSF, VEGF, IL-1beta, IL-6, IL-8, IL-11, IL-12, IL-15, SDF-1alpha, and HGF. When USSC were stimulated with IL-1beta, G-CSF was released. Production of SCF and LIF were significantly higher in USSC compared to BMMSC. At 1, 2, 3, and 4 weeks, cocultivation of CD34+ cells on the USSC layer resulted in a 14.6-fold +/- 1.1-fold, 110.1-fold +/- 17.9-fold, 151.8-fold +/- 39.7-fold, and 183.6-fold +/- 40.4-fold amplification of total cells and in a 30.6-fold +/- 4.4-fold, 101.4-fold +/- 27.5-fold, 64.7-fold +/- 15.8-fold, and 29.4-fold +/- 3.1-fold amplification of CFC, respectively. LTC-IC expansion at 1 and 2 weeks was, with 2.0-fold +/- 0.1-fold and 2.5-fold +/- 0.3-fold, significantly higher for USSC than BMMSC (1.1-fold +/- 0.03-fold and 1.1-fold +/- 0.1-fold), but declined after day 21. Transwell cocultures of USSC did not significantly alter total cell or CFC expansion. USSC produce functionally significant amounts of hematopoiesis-supporting cytokines and are superior to BMMSC in expansion of CD34+ cells from CB. USSC is therefore a suitable candidate for stroma-driven ex vivo expansion of hematopoietic CB cells for short-term reconstitution.

Cell processing engineering for ex-vivo expansion of hematopoietic cells

The cell processing engineering for ex vivo expansion of hematopoietic cells is reviewed. All hematopoietic cells of different lineages and/or at various stages of differentiation are derived from the same precursor, pluripotent hematopoietic stem cells. Bone marrow stromal cells promote and regulate the self-renewal, commitment, differentiation, and proliferation of stem cells and progenitors through their secreted extracellular matrices and cytokine environment in the hematopoietic microenvironment. Although stroma-mediated hematopoiesis has been studied in vitro using the Dexter culture system in tissue culture flasks, hematopoiesis in the Dexter culture system is almost limited to a granulocyte lineage and the system could not expand primitive cells. The addition of large amounts of cytokines to the culture of hematopoietic cells enabled their expansion, but is too expensive. Some clonal stromal cell lines have been established from the Dexter culture of murine bone marrow cells in order to simplify and stimulate the ex vivo expansion of hematopoietic cells. In order to solve the problem regarding the usage of exogeneic stromal cell lines, a novel membrane-separated coculture system, in which stromal cells adhere onto the lower surface of a porous membrane and hematopoietic cells are incubated on the upper surface of the membrane, was proposed. In order to mimic the contact between stromal and hematopoietic cells in vivo in the bone marrow, several types of three-dimensional (3-D) culture of hematopoietic cells were developed. The 3-D coculture of hematopoietic cells with spatial development of stromal cells in nonwoven fabrics enabled the expansion of progenitors without cytokine addition. Progenitors in cord blood mononucleated cells were also successfully expanded without the addition in the 3-D coculture with primary human bone marrow stromal cells in 3-D. Heparin addition to the 3-D coculture and coating the nonwoven fabrics with N-(O-beta-(6-O-sulfogalactopyranosyl)-6-oxyhexyl)-3,5-bis(dodecyloxy)-benzamide further increased the number of progenitors.

Cord Blood Processing by Using a Standard Manual Technique and Automated Closed System "Sepax" (Kit CS-530)

Stem Cells and DevelopmentVol. 14, No. 1 CorrespondenceCord Blood Processing by Using a Standard Manual Technique and Automated Closed System "Sepax" (Kit CS-530)Bernard Dazey, Pascale Duchez, Catherine Letellier, Gerard Vezon, and Dr. Zoran IvanovicBernard DazeySearch for more papers by this author, Pascale DuchezSearch for more papers by this author, Catherine LetellierSearch for more papers by this author, Gerard VezonSearch for more papers by this author, and Dr. Zoran IvanovicSearch for more papers by this authorPublished Online:22 Feb 2005https://doi.org/10.1089/scd.2005.14.6AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byDiscarded plasma obtained after cord blood volume reduction as an alternative for fetal calf serum in mesenchymal stromal cells cultures6 August 2020 | Transfusion, Vol. 3Umbilical cord stem cells: Background, processing and applicationsTissue and Cell, Vol. 65Automated thawing increases recovery of colony-forming units from banked cord blood unit grafts11 October 2018 | Transfusion, Vol. 58, No. 12Cord Blood Graft Thawing14 October 2017Comparison of human umbilical cord blood processing with or without hydroxyethyl starch23 August 2017 | Transfusion, Vol. 57, No. 11Cord blood collection and processing with hydroxyethyl starch or non–hydroxyethyl starchCytotherapy, Vol. 18, No. 5Cell BankingCurrent thawing and infusion practice of cryopreserved cord blood: the impact on graft quality, recipient safety, and transplantation outcomes4 June 2014 | Transfusion, Vol. 54, No. 11A Novel Procedure to Improve Functional Preservation of Hematopoietic Stem and Progenitor Cells in Cord Blood Stored at +4°C Before Cryopreservation Jean Chevaleyre, Laura Rodriguez, Pascale Duchez, Marie Plainfossé, Bernard Dazey, Véronique Lapostolle, Marija Vlaski, Philippe Brunet de la Grange, Bruno Delorme, and Zoran Ivanovic7 April 2014 | Stem Cells and Development, Vol. 23, No. 15Cord blood banking activity in Iran National Cord Blood Bank: A two years experienceTransfusion and Apheresis Science, Vol. 50, No. 1Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cellsJournal of Translational Medicine, Vol. 12, No. 1Nutrient Regulation by Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids18 October 2013 | PLoS ONE, Vol. 8, No. 10Cryopreservation of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34+ cells11 December 2012 | Transfusion, Vol. 53, No. 9Functional Stability (at +4°C) of Hematopoietic Stem and Progenitor Cells Amplified Ex Vivo from Cord Blood CD34 + Cells1 August 2013 | Cell Transplantation, Vol. 22, No. 8Potential Application of Cord Blood-Derived Stromal Cells in Cellular Therapy and Regenerative MedicineJournal of Blood Transfusion, Vol. 2012Definitive Setup of Clinical Scale Procedure for Ex Vivo Expansion of Cord Blood Hematopoietic Cells for Transplantation1 November 2012 | Cell Transplantation, Vol. 21, No. 11Human Umbilical Cord Blood-Derived Very-Small-Embryonic-Like Stem Cells with Maximum Regenerative Potential? Zoran Ivanovic15 March 2012 | Stem Cells and Development, Vol. 21, No. 14Analysis of 120 pediatric patients with nonmalignant disorders transplanted using unrelated plasma-depleted or -reduced cord blood21 November 2011 | Transfusion, Vol. 52, No. 6Processing of hematopoietic stem cells from peripheral blood before cryopreservation: use of a closed automated system20 May 2011 | Transfusion, Vol. 51, No. 12Cord blood banking and transplantation20 October 2011 | ISBT Science Series, Vol. 6, No. 2Cell recovery comparison between plasma depletion/reduction- and red cell reduction-processing of umbilical cord bloodCytotherapy, Vol. 13, No. 9Clinical-Scale Cultures of Cord Blood CD34 + Cells to Amplify Committed Progenitors and Maintain Stem Cell Activity1 October 2011 | Cell Transplantation, Vol. 20, No. 9Cord (placental) blood storage: extent and functional aspects6 September 2011 | Transfusion, Vol. 51, No. 9High recovery of mesenchymal progenitor cells with non-density gradient separation of human bone marrowCytotherapy, Vol. 12, No. 5Expansion ex vivo des cellules hematopoiétiques : concept et utilité cliniqueTransfusion Clinique et Biologique, Vol. 16, No. 5-6A new automatic device for routine cord blood banking: critical analysis of different volume reduction methodologiesCytotherapy, Vol. 11, No. 8A clinical study on the feasibility of autologous cord blood transfusion for anemia of prematurityTransfusion, Vol. 48, No. 8Good Manufacturing Practice-Compliant Expansion of Marrow-Derived Stem and Progenitor Cells for Cell Therapy22 June 2017 | Cell Transplantation, Vol. 16, No. 7 Volume 14Issue 1Feb 2005 InformationCopyright 2005, Mary Ann Liebert, Inc.To cite this article:Bernard Dazey, Pascale Duchez, Catherine Letellier, Gerard Vezon, and Dr. Zoran Ivanovic.Cord Blood Processing by Using a Standard Manual Technique and Automated Closed System "Sepax" (Kit CS-530).Stem Cells and Development.Feb 2005.6-10.http://doi.org/10.1089/scd.2005.14.6Published in Volume: 14 Issue 1: February 22, 2005PDF download

Cell therapy for the treatment of accidental radiation overexposure

Irradiation kills cells directly or indirectly, and the basic issue is to replace them. To do so, the cells required to heal the injured compartment can be obtained from a non- (or less) injured part of the body or from an allogeneic source. Because the number of cells necessary to treat an injury can be very high, the cells have to be expanded ex vivo. Owing to their transdifferentiation or plasticity property, cells from some tissues could even possibly repopulate another system. Human bone marrow contains two cell compartments: the haematopoietic cell compartment, and the stromal cell compartment that comprises mesenchymal stem cells (MSCs). Both compartments could be used for cell therapy. We will focus on four specific questions. Is it possible to use ex vivo expansion of haematopoietic cells to treat bone marrow aplasia? Are human MSCs homing in the injured tissues following irradiation? Is it possible to separate MSC haematopoietic support from MSC homing ability following irradiation? Is it possib...

EVI1 Abrogates Interferon-α Response by Selectively Blocking PML Induction

EVI1 is an oncogene frequently associated with chronic and acute myeloid leukemia. In hematopoietic cells, EVI1 impairs several pathways including proliferation, differentiation, and apoptosis. Interferon-alpha (IFN-alpha) is a powerful cytokine that controls the immune response and limits the expansion of several tissues including bone marrow. These properties contribute to the effectiveness of IFN-alpha in the treatment of many neoplastic disorders especially chronic myeloid leukemia. We report here that in murine hematopoietic progenitors the expression of EVI1 completely abrogates the antiproliferative and apoptotic effects of IFN-alpha. EVI1 does not repress the JAK/STAT signaling pathway or the activation of many IFN-responsive genes. On the contrary, EVI1 prolongs the phosphorylation of STAT1 and the activation of an IFN-dependent reporter gene. However, EVI1 specifically represses the IFN-dependent induction of the tumor suppressor PML and blocks the apoptotic pathways activated by PML. We show that the position of the ISRE, which is located within the first exon of PML, is critical to block PML induction by IFN-alpha. The relocation of the ISRE to a position upstream of the transcription start site is sufficient to re-establish the response to IFN in the presence of EVI1. Our data suggest that stabilized STAT1 phosphorylation and prolonged binding of the STAT1 complex to the first exon could impair PML transcription and inhibit the activation of PML-dependent apoptotic pathways resulting in loss of IFN response. These results point to a novel mechanism utilized by an oncogene to escape normal cell response to growth-controlling cytokines.

Expression Profiling of Murine Acute Promyelocytic Leukemia Cells Reveals Multiple Model-Dependent Progression Signatures

Leukemia results from the expansion of self-renewing hematopoietic cells that are thought to contain mutations that contribute to disease initiation and progression. Studies of the gene expression profiles of human acute myeloid leukemia samples has allowed their classification based on the presence of translocations and French-American-British subtypes, but it is not yet clear whether their molecular signatures reflect the initiating mutations or mutations acquired during progression. To begin to address this question, we examined the expression profiles of normal murine promyelocyte-enriched samples, nontransformed murine promyelocytes expressing human promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) fusion gene, and primary acute promyelocytic leukemia cells. The expression profile of nontransformed cells expressing PML-RARalpha was remarkably similar to that of wild-type promyelocytes. In contrast, the expression profiles of fully transformed cells from three acute promyelocytic leukemia model systems were all different, suggesting that the expression signature of acute promyelocytic leukemia cells reflects the genetic changes that contributed to progression. To further evaluate these progression events, we compared two high-penetrance acute promyelocytic leukemia models that both commonly acquire an interstitial deletion of chromosome 2 during progression. The two models exhibited distinct gene expression profiles, suggesting that the dominant molecular signatures of murine acute promyelocytic leukemia can be influenced by several independent progression events.

Three‐Dimensional Bioreactor Cultures: A Useful Dynamic Model for the Study of Cellular Interactions

The ex vivo expansion of hematopoietic cells is a developing area with emphasis on bioreactor systems for amelioration of culture conditions. A rational design of bioreactors, especially those allowing microgravity, could permit the production of stem cells and will offer new approaches for studying the mechanisms of proliferation, differentiation, and signal transduction of cultured cells. The efficacy of two commercially available bioreactors (rotating-vessel miniPERM and static INTEGRA CL 350) to support long-term bone marrow cell cultures (LTBMCC) and three-dimensional growth of Hodgkin's lymphoma HD-MY-Z cells was investigated. In the miniPERM system, the growth of LTBMCC spheroids (containing 30-40 cells) was obtained. An essentially higher content of hematopoietic precursor cells (colony-forming units-granulocyte macrophage) was registered in the rotating-vessel system. In this bioreactor, a growth of large HD-MY-Z spheroids (containing 100-200 cells) was achieved. The composed mathematical models of the physicomechanical behavior of spheroids enabled the evaluation of the revolution frequency increase schedule. The differential equations took into account all inertial effects caused by the production module rotation movement as well as those caused by the relative movement of the spheroid in the fluid. The models aimed at the optimization of the rotation frequency increase schedule for different types of cells to reduce shear stress, augment productivity, and tolerate the growth of large spheroids. The models were numerically tested using MATLAB-SIMULINK software, and the trajectories of prestained HD-MY-Z spheroids were filmed. The coincidence of the theoretical and experimental trajectories was sufficient.

Characterization of expanded intermediate cell mass in zebrafish chordin morphant embryos

We investigated the mechanisms of intermediate cell mass (ICM) expansion in zebrafish chordin ( Chd) morphant embryos and examined the role of BMPs in relation to this phenotype. At 24 h post-fertilization (hpf), the expanded ICM of embryos injected with chd morpholino (MO) (Chd MO embryos) contained a monotonous population of hematopoietic progenitors. In situ hybridization showed that hematopoietic transcription factors were ubiquitously expressed in the ICM whereas vascular gene expression was confined to the periphery. BMP4 (but not BMP2b or 7) and smad5 mRNA were ectopically expressed in the Chd MO ICM. At 48 hpf, monocytic cells were evident in both the ICM and circulation of Chd MO but not WT embryos. While injection of BMP4 MO had no effect on WT hematopoiesis, co-injecting BMP4 with chd MOs significantly reduced ICM expansion. Microarray studies revealed a number of genes that were differentially expressed in Chd MO and WT embryos and their roles in hematopoiesis has yet to be determined. In conclusion, the expanded ICM in Chd MO embryos represented an expansion of embryonic hematopoiesis that was skewed towards a monocytic lineage. BMP4, but not BMP2b or 7, was involved in this process. The results provide ground for further research into the mechanisms of embryonic hematopoietic cell expansion.

Clinical Grade Ex-Vivo Expansion of Granulomonocytic Cells and Progenitors Using High-Dose SCF and G-CSF without Exhaustion of Stem Cell Activity.

Ex-vivo expansion of mature hematopoietic cells and progenitors is a major cell therapy technology aiming to shorten chemotherapy-induced cytopenias. The goal of amplifying the most primitive hematopoietic stem cell populations in clinically acceptable conditions is currently difficult, due to the absence of clinical grade early acting-cytokines and lack of adequate stem cell assays. Stem cell factor (SCF) and granulocyte colony stimulating factor (G-CSF) are both clinically applicable and they have been shown to induce a dose-response effect with regard to the expansion of mature cell populations when used in combination with early- acting cytokines. The combined effects of high concentrations of G-CSF and SCF on hematopoietic cells and progenitors and the effects of this procedure on the more primitive stem cell subsets have not been studied. We have developed a preclinical protocol to evaluate the feasibility of expansion of granulomonocytic cells using SCF and G-CSF with the goal of translating this protocol to the clinical application. In preliminary assays, the effects of low and high concentrations of both cytokines on total nucleated cell numbers and progenitors from purifed CD34+ cells (50 104cells/ml) have been tested. As compared to low concentrations (G-CSF 10 ng/ml and SCF 50 ng/ml) high concentrations of G-CSF and SCF (100 ng/ml and 300 ng/ml respectively) induced a two-fold increase of the nucleated cells and progenitors after 9 days of culture (n=7 experiments). High concentrations were therefore chosen for further experiments. CD34+ cells purified from mobilized peripheral blood grafts (purity 99%) have been cultured in a clinically acceptable medium containing clinical grade fetal calf serum ( 10 %), G-CSF (100 ng/ml) and SCF (300 ng/ml) for 9 days. Cells were analyzed using apoptosis tests, immunophenotyping and clonogenic assays at day 0 and day+9. In several experiments ( n= 9) a mean 21-fold expansion of total viable cell numbers was obtained, with a mean 4-fold expansion of clonogenic progenitors. Expanded cells had consistently CD11b+, CD13+, CD15+ phenotype and 2–4% of them remained CD34+. In a scale-up experiment started with 23.106 CD34+ cells, the total cell numbers expanded 16-fold at day+9, with generation of high numbers of CD11b + (18%), CD13+(98%) and CD15+ cells (91%) demonstrating the feasibility of the protocol in clinical scale. To determine if ex-vivo expansion would lead to a significant exhaustion of more primitive stem cells, we have evaluated the NOD/SCID-repopulating cell (RC) contents of the cultures before (day 0) and after ex-vivo expansion (day 9), in mice transplanted with day 0 and day+9 cells. In two experiments, the numbers of NOD/SCID mice engrafted with CD45+ human cells have been found increase from 30% ( 3/10) at day 0 to 100 % ( 10/10 ) at day 9 (Exp1 ) and from 50 % (3/6) at day 0 to 90 % ( 7/8 ) at day9 (Exp 2), demonstrating the persistence of NOD/SCID-RC potential after ex-vivo expansion. Overall the large-scale granulomonocytic cell production protocol that we developed could be of major interest in order to maintain the dose-intensity in high dose chemotherapy regimens and to shorten neutropenia after autologous PBSC transplantation while maintaing the stem cell potential of the graft.

Blood from Mesenchymal Stem Cell.

Mesenchymal stem cells (MSC) have been described to support the maintainance and expansion of clonogenic hematopoietic cells in vitro and as progenitors for nonhematopoietic tissues. They clearly can be distinguished from hematopoietic cells by the surface markers CD45 and CD34. We investigated the potential of human MSC to form blood and endothelial cells. Human MSC in expansion cultures expressed RNA for CD117 and EpoR but not for other hematopoietic antigens. Many transcription factors essential for blood cell differentiation, e.g. c-mpl, Notch-1, Gata-2 and -3, Runx-1 and SCL, were detected in these cells. We used different differentiation strategies: A) Hematopoietic differentiation: 1. Prestimulation in IMDM + fetal calf serum (FCS) + horse serum (HS), mixed with methylcellulose (MC, containing cytokines SCF, IL-3, IL-6, G-CSF, GM-CSF and Epo) and differentiation in MC; 2. Prestimulation in serum free medium supplemented with SCF, TPO, GM-CSF and flt.- 3 ligand and differentiation in serum free MC; B) Endothelial differentiation: Prestimulation in serum free medium supplemented with SCF, TPO, and flt-3L and differentiation in medium + VEGF + bFGF + IGF. Using FACS-analysis, we detected up to 1.8 % CD34-positive cells within 2 weeks. Immunochemically we observed cells positive for CD45, CD34, CD133, CD14, CD16 and CD41. Although hMSC do not form colonies after seeding in MC, after prestimulation we observed colonies with the typical appearance of BFU-E which were GlycophorinA positive. Under endothelial conditions we detected cells positive for CD34 and KDR or CD31 and vWF. However, cell numbers of positive cells were low and therefore RT-PCR results were not very reliable. To confirm the differentiation capacity of MSC we generated CD45.2 mouse MSC (P9 what took about 6 month) and transplanted into lethally irradiated CD45.1 recipient mice. Four weeks after transplantation mice showed normal white blood cell counts but still decreased platelet counts. Analysis of donor chimerism 29 weeks after transplantation revealed up to 36 % (14.3±7.9%) donor cells in peripheral blood, mainly in the CD11b positive myeloid population but not in lymphoid cells. In thymus and bone marrow, marginal donor chimerism (3±0.8%) was detected, but the number in c-kit+ cells reached up to 28.2% (7.9±6.8%). In B-cells and erythroid progenitors chimerism was low but stable, but not in CD3+ cells. Taken together, we have shown that under the right conditions MSC are able to form blood and endothelial cells and reconstitute animals. We suggest that MSC could be an yet unrecognised precursor for both cell types and may contain the long searched hemangioblast in adult organisms.