163 results on '"Stephen G. Emerson"'
Search Results
2. Identification of a Multipotent Progenitor Population in the Spleen That Is Regulated by NR4A1
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Jennifer A. Punt, Stephen G. Emerson, Elizabeth D. Lynch, Sophia B. Golec, Melanie Mumau, and Ashley N. Vanderbeck
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0301 basic medicine ,Myeloid ,Immunology ,Population ,Spleen ,Biology ,Article ,Thrombopoiesis ,Mice ,03 medical and health sciences ,Antigens, CD ,Receptors, Transferrin ,Nuclear Receptor Subfamily 4, Group A, Member 1 ,medicine ,Animals ,Immunology and Allergy ,Erythropoiesis ,Myeloid Cells ,Progenitor cell ,education ,Mice, Knockout ,education.field_of_study ,CD24 Antigen ,Hematopoietic Stem Cells ,Cell biology ,Mice, Inbred C57BL ,Proto-Oncogene Proteins c-kit ,Haematopoiesis ,030104 developmental biology ,medicine.anatomical_structure ,Bone marrow ,Stem cell - Abstract
The developmental fate of hematopoietic stem and progenitor cells is influenced by their physiological context. Although most hematopoietic stem and progenitor cells are found in the bone marrow of the adult, some are found in other tissues, including the spleen. The extent to which the fate of stem cells is determined by the tissue in which they reside is not clear. In this study, we identify a new progenitor population, which is enriched in the mouse spleen, defined by cKit+CD71lowCD24high expression. This previously uncharacterized population generates exclusively myeloid lineage cells, including erythrocytes, platelets, monocytes, and neutrophils. These multipotent progenitors of the spleen (MPPS) develop from MPP2, a myeloid-biased subset of hematopoietic progenitors. We find that NR4A1, a transcription factor expressed by myeloid-biased long term-hematopoietic stem cells, guides the lineage specification of MPPS. In vitro, NR4A1 expression regulates the potential of MPPS to differentiate into erythroid cells. MPPS that express NR4A1 differentiate into a variety of myeloid lineages, whereas those that do not express NR4A1 primarily develop into erythroid cells. Similarly, in vivo, after adoptive transfer, Nr4a1-deficient MPPS contribute more to erythrocyte and platelet populations than do wild-type MPPS. Finally, unmanipulated Nr4a1−/− mice harbor significantly higher numbers of erythroid progenitors in the spleen compared with wild-type mice. Together, our data show that NR4A1 expression by MPPS limits erythropoiesis and megakaryopoeisis, permitting development to other myeloid lineages. This effect is specific to the spleen, revealing a unique molecular pathway that regulates myeloid bias in an extramedullary niche.
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- 2018
3. Clinical and immunologic impact of CCR5 blockade in graft-versus-host disease prophylaxis
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David L. Porter, Ximi K. Wang, Ran Reshef, Ryan H. Moy, Lee P. Richman, Austin P. Huffman, James A. Hoxie, Stephen G. Emerson, Yi Zhang, Robert H. Vonderheide, Lisa Crisalli, and Rosemarie Mick
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Male ,0301 basic medicine ,viruses ,T-Lymphocytes ,medicine.medical_treatment ,Graft vs Host Disease ,Pancreatitis-Associated Proteins ,Hematopoietic stem cell transplantation ,Lymphocyte Activation ,Biochemistry ,chemistry.chemical_compound ,immune system diseases ,Interleukin-15 ,Immunity, Cellular ,Hematopoietic Stem Cell Transplantation ,virus diseases ,Hematology ,Middle Aged ,Treatment Outcome ,surgical procedures, operative ,CCR5 Receptor Antagonists ,CXCL9 ,Female ,Adult ,Receptors, CCR5 ,Immunology ,chemical and pharmacologic phenomena ,CCR5 receptor antagonist ,Young Adult ,03 medical and health sciences ,Immune system ,Antigens, Neoplasm ,Biomarkers, Tumor ,medicine ,Humans ,Transplantation, Homologous ,Lectins, C-Type ,Aged ,Maraviroc ,Transplantation ,business.industry ,Cell Biology ,medicine.disease ,Blockade ,030104 developmental biology ,Graft-versus-host disease ,chemistry ,business - Abstract
Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Lymphocyte trafficking via chemokine receptors such as CCR5 plays a critical role in alloreactive responses, and previous data suggest that CCR5 blockade with maraviroc results in a low incidence of visceral GVHD. However, the full scope of clinical and immunologic effects of CCR5 blockade in HSCT has not been described. We compared a cohort of patients enrolled on a trial of reduced-intensity allo-HSCT with standard GVHD prophylaxis plus maraviroc to a contemporary control cohort receiving standard GVHD prophylaxis alone. Maraviroc treatment was associated with a lower incidence of acute GVHD without increased risk of disease relapse, as well as reduced levels of gut-specific markers. At day 30, maraviroc treatment increased CCR5 expression on T cells and dampened T-cell activation in peripheral blood without impairing early immune reconstitution or increasing risk for infections. Patients who developed acute GVHD despite maraviroc prophylaxis showed increased T-cell activation, naive T-cell skewing, and elevated serum CXCL9 and CXCL10 levels. Collectively, these data suggest that maraviroc effectively protects against GVHD by modulating alloreactive donor T-cell responses, and that CXCR3 signaling may be an important resistance mechanism to CCR5 blockade in GVHD.
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- 2017
4. Infusion of CD3/CD28 costimulated umbilical cord blood T cells at the time of single umbilical cord blood transplantation may enhance engraftment
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Noelle V. Frey, Elizabeth O. Hexner, Selina M. Luger, Grace R. Jeschke, James K. Mangan, Nicole A. Aqui, Lee P. Richman, Edward A. Stadtmauer, Dale Frank, Ran Reshef, Carl H. June, Yi Zhang, David L. Porter, Bruce L. Levine, Robert H. Vonderheide, Misha Rosenbach, Stephen G. Emerson, Alison W. Loren, and Anne Chew
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Neutrophil Engraftment ,Myeloid ,business.industry ,Umbilical Cord Blood Transplantation ,T cell ,Hematology ,Cord Blood Stem Cell Transplantation ,Umbilical cord ,Transplantation ,03 medical and health sciences ,surgical procedures, operative ,0302 clinical medicine ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Immunology ,Medicine ,Transplantation Conditioning ,business ,030215 immunology - Abstract
Limited cell numbers in umbilical cord blood (UCB) grafts present a major impediment to favorable outcomes in adult transplantation, largely related to delayed or failed engraftment. The advent of UCB transplantation (UCBT) using two grafts successfully circumvents this obstacle, despite the engraftment of only one unit. Preclinical models suggested that the addition of UCB T cells at the time of transplant can enhance engraftment. We tested whether ex vivo activation by CD3/CD28 costimulation and expansion of T cells from a single UCB graft would be safe and feasible in adults with advanced hematologic malignancies, with an overall objective of optimizing engraftment in single unit UCBT. In this phase 1 study, recipients of single UCB units were eligible if the unit was stored in two adequate fractions. Dose limiting toxicity was defined as grade 3 or grade 4 GVHD within 90 days of UCBT. Four patients underwent UCBT; all were treated at the first dose level (10(5) cells/kg). At the 10(5) cells/kg dose level two subjects experienced grade 3 intestinal GVHD, thus meeting stopping criteria. For three subjects, neutrophil engraftment was early (12, 17, and 20 days), while one subject experienced primary graft failure. We observed early donor T cell trafficking and found that expanded T cells produced supraphysiologic levels of cytokines relevant to engraftment and to lymphoid differentiation and function. Taken together, these preliminary data suggest rapid engraftment in recipients of a single UCBT combined with relatively low doses of activated T cells, though potentially complicated by severe GVHD.
- Published
- 2016
5. Decreased Naïve T-cell Production Leading to Cytokine Storm as Cause of Increased COVID-19 Severity with Comorbidities
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Stephen G. Emerson, Michael D Schwartz, Willow D Goff, and Jennifer A. Punt
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0301 basic medicine ,Coronavirus disease 2019 (COVID-19) ,Naive T cell ,medicine.medical_treatment ,Type 2 diabetes ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Interferon ,medicine ,naïve T cells ,SARS-CoV-2 ,business.industry ,COVID-19 ,Cell Biology ,medicine.disease ,030104 developmental biology ,Increased risk ,Cytokine ,medicine.anatomical_structure ,cytokine storm ,Immunology ,Commentary ,type 2 diabetes ,Neurology (clinical) ,Geriatrics and Gerontology ,Cytokine storm ,business ,030217 neurology & neurosurgery ,medicine.drug ,Respiratory tract - Abstract
Aging, type 2 diabetes, and male gender are major risk factors leading to increased COVID-19 morbidity and mortality. Thymic production and the export of naïve T cells decrease with aging through the effects of androgens in males and in type 2 diabetes. Furthermore, with aging, recovery of naïve T-cell populations after bone marrow transplantation is delayed and associated with an increased risk of chronic graft vs. host disease. Severe COVID-19 and SARS infections are notable for severe T-cell depletion. In COVID-19, there is unique suppression of interferon signaling by infected respiratory tract cells with intact cytokine signaling. A decreased naïve T-cell response likely contributes to an excessive inflammatory response and increases the odds of a cytokine storm. Treatments that improve naïve T-cell production may prove to be vital COVID-19 therapies, especially for these high-risk groups.
- Published
- 2020
6. The Orphan Nuclear Receptor NR4A1 Specifies a Distinct Subpopulation of Quiescent Myeloid-Biased Long-Term HSCs
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Jennifer A. Punt, Anna K. Rayne, Jian Huang, Matthew Gross, Stephen G. Emerson, Ruben H. Land, Sophie Eiger, Trevor S. Barlowe, Shwetha Manjunath, Nicole R. Cunningham, Ashley N. Vanderbeck, and Peter S. Klein
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Myeloid ,Cellular differentiation ,Population ,Biology ,Cell fate determination ,Article ,Green fluorescent protein ,Mice ,Nuclear Receptor Subfamily 4, Group A, Member 1 ,medicine ,Animals ,education ,Cell Proliferation ,education.field_of_study ,Hematopoietic stem cell ,Cell Differentiation ,hemic and immune systems ,Cell Biology ,Hematopoietic Stem Cells ,Cell biology ,Mice, Inbred C57BL ,Haematopoiesis ,medicine.anatomical_structure ,Immunology ,Molecular Medicine ,Stem cell ,Developmental Biology - Abstract
Hematopoiesis is maintained throughout life by self-renewing hematopoietic stem cells (HSCs) that differentiate to produce both myeloid and lymphoid cells. The NR4A family of orphan nuclear receptors, which regulates cell fate in many tissues, appears to play a key role in HSC proliferation and differentiation. Using a NR4A1GFP BAC transgenic reporter mouse we have investigated NR4A1 expression and its regulation in early hematopoiesis. We show that NR4A1 is most highly expressed in a subset of Lin−Sca-1+c-Kit+ CD48−CD150+ long-term (LT) HSCs, and its expression is tightly associated with HSC quiescence. We also show that NR4A1 expression in HSCs is induced by PGE2, a known enhancer of stem cell engraftment potential. Finally, we find that both NR4A1GFP+ and NR4A1GFP− HSCs successfully engraft primary and secondary irradiated hosts; however, NR4A1GFP+ HSCs are distinctly myeloid-biased. These results show that NR4A1 expression identifies a highly quiescent and distinct population of myeloid-biased LT-HSCs. Stem Cells 2015;33:278–288
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- 2014
7. Implementation of next generation sequencing into pediatric hematology-oncology practice: moving beyond actionable alterations
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Helen Remotti, Andrew T. Turk, Andrew L. Kung, Danielle Pendrick, Hanina Hibshoosh, Stuart J. Andrews, Wendy K. Chung, Filemon S. Dela Cruz, Daniel Diolaiti, Peter L. Nagy, Jennifer A. Oberg, Rebecca J. Zylber, Julia L. Glade Bender, Anthony N. Sireci, Carrie Koval, Jiuhong Pang, Maria Luisa Sulis, Darrell J. Yamashiro, Mahesh M. Mansukhani, Susan J. Hsiao, James Garvin, and Stephen G. Emerson
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0301 basic medicine ,Oncology ,Male ,medicine.medical_specialty ,Adolescent ,Oncogene Proteins, Fusion ,Pediatric Hematology/Oncology ,Pediatric oncology ,Bioinformatics ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Neoplasms ,Genetics ,Medicine ,Humans ,Genetics(clinical) ,RNA, Neoplasm ,Child ,Molecular Biology ,Genetics (clinical) ,Exome sequencing ,business.industry ,Research ,Precision medicine ,Whole exome sequencing ,Infant, Newborn ,Cancer ,High-Throughput Nucleotide Sequencing ,Infant ,RNA sequencing ,medicine.disease ,Pediatric cancer ,Hematologic Diseases ,030104 developmental biology ,Hematologic disease ,030220 oncology & carcinogenesis ,Child, Preschool ,Medical genetics ,Molecular Medicine ,Female ,business ,Personal genomics - Abstract
Background Molecular characterization has the potential to advance the management of pediatric cancer and high-risk hematologic disease. The clinical integration of genome sequencing into standard clinical practice has been limited and the potential utility of genome sequencing to identify clinically impactful information beyond targetable alterations has been underestimated. Methods The Precision in Pediatric Sequencing (PIPseq) Program at Columbia University Medical Center instituted prospective clinical next generation sequencing (NGS) for pediatric cancer and hematologic disorders at risk for treatment failure. We performed cancer whole exome sequencing (WES) of patient-matched tumor-normal samples and RNA sequencing (RNA-seq) of tumor to identify sequence variants, fusion transcripts, relative gene expression, and copy number variation (CNV). A directed cancer gene panel assay was used when sample adequacy was a concern. Constitutional WES of patients and parents was performed when a constitutionally encoded disease was suspected. Results were initially reviewed by a molecular pathologist and subsequently by a multi-disciplinary molecular tumor board. Clinical reports were issued to the ordering physician and posted to the patient’s electronic medical record. Results NGS was performed on tumor and/or normal tissue from 101 high-risk pediatric patients. Potentially actionable alterations were identified in 38% of patients, of which only 16% subsequently received matched therapy. In an additional 38% of patients, the genomic data provided clinically relevant information of diagnostic, prognostic, or pharmacogenomic significance. RNA-seq was clinically impactful in 37/65 patients (57%) providing diagnostic and/or prognostic information for 17 patients (26%) and identified therapeutic targets in 15 patients (23%). Known or likely pathogenic germline alterations were discovered in 18/90 patients (20%) with 14% having germline alternations in cancer predisposition genes. American College of Medical Genetics (ACMG) secondary findings were identified in six patients. Conclusions Our results demonstrate the feasibility of incorporating clinical NGS into pediatric hematology-oncology practice. Beyond the identification of actionable alterations, the ability to avoid ineffective/inappropriate therapies, make a definitive diagnosis, and identify pharmacogenomic modifiers is clinically impactful. Taking a more inclusive view of potential clinical utility, 66% of cases tested through our program had clinically impactful findings and samples interrogated with both WES and RNA-seq resulted in data that impacted clinical decisions in 75% of cases. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0389-6) contains supplementary material, which is available to authorized users.
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- 2016
8. Brief Report: Long-Term Functional Engraftment of Mesenchymal Progenitor Cells in a Mouse Model of Accelerated Aging
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Yi Zhang, Robert J. Pignolo, F. Brad Johnson, Emily A. Mcmillan, Qijun Chen, Stephen G. Emerson, Lakshman Singh, Jung Hoon Kim, Kevin P. Egan, Tracy A. Brennan, and Kurt D. Hankenson
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Senescence ,Mesenchymal stem cell ,Stem cell theory of aging ,Osteoblast ,Cell Biology ,Biology ,Transplantation ,medicine.anatomical_structure ,Immunology ,Cancer research ,medicine ,Molecular Medicine ,Progenitor cell ,Cell aging ,Whole Bone Marrow ,Developmental Biology - Abstract
Age-related osteoporosis is characterized by a decrease in bone-forming capacity mediated by defects in the number and function of osteoblasts. An important cellular mechanism that may in part explain osteoblast dysfunction that occurs with aging is senescence of mesenchymal progenitor cells (MPCs). In the telomere-based Wrn−/−Terc−/− model of accelerated aging, the osteoporotic phenotype of these mice is also associated with a major decline in MPC differentiation into osteoblasts. To investigate the role of MPC aging as a cell-autonomous mechanism in senile bone loss, transplantation of young wild-type whole bone marrow into Wrn−/−Terc−/− mutants was performed and the ability of engrafted cells to differentiate into cells of the osteoblast lineage was assessed. We found that whole bone marrow transplantation in Wrn−/−Terc−/− mice resulted in functional engraftment of MPCs up to 42 weeks, which was accompanied by a survival advantage as well as delays in microarchitectural features of skeletal aging.
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- 2013
9. Osteoblasts and The Hematopoietic Microenvironment
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Russell S. Taichman, Marcelle J. Reilly, and Stephen G. Emerson
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CD34 ,Hematology ,Hematopoietic organ ,Biology ,Cell biology ,03 medical and health sciences ,Haematopoiesis ,0302 clinical medicine ,medicine.anatomical_structure ,In vivo ,030220 oncology & carcinogenesis ,medicine ,Bone marrow ,Progenitor cell ,Stem cell ,030215 immunology - Abstract
Cells of the bone marrow are chiefly dedicated to two processes; the production of blood, and the production of bone that houses the hematopoietic organ. The majority of our understanding of these processes comes from data focused on one of these functions. Yet, in vivo the processes are intermixed. Our recent data demonstrate that human osteoblast-like cells have important accessory roles in hematopoiesis. These data include the demonstration that human osteoblast-like cells; support the growth of primitive human hematopoietic progenitors (CD34(+) cells) in short and long term cultures and, synthesize multiple cytokines believed to regulate hematopoiesis. Based upon anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, and these findings, we hypothesize that osteoblasts play a critical role in hematopoietic cells development in vivo.
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- 2016
10. 7. Engaging Undergraduate Students in Research: Sustaining Our Nation’s Pipeline of Future Biomedical Investigators
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Jennifer A. Punt, Stephen G. Emerson, and Philip Meneely
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Political science ,Engineering ethics ,Pipeline (software) - Published
- 2016
11. Bone and Blood Vessels: The Hard and the Soft of Hematopoietic Stem Cell Niches
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Russell Garrett and Stephen G. Emerson
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Myelopoiesis ,Osteoblasts ,Lymphopoiesis ,Mesenchymal stem cell ,Niche ,Hematopoietic stem cell ,Osteoblast ,Cell Biology ,Biology ,Hematopoietic Stem Cells ,Bone and Bones ,Cell biology ,Haematopoiesis ,medicine.anatomical_structure ,Immunology ,medicine ,Genetics ,Animals ,Blood Vessels ,Humans ,Molecular Medicine ,Stem Cell Niche ,Stem cell - Abstract
Osteoblasts are important regulators of myelopoiesis and lymphopoiesis, producing several necessary soluble and membrane-associated factors. In addition, they may play important roles, along with other mesenchymal populations, in constructing an environment that is suitable for development of sinusoidal niches capable of supporting hematopoietic stem cells.
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- 2009
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12. Lestaurtinib (CEP701) is a JAK2 inhibitor that suppresses JAK2/STAT5 signaling and the proliferation of primary erythroid cells from patients with myeloproliferative disorders
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Elizabeth O. Hexner, Thelma S. Angeles, Pawel Dobrzanski, Cynthia Serdikoff, Martin Carroll, Mahfuza Jan, Bruce Ruggeri, Shi Yang, Stephen G. Emerson, Cezary R. Swider, and Candy Robinson
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Immunology ,Carbazoles ,Mice, Nude ,Bone Marrow Cells ,Biology ,Biochemistry ,Mice ,Myeloproliferative Disorders ,Erythroid Cells ,hemic and lymphatic diseases ,STAT5 Transcription Factor ,medicine ,Animals ,Humans ,Phosphorylation ,Progenitor cell ,Kinase activity ,Furans ,Cells, Cultured ,Cell growth ,Lestaurtinib ,Wild type ,Cell Biology ,Hematology ,Janus Kinase 2 ,Hematopoietic Stem Cells ,Xenograft Model Antitumor Assays ,Phenotype ,Mutation ,Cancer research ,Signal transduction ,Tyrosine kinase ,Cell Division ,Signal Transduction ,medicine.drug - Abstract
Recent studies have demonstrated that patients with myeloproliferative disorders (MPDs) frequently have acquired activating mutations in the JAK2 tyrosine kinase. A multikinase screen determined that lestaurtinib (formerly known as CEP-701) inhibits wild type JAK2 kinase activity with a concentration that inhibits response by 50% (IC50) of 1 nM in vitro. We hypothesized that lestaurtinib would inhibit mutant JAK2 kinase activity and suppress the growth of cells from patients with MPDs. We found that lestaurtinib inhibits the growth of HEL92.1.7 cells, which are dependent on mutant JAK2 activity for growth in vitro and in xenograft models. Erythroid cells expanded from primary CD34+ cells from patients with MPDs were inhibited by lestaurtinib at concentrations of 100 nM or more in 15 of 18 subjects, with concomitant inhibition of phosphorylation of STAT5 and other downstream effectors of JAK2. By contrast, growth of erythroid cells derived from 3 healthy controls was not significantly inhibited. These results demonstrate that lestaurtinib, in clinically achievable concentrations, inhibits proliferation and JAK2/STAT5 signaling in cells from patients with MPDs, and therefore holds promise as a therapeutic agent for patients with these disorders.
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- 2008
13. Annexin II expressed by osteoblasts and endothelial cells regulates stem cell adhesion, homing, and engraftment following transplantation
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Yan Xi Sun, Jingcheng Wang, Younghun Jung, Jianhua Wang, Junhui Song, Paul H. Krebsbach, Zhuo Wang, Yusuke Shiozawa, Aaron M. Havens, Stephen G. Emerson, and Russell S. Taichman
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Immunology ,Biology ,Biochemistry ,Mice ,Bone Marrow ,Cell Movement ,Cell Adhesion ,medicine ,Animals ,Cell adhesion ,Annexin A2 ,Mice, Knockout ,Osteoblasts ,Graft Survival ,Hematopoietic Stem Cell Transplantation ,Endothelial Cells ,Osteoblast ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Hematopoiesis ,Cell biology ,Endothelial stem cell ,Haematopoiesis ,medicine.anatomical_structure ,Bone marrow ,Stem cell ,Homing (hematopoietic) - Abstract
Differentiation of hematopoietic stem cells (HSCs) after birth is largely restricted to the bone marrow cavity, where HSCs are associated closely with osteoblasts (OBs). How OBs localize HSCs to the endosteal niche remains unclear. To explore adhesive interactions between HSCs and OBs, a cell blot analysis was used that revealed 2 major bands that corresponded to monomers and multimers of annexin II (Anxa2). Immunohistochemistry revealed that OBs and marrow endothelial cells express Anxa2 at high levels. Function-blocking studies confirmed that Anxa2 mediates HSC adhesion mainly via the N-terminal portion of the Anxa2 peptide. Adhesion of HSCs to OBs derived from Anxa2-deficient animals (Anxa2−/−) was significantly impaired compared with OBs obtained from wild-type animals (Anxa2+/+). Moreover, fewer HSCs were found in the marrow of Anxa2−/− versus Anxa2+/+ animals. Short-term lodging, engraftment, and survival of irradiated mice with whole marrow cells were substantially inhibited by N-terminal peptide fragments of Anxa2 or anti-Anxa2 antibodies. Similar findings were noted in long-term competitive repopulation studies. Collectively, these findings reveal that Anxa2 regulates HSC homing and binding to the bone marrow microenvironment and suggest that Anxa2 is crucial for determining the bone marrow niche of HSCs.
- Published
- 2007
14. c-Myb Contributes to G2/M Cell Cycle Transition in Human Hematopoietic Cells by Direct Regulation of Cyclin B1 Expression
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Yuji Nakata, Anna Kalota, Stephen I. Rudnick, Alan M. Gewirtz, Susan E. Shetzline, Stephen G. Emerson, Yi Zhang, Ravikumar Rallapalli, and Chizuko Sakashita
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G2 Phase ,animal structures ,Cell cycle checkpoint ,Hematopoietic System ,T-Lymphocytes ,Cyclin D ,Cyclin A ,Cyclin B ,Antigens, CD34 ,Proto-Oncogene Proteins c-myb ,Cyclin-dependent kinase ,Humans ,Cyclin B1 ,Phytohemagglutinins ,RNA, Small Interfering ,Molecular Biology ,Cells, Cultured ,Base Sequence ,biology ,fungi ,Articles ,Cell Biology ,Cell cycle ,Molecular biology ,Cell biology ,Gene Expression Regulation ,biology.protein ,Interleukin-2 ,Cell Division ,Cyclin A2 - Abstract
Myb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G(1)/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G(2)/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-myb expression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G(2)/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-myb functions in regulating human hematopoiesis.
- Published
- 2007
15. Hematopoietic Stem-Cell Contribution to Ectopic Skeletogenesis
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Yi Zhang, Stephen G. Emerson, Robert J. Pignolo, Frederick S. Kaplan, Stephen J. Forman, Eileen M. Shore, David L. Glaser, Meiqi Xu, and David Senitzer
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Adult ,Male ,Pathology ,medicine.medical_specialty ,Population ,DNA Mutational Analysis ,Mice, Transgenic ,Bone Morphogenetic Protein 4 ,Mice ,medicine ,Animals ,Humans ,Orthopedics and Sports Medicine ,Aplastic anemia ,education ,Bone Marrow Transplantation ,education.field_of_study ,integumentary system ,business.industry ,Ossification, Heterotopic ,Hematopoietic stem cell ,Anemia, Aplastic ,Sequence Analysis, DNA ,General Medicine ,medicine.disease ,Transplantation ,medicine.anatomical_structure ,Myositis Ossificans ,Fibrodysplasia ossificans progressiva ,Bone Morphogenetic Proteins ,Heterotopic ossification ,Surgery ,Bone marrow ,Stem cell ,business ,Activin Receptors, Type I - Abstract
Background: Fibrodysplasia ossificans progressiva is a rare genetic disorder of ectopic skeletogenesis associated with dysregulation of bone morphogenetic protein (BMP) signaling. Hematopoietic cells have been implicated in the ectopic skeletogenesis of fibrodysplasia ossificans progressiva, and their replacement has been postulated as a possible cure. However, the definitive contribution of hematopoietic cells to the pathogenesis of ectopic skeletogenesis remains obscure. Methods: We employed both careful clinical observation and in vivo murine transplantation studies to more precisely determine the contribution of hematopoietic cells to ectopic skeletogenesis. We identified a patient with fibrodysplasia ossificans progressiva who had undergone bone marrow transplantation for the treatment of intercurrent aplastic anemia twenty-five years earlier and investigated whether the clinical course of the fibrodysplasia ossificans progressiva had been influenced by bone marrow replacement or immunosuppression, or both. In complementary studies, we transplanted hematopoietic stem cells from constitutively expressing LacZ transgenic mice to identify the contribution of hematopoietic cells to BMP4-induced heterotopic ossification, a histopathologic model of fibrodysplasia ossificans progressiva. Results: We found that replacement of hematopoietic cells was not sufficient to prevent ectopic skeletogenesis in the patient with fibrodysplasia ossificans progressiva but pharmacologic suppression of the apparently normal donor immune system following transplantation in the new host modulated the activity of the fibrodysplasia ossificans progressiva and diminished the expression of skeletal ectopia. In complementary murine transplantation studies, we found that cells of hematopoietic origin contributed to the early inflammatory and late marrow-repopulating stages of BMP4-induced heterotopic ossification but were not represented in the fibroproliferative, chondrogenic, or osteogenic stages of heterotopic ossification. Interestingly, both recombinant human BMP4 induction in an animal model and the dysregulated BMP signaling pathway in a patient with fibrodysplasia ossificans progressiva were sufficient to recruit at least two populations of cells, one of hematopoietic origin and at least one of non-hematopoietic origin, that contribute to the formation of an ectopic skeleton. Conclusions: Taken together, these findings demonstrate that bone marrow transplantation did not cure fibrodysplasia ossificans progressiva in the patient in this study, most likely because the hematopoietic cell population is not the site, or at least not the dominant site, of the intrinsic dysregulation of the BMP signaling pathway in fibrodysplasia ossificans progressiva. However, following transplantation of bone marrow from a presumably normal donor, immunosuppression of the immune system appeared to ameliorate activation of ectopic skeletogenesis in a genetically susceptible host. Thus, cells of hematopoietic origin may contribute to the formation of an ectopic skeleton, although they are not sufficient to initiate the process alone. Clinical Relevance: Therapeutic regulation of hematopoietic and osteogenic cell populations involved in fibrodysplasia ossificans progressiva lesions holds promise for treatment of fibrodysplasia ossificans progressiva and possibly other disorders of heterotopic ossification.
- Published
- 2007
16. Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells
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Russell S. Taichman, Jingcheng Wang, Yi Zhang, Younghun Jung, Elizabeth O. Hexner, Stephen G. Emerson, Russell Garrett, Gerard Joe, Nacksung Kim, Yongwon Choi, and Jiang Zhu
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Myeloid ,Cellular differentiation ,Immunology ,Mice, Transgenic ,Biology ,Biochemistry ,Birds ,Mice ,Bursa of Fabricius ,medicine ,Animals ,Lymphopoiesis ,Cells, Cultured ,B-Lymphocytes ,Osteoblasts ,Hematopoietic stem cell ,Cell Differentiation ,Osteoblast ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Antigens, Differentiation ,Coculture Techniques ,Rats ,Cell biology ,Haematopoiesis ,medicine.anatomical_structure ,Cytokines ,Myelopoiesis ,Stem cell - Abstract
Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin−Sca-1+Rag-2− BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Δ-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.
- Published
- 2007
17. A phase 1 trial of donor lymphocyte infusions expanded and activated ex vivo via CD3/CD28 costimulation
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Selina M. Luger, Julie A. Phillips, David L. Porter, Alexander E. Perl, Ambika Sohal, Stephen J. Schuster, Steven A. Goldstein, Nancy Bunin, Carl H. June, Sunita D. Nasta, Donald E. Tsai, Elizabeth Veloso, Edward A. Stadtmauer, Alison W. Loren, Bruce L. Levine, and Stephen G. Emerson
- Subjects
Adult ,Male ,Adoptive cell transfer ,Adolescent ,Lymphoma ,CD8 Antigens ,Chronic lymphocytic leukemia ,Immunology ,Lymphocyte Activation ,Biochemistry ,Donor lymphocyte infusion ,Myelogenous ,CD28 Antigens ,Antigens, CD ,hemic and lymphatic diseases ,Acute lymphocytic leukemia ,medicine ,Humans ,Transplantation, Homologous ,Child ,Leukemia ,business.industry ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,Transplantation ,Lymphocyte Transfusion ,Female ,business ,Ex vivo ,Stem Cell Transplantation - Abstract
Donor lymphocyte infusions (DLIs) induce potent graft versus tumor (GVT) effects for relapsed chronic myelogenous leukemia (CML) after allogeneic stem cell transplantation (SCT) but are disappointing for other diseases. Disease resistance can occur if donor T cells are not appropriately activated in vivo. Ex vivo T-cell activation might overcome disease-induced anergy and augment GVT activity. We performed a phase 1 trial of ex vivo–activated DLI (aDLI) for 18 patients with relapse after SCT. Activated donor T cells are produced through costimulation with anti-CD3– and anti-CD28–coated beads. Patients with aggressive malignancies received induction chemotherapy, and all patients received conventional DLI (median, 1.5 × 108 mononuclear cells/kg) followed 12 days later by aDLI. Activated DLI was dose escalated from 1 × 106 to 1 × 108 CD3+ cells per kilogram in 5 levels. Seven patients developed acute graft versus host disease (GVHD) (5 grade I-II, 2 grade III), and 4 developed chronic GVHD. Eight patients achieved complete remission, including 4 of 7 with acute lymphocytic leukemia (ALL), 2 of 4 with acute myelogenous leukemia (AML), 1 with chronic lymphocytic leukemia (CLL), and 1 of 2 with non-Hodgkin lymphoma (NHL). Four complete responders relapsed while 4 remain alive in remission a median 23 months after aDLI. Overall, 10 of 18 remain alive 11 to 53 months after aDLI. Adoptive transfer of costimulated activated allogeneic T cells is feasible, does not result in excessive GVHD, and may contribute to durable remissions in diseases where conventional DLI has been disappointing.
- Published
- 2006
18. Alloreactive Memory T Cells Are Responsible for the Persistence of Graft-versus-Host Disease
- Author
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Yi Zhang, Elizabeth O. Hexner, Jiang Zhu, Gerard Joe, and Stephen G. Emerson
- Subjects
CD4-Positive T-Lymphocytes ,Time Factors ,Cell Survival ,T cell ,Immunology ,Graft vs Host Disease ,Biology ,Lymphocyte Activation ,Minor Histocompatibility Antigens ,Mice ,Interleukin 21 ,T-Lymphocyte Subsets ,medicine ,Animals ,Transplantation, Homologous ,Immunology and Allergy ,Cytotoxic T cell ,IL-2 receptor ,Bone Marrow Transplantation ,Mice, Inbred BALB C ,Mice, Inbred C3H ,Cell Differentiation ,medicine.disease ,Mice, Inbred C57BL ,Transplantation ,Graft-versus-host disease ,medicine.anatomical_structure ,Immunologic Memory ,Memory T cell ,CD8 - Abstract
Graft-vs-host disease (GVHD) is caused by a donor T cell anti-host reaction that evolves over several weeks to months, suggesting a requirement for persistent alloreactive T cells. Using the C3H.SW anti-C57BL/6 (B6) mouse model of human GVHD directed against minor histocompatibility Ags, we found that donor CD8+ T cells secreting high levels of IFN-γ in GVHD B6 mice receiving C3H.SW naive CD8+ T cells peaked by day 14, declined by day 28 after transplantation, and persisted thereafter, corresponding to the kinetics of a memory T cell response. Donor CD8+ T cells recovered on day 42 after allogeneic bone marrow transplantation expressed the phenotype of CD44highCD122highCD25low, were able to homeostatically survive in response to IL-2, IL-7, and IL-15 and rapidly proliferated upon restimulation with host dendritic cells. Both allogeneic effector memory (CD44highCD62Llow) and central memory (CD44highCD62Lhigh) CD8+ T cells were identified in B6 mice with ongoing GVHD, with effector memory CD8+ T cells as the dominant (>80%) population. Administration of these allogeneic memory CD8+ T cells into secondary B6 recipients caused virulent GVHD. A similar allogeneic memory CD4+ T cell population with the ability to mediate persistent GVHD was also identified in BALB/b mice receiving minor histocompatibility Ag-mismatched B6 T cell-replete bone marrow transplantation. These results indicate that allogeneic memory T cells are generated in vivo during GVH reactions and are able to cause GVHD, resulting in persistent host tissue injury. Thus, in vivo blockade of both alloreactive effector and memory T cell-mediated host tissue injury may prove to be valuable for GVHD prevention and treatment.
- Published
- 2005
19. Molecular pathways regulating the self-renewal of hematopoietic stem cells
- Author
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Matthew I. Stein, Stephen G. Emerson, and Jiang Zhu
- Subjects
Cancer Research ,Cellular differentiation ,Stem cell factor ,Biology ,S Phase ,Mice ,Cancer stem cell ,Genetics ,Animals ,Humans ,Molecular Biology ,Transcription factor ,Mice, Knockout ,G1 Phase ,Cell Differentiation ,Cell Biology ,Hematology ,Cell cycle ,Hematopoietic Stem Cells ,Cell biology ,Repressor Proteins ,Endothelial stem cell ,Haematopoiesis ,Gene Expression Regulation ,Stem cell ,Cell Division ,Signal Transduction ,Transcription Factors - Abstract
Hematopoietic stem cells (HSC) self-renew throughout life, but the molecular mechanisms by which this process occurs and is regulated are imprecisely understood. We review the published data from overexpression and knockout studies describing genes that influence stem cell self-renewal, including transcription factors, cell cycle regulators, and genes that influence chromosome structure. One model suggesting how some of these disparate classes of molecular regulators might be integrated is presented, focusing on the role of G1/S progression in the developmental switch toward stem cell self-renewal vs differentiation. Experimental exploration of this model and other related hypotheses will hopefully lead to a more complete description of HSC self-renewal and its regulation, both in normal physiology and in applied therapeutics.
- Published
- 2004
20. Graft-versus-leukemia in a retrovirally induced murine CML model: mechanisms of T-cell killing
- Author
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James H. Cormier, Stephen G. Emerson, Jinli Liu, Britt E. Anderson, Warren D. Shlomchik, Catherine C. Matte, Warren S. Pear, and Ioanna Athanasiadis
- Subjects
CD4-Positive T-Lymphocytes ,Male ,T cell ,Immunology ,Fusion Proteins, bcr-abl ,Graft vs Leukemia Effect ,CD8-Positive T-Lymphocytes ,Biology ,Receptor, Nerve Growth Factor ,Biochemistry ,Receptors, Tumor Necrosis Factor ,Epitopes ,Mice ,Mice, Inbred AKR ,Myelogenous ,Antigen ,Antigens, CD ,immune system diseases ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,hemic and lymphatic diseases ,medicine ,Animals ,Receptors, Tumor Necrosis Factor, Type II ,fas Receptor ,Mice, Inbred BALB C ,Mice, Inbred C3H ,Cell Biology ,Hematology ,medicine.disease ,Fusion protein ,Mice, Mutant Strains ,Histocompatibility ,Disease Models, Animal ,Leukemia ,Retroviridae ,surgical procedures, operative ,medicine.anatomical_structure ,Receptors, Tumor Necrosis Factor, Type I ,Female ,Tumor necrosis factor alpha ,Chronic myelogenous leukemia - Abstract
The graft-versus-leukemia (GVL) effect, mediated by donor T cells, has revolutionized the treatment of leukemia. However, effective GVL remains difficult to separate from graft-versus-host disease (GVHD), and many neoplasms are GVL resistant. Murine studies aimed at solving these problems have been limited by the use of leukemia cell lines with limited homology to human leukemias and by the absence of loss-of-function leukemia variants. To address these concerns, we developed a GVL model against murine chronic-phase chronic myelogenous leukemia (mCP-CML) induced with retrovirus expressing the bcr-abl fusion cDNA, the defining genetic abnormality of chronic-phase CML (CP-CML). By generating mCP-CML in gene-deficient mice, we have studied GVL T-cell effector mechanisms. mCP-CML expression of Fas or tumor necrosis factor (TNF) receptors is not required for CD8-mediated GVL. Strikingly, maximal CD4-mediated GVL requires cognate interactions between CD4 cells and mCP-CML cells as major histocompatibility complex-negative (MHC II-/-) mCP-CML is relatively GVL resistant. Nevertheless, a minority of CD4 recipients cleared MHC II-/- mCP-CML; thus, CD4 cells can also kill indirectly. CD4 GVL did not require target Fas expression. These results suggest that CPCML's GVL sensitivity may in part be explained by the minimal requirements for T-cell killing, and GVL-resistance may be related to MHC II expression. (Blood. 2004;103:4353-4361)
- Published
- 2004
21. Dendritic cell–activated CD44hiCD8+ T cells are defective in mediating acute graft-versus-host disease but retain graft-versus-leukemia activity
- Author
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Elizabeth O. Hexner, Jiang Zhu, Carl H. June, Richard G. Carroll, Yi Zhang, Gerard Joe, Bruce L. Levine, and Stephen G. Emerson
- Subjects
Cytotoxicity, Immunologic ,Immunology ,Graft vs Host Disease ,Graft vs Leukemia Effect ,Mice, Inbred Strains ,CD8-Positive T-Lymphocytes ,Biology ,Biochemistry ,Lymphocyte Depletion ,Cell therapy ,Mice ,Interleukin 21 ,T-Lymphocyte Subsets ,immune system diseases ,medicine ,Animals ,Cytotoxic T cell ,fas Receptor ,IL-2 receptor ,Antigen-presenting cell ,Bone Marrow Transplantation ,Antigen Presentation ,Dendritic Cells ,Cell Biology ,Hematology ,Dendritic cell ,medicine.disease ,Hyaluronan Receptors ,surgical procedures, operative ,Graft-versus-host disease ,Acute Disease ,CD8 - Abstract
Graft versus host disease (GVHD) is triggered by host antigen-presenting cells (APCs) that activate donor T cells to proliferate and differentiate, but which APC-activated donor T-cell subsets mediate GVHD versus beneficial antitumor effects is not known. Using a CD8+ T cell–dependent mouse model of human GVHD, we found that host dendritic cell (DC)–induced CD44hiCD8+ effector/memory T cells were functionally defective in inducing GVHD, whereas CD44loCD8+ naive phenotype T cells were extremely potent GVHD inducers. Depletion of CD44loCD8+ T cells from host DC-stimulated T cells before transplantation prevented GVHD without impairing their antitumor activity in vivo. Compared with CD44loCD8+ T cells, CD44hiCD8+ T cells expressed high levels of Fas and were efficiently deleted in vivo following transplantation. These results suggest that ex vivo allogeneic DC stimulation of donor CD8+ T cells may be useful for the prevention of GVHD and for optimizing antitumor therapies in vivo.
- Published
- 2004
22. Stem Cell Transplantation (Cord Blood Transplants)
- Author
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Nelson J. Chao, Kenneth I. Weinberg, and Stephen G. Emerson
- Subjects
Adult ,Male ,Placenta ,T-Lymphocytes ,medicine.medical_treatment ,Cell Culture Techniques ,Cell Separation ,Hematopoietic stem cell transplantation ,Bioinformatics ,Umbilical cord ,Pregnancy ,Risk Factors ,medicine ,Humans ,Progenitor cell ,Immunosuppression Therapy ,Umbilical Cord Blood Transplantation ,business.industry ,Histocompatibility Testing ,Cell Differentiation ,Hematology ,Fetal Blood ,Hematopoietic Stem Cells ,medicine.disease ,Hematopoiesis ,Haematopoiesis ,Treatment Outcome ,medicine.anatomical_structure ,Graft-versus-host disease ,Cord blood ,Female ,Cord Blood Stem Cell Transplantation ,Stem cell ,business ,Signal Transduction - Abstract
Allogeneic stem cell transplantation is an accepted treatment modality for selected malignant and non-malignant diseases. However, the ability to identify suitably matched related or unrelated donors can be difficult in some patients. Alternative sources of stem cells such as cord blood provide a readily available graft for such patients. Data accumulated over the past several years have demonstrated that the use of cord blood is an accepted source of stem cells for pediatric patients. Since the cell numbers of hematopoietic progenitors in cord blood is limited and the collection can occur only in a single occasion, its use in adult patients can be more problematic. Here, new developments in the use of cord blood for adults and studies aimed at expansion of cord blood cells and immune reconstitution are described.In Section I, Dr. Nelson Chao describes the early data in cord blood transplantation in adult patients. The patient outcomes are reviewed and analyzed for various factors such as cell dose, HLA typing, and patient selection that could have contributed to the final outcome of these adult patients. Myeloablative as well as nonmyeloablative approaches are presented. Discussion of the various benefits and risks are presented. More recent data from multiple single institutions as well as larger registry data comparisons are also provided. Analyses of these studies suggest methods to improve on the outcome. These newer data should lead to a logical progression in the use of cord blood cells in adult patients.In Section II, Dr. Stephen Emerson describes the historical efforts associated with expansion of hematopoietic stem cells, specifically with cord blood cells. These efforts to expand cord blood cells continue with novel methods. Moreover, a better understanding of stem cell biology and signaling is critical if we are to be able to effectively expand these cells for clinical use. An alternative, more direct, approach to expanding stem cells could be achieved by specific genetic pathways known or believed to support primitive HSC proliferation such as Notch-1 receptor activation, Wnt/LEF-1 pathway induction, telomerase or the Homeobox (Hox) gene products. The clinical experience with the use of expanded cord blood cells is also discussed.In Section III, Dr. Kenneth Weinberg describes immune reconstitution or lack thereof following cord blood transplantation. One of the hallmarks of successful hematopoietic stem cell transplantation is the ability to fully reconstitute the immune system of the recipient. Thus, the relationship between stem cell source and the development of T lymphocyte functions required for protection of the recipient from infection will be described, and cord blood recipients will be compared with those receiving other sources of stem cells. T cell development is described in detail, tracking from prethymic to postthymic lymphocytes with specific attention to umbilical cord blood as the source of stem cells. Moreover, a discussion of the placenta as a special microenvironment for umbilical cord blood is presented. Strategies to overcome the immunological defects are presented to improve the outcome of these recipients.
- Published
- 2004
23. Clinical application of hematopoietic progenitor cell expansion: current status and future prospects
- Author
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S M Devine, Hillard M. Lazarus, and Stephen G. Emerson
- Subjects
Oncology ,Transplantation ,medicine.medical_specialty ,Hematology ,business.industry ,Cell Culture Techniques ,Hematopoietic stem cell ,Hematopoietic Stem Cells ,Umbilical cord ,Hematopoietic Stem Cell Mobilization ,Tissue Donors ,Clinical trial ,Haematopoiesis ,medicine.anatomical_structure ,Internal medicine ,Immunology ,medicine ,Humans ,Bone marrow ,Stem cell ,business ,Ex vivo ,Stem Cell Transplantation - Abstract
In the past decade, we have witnessed significant advances in ex vivo hematopoietic stem cell culture expansion, progressing to the point where clinical trials are being designed and conducted. Preclinical milestone investigations provided data to enable expansion of portions of hematopoietic grafts in a clinical setting, indicating safety and feasibility of this approach. Data derived from current clinical trials indicate successful reconstitution of hematopoiesis after myeloablative chemoradiotherapy using infusion of ex vivo-expanded perfusion cultures. Future avenues of exploration will focus upon refining preclinical and clinical studies in which cocktails of available cytokines, novel molecules and sophisticated expansion systems will explore expansion of blood, marrow and umbilical cord blood cells.
- Published
- 2003
24. Selective T-cell subset ablation demonstrates a role for T1 and T2 cells in ongoing acute graft-versus-host disease: a model system for the reversal of disease
- Author
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Warren D. Shlomchik, Jennifer M. McNiff, Mark J. Shlomchik, Marie E. Robert, Britt E. Anderson, Jinli Liu, and Stephen G. Emerson
- Subjects
Male ,T-Lymphocytes ,medicine.medical_treatment ,Immunology ,Graft vs Host Disease ,Graft vs Leukemia Effect ,Mice, Transgenic ,Thymus Gland ,Biology ,Weight Gain ,Thymidine Kinase ,Biochemistry ,Mice ,immune system diseases ,Aldesleukin ,medicine ,Animals ,Simplexvirus ,Promoter Regions, Genetic ,Ganciclovir ,Cell Death ,Genetic transfer ,Cell Biology ,Hematology ,Immunotherapy ,Suicide gene ,Flow Cytometry ,Hematopoietic Stem Cells ,medicine.disease ,Lymphocyte Subsets ,Hematopoiesis ,Transplantation ,Disease Models, Animal ,surgical procedures, operative ,Graft-versus-host disease ,Cytokine ,Cytokines ,Interleukin-2 ,Interleukin-4 ,Stem cell ,Spleen - Abstract
Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality of allogeneic stem cell transplantation. Strategies to control GVHD while maintaining graft versus leukemia (GVL) include herpes simplex virus thymidine kinase (HSV-tk) gene transduction of donor T cells followed by treatment with ganciclovir (GCV). Alternatively, GVHD and GVL may be mediated by distinct processes. In this regard, whether cytokine polarization occurs and to what degrees various subsets of cytokine-producing T cells mediate GVHD or GVL has been an active area of research using cytokine or cytokine antibody infusion or genetically deficient mice. This study takes a different approach that allows simultaneous investigation into both the mechanisms underlying GVHD reactions and the efficacy of HSV-tk suicide gene-based T-cell deletion. A source of donor T cells, splenocytes from mice transgenic for HSV-tk controlled by elements of either the interleukin-2 (IL-2) or IL-4 promoters (IL-2-tk and IL-4-tk, respectively) was used, thus allowing investigation into the roles of T1 and T2 cells in ongoing GVHD reactions. To assess treatment rather than prevention of GVHD, GCV was started at peak disease. Remarkably, treatment at this late time point rescued mice from the clinical effects of GVHD caused by T cells expressing either transgene. Thus, both T1 and T2 cells play an important role in clinical GVHD in a minor histocompatibility antigen-mismatched setting. In addition, because clinical disease was reversible even at its maximum, these observations provide controlled evidence that this strategy of treating ongoing GVHD could be effective clinically.
- Published
- 2001
25. Answer: Marrow Stem Cell Transplantation in Fibrodysplasia Ossificans Progressiva
- Author
-
Frederick S. Kaplan and Stephen G. Emerson
- Subjects
Pathology ,medicine.medical_specialty ,business.industry ,Hematopoietic Stem Cell Transplantation ,General Medicine ,medicine.disease ,Transplantation ,Treatment Outcome ,Fibrodysplasia ossificans progressiva ,medicine ,Humans ,Orthopedics and Sports Medicine ,Surgery ,Stem cell ,business ,Bone Marrow Transplantation - Published
- 2001
26. Numerous growth factors, cytokines, and chemokines are secreted by human CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner
- Author
-
Karen Ehrenman, Alan M. Gewirtz, Anna Janowska-Wieczorek, Stephen G. Emerson, Janina Ratajczak, M. Anna Kowalska, Marcin Majka, Mariusz Z. Ratajczak, and Zbigniew Pietrzkowski
- Subjects
Erythroblasts ,Cell Survival ,medicine.medical_treatment ,T cell ,Immunology ,Gene Expression ,Antigens, CD34 ,Stem cell factor ,Cell Separation ,Biology ,Biochemistry ,Paracrine signalling ,medicine ,Homeostasis ,Humans ,RNA, Messenger ,Growth Substances ,Autocrine signalling ,Cells, Cultured ,Rh-Hr Blood-Group System ,Chemotaxis ,Growth factor ,Monocyte ,Cell Biology ,Hematology ,Flow Cytometry ,Hematopoietic Stem Cells ,Hematopoiesis ,Cytokine ,medicine.anatomical_structure ,Culture Media, Conditioned ,Cancer research ,Cytokines ,Hepatocyte growth factor ,Chemokines ,Megakaryocytes ,Cell Division ,Granulocytes ,medicine.drug - Abstract
The aim of this study was to explore further the hypothesis that early stages of normal human hematopoiesis might be coregulated by autocrine/paracrine regulatory loops and by cross-talk among early hematopoietic cells. Highly purified normal human CD34+cells and ex vivo expanded early colony-forming unit–granulocyte-macrophage (CFU-GM)–derived, burst forming unit–erythroid (BFU-E)–derived, and CFU–megakaryocyte (CFU-Meg)–derived cells were phenotyped for messenger RNA expression and protein secretion of various growth factors, cytokines, and chemokines to determine the biological significance of this secretion. Transcripts were found for numerous growth factors (kit ligand [KL], FLT3 ligand, fibroblast growth factor–2 [FGF-2], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulinlike growth factor–1 [IGF-1], and thrombopoietin [TPO]); cytokines (tumor necrosis factor–α, Fas ligand, interferon α, interleukin 1 [IL-1], and IL-16); and chemokines (macrophage inflammatory protein–1α [MIP-1α], MIP-1β, regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein–3 [MCP-3], MCP-4, IL-8, interferon-inducible protein–10, macrophage-derived chemokine [MDC], and platelet factor–4 [PF-4]) to be expressed by CD34+ cells. More importantly, the regulatory proteins VEGF, HGF, FGF-2, KL, FLT3 ligand, TPO, IL-16, IGF-1, transforming growth factor–β1 (TGF-β1), TGF-β2, RANTES, MIP-1α, MIP-1β, IL-8, and PF-4 were identified in media conditioned by these cells. Moreover, media conditioned by CD34+ cells were found to inhibit apoptosis and slightly stimulate the proliferation of other freshly isolated CD34+ cells; chemo-attract CFU-GM– and CFU-Meg–derived cells as well as other CD34+ cells; and, finally, stimulate the proliferation of human endothelial cells. It was also demonstrated that these various hematopoietic growth factors, cytokines, and chemokines are expressed and secreted by CFU-GM–, CFU-Meg–, and BFU-E–derived cells. It is concluded that normal human CD34+ cells and hematopoietic precursors secrete numerous regulatory molecules that form the basis of intercellular cross-talk networks and regulate in an autocrine and/or a paracrine manner the various stages of normal human hematopoiesis.
- Published
- 2001
27. Hybrid HIV/MSCV LTR Enhances Transgene Expression of Lentiviral Vectors in Human CD34 + Hematopoietic Cells
- Author
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John K. Choi, Patricia D. Conrad, Antonina M. Vilardi, Alan M. Gewirtz, Nghia Hoang, and Stephen G. Emerson
- Subjects
Time Factors ,Recombinant Fusion Proteins ,viruses ,Genetic Vectors ,Green Fluorescent Proteins ,Antigens, CD34 ,Bone Marrow Cells ,Cell Separation ,Mice, SCID ,Polymerase Chain Reaction ,Avian sarcoma virus ,Virus ,Cell Line ,Viral vector ,Mice ,Murine leukemia virus ,Animals ,Humans ,Transgenes ,Vector (molecular biology) ,Promoter Regions, Genetic ,Cells, Cultured ,HIV Long Terminal Repeat ,Models, Genetic ,biology ,Lentivirus ,Gene Transfer Techniques ,HIV ,virus diseases ,Cell Biology ,Fetal Blood ,Flow Cytometry ,Hematopoietic Stem Cells ,biology.organism_classification ,Virology ,Molecular biology ,Leukemia Virus, Murine ,Luminescent Proteins ,Avian Sarcoma Viruses ,Molecular Medicine ,Stem cell ,K562 Cells ,HeLa Cells ,Developmental Biology - Abstract
HIV-based lentiviral vectors can transduce nondividing cells, an important advantage over murine leukemia virus (MLV)-based vectors when transducing slowly dividing hematopoietic stem cells. However, we find that in human CD34(+) hematopoietic cells, the HIV-based vectors with an internal cytomegalovirus (CMV) promoter express transgenes 100- to 1,000-fold less than the MLV-based retroviral vector murine stem cell virus (MSCV). To increase the expression of the integrated lentivirus, we replaced CMV promoter with that of the Rous sarcoma virus or MSCV and obtained a modest augmentation in expression. A more dramatic effect was seen when the CMV enhancer/promoter was removed and the HIV long-terminal repeat (LTR) was replaced by a novel HIV/MSCV hybrid LTR. This vector retains the ability to transduce nondividing cells but now expresses its transgene (enhanced green fluorescent protein) 10- to 100-fold greater than the original HIV-based vector. When compared under identical conditions, the HIV vector with the hybrid LTR transduced a higher percentage of CD34(+) cells than the MSCV-based retroviral vector (19.4% versus 2.4%). The number of transduced cells and level of transgene expression remain constant over 5-8 weeks as determined by long-term culture-initiating cells, fluoresence-activated cell sorting, and nonobese diabetic/severe combined immunodeficiency repopulation assay.
- Published
- 2001
28. Hepatocyte growth factor is secreted by osteoblasts and cooperatively permits the survival of haematopoietic progenitors
- Author
-
Stephen G. Emerson, Karen Ehrenman, Russell S. Taichman, Marcelle J. Reilly, and Rama Shanker Verma
- Subjects
medicine.medical_specialty ,medicine.medical_treatment ,Growth factor ,Osteoblast ,Hematology ,Biology ,Cell biology ,Haematopoiesis ,Cytokine ,medicine.anatomical_structure ,Endocrinology ,Internal medicine ,medicine ,Hepatocyte growth factor ,Bone marrow ,Progenitor cell ,Stem cell ,medicine.drug - Abstract
Human osteoblasts (HOBs) support the growth of human haematopoietic progenitor cells, and support the survival and limited expansion of long-term culture-initiating cells. Using human CD34+ cells and the murine myelomonocytic cell line NFS-60 as targets, we previously found that one component of HOB-derived haematopoietic activity is cell-associated granulocyte colony-stimulating factor (G-CSF). However, antibody failed to neutralize all the activity, suggesting that more than one factor supports haematopoietic cells. In the present investigations, we asked whether the HOB-derived, non-G-CSF secreted activity was as a result of other known growth factors. We found that, among the cytokines expressed by HOBs, only hepatocyte growth factor (HGF) and G-CSF stimulated NFS-60 cell proliferation. HOB cells and osteosarcoma cells secreted biologically active HGF, although the levels varied considerably. Moreover, addition of neutralizing HGF antibody to CD34+ cell/HOB co-cultures resulted in a significant reduction (≈50%) in the ability of the HOBs to support haematopoietic progenitor cells. These results suggest that a major component of osteoblast-derived haematopoietic activity is HGF. Secretion of HGF, in concert with cell-associated cytokines such as G-CSF, may account for the stem cell-stimulating activities of osteogenic cells and, thereby, the unique stem cell-supporting role of the osteoblasts within the bone marrow microenvironment.
- Published
- 2001
29. ETK2 receptor tyrosine kinase promotes survival of factor-dependent FDC-P1 progenitor cells
- Author
-
Stephen G. Emerson, Matthew S. Couzens, Diane Giannola, and Christine Darby
- Subjects
Cancer Research ,Cell Survival ,Recombinant Fusion Proteins ,Genetic Vectors ,Apoptosis ,Transfection ,Proto-Oncogene Mas ,Tropomyosin receptor kinase C ,Culture Media, Serum-Free ,Receptor tyrosine kinase ,Mice ,Growth factor receptor ,Genes, Synthetic ,Genetics ,Animals ,Phosphorylation ,Molecular Biology ,Cells, Cultured ,Sequence Deletion ,Leukemia ,biology ,Cell Cycle ,Granulocyte-Macrophage Colony-Stimulating Factor ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Culture Media ,Protein Structure, Tertiary ,Cell biology ,Cell Transformation, Neoplastic ,Retroviridae ,ROR1 ,biology.protein ,Cancer research ,Interleukin-3 ,Janus kinase ,Protein Processing, Post-Translational ,Tyrosine kinase ,Platelet-derived growth factor receptor ,Signal Transduction ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Objective By virtue of its high expression in both developing hematopoietic tissues and many myeloid leukemia cells lines, the embryonic tyrosine kinase receptor ETK2 (also known as Tyro3, Sky, and Rse) has been postulated to play a role in early hematopoiesis. To investigate this role, we expressed murine ETK2 in the interleukin 3 (IL-3) dependent myeloid progenitor cell line FDC-P1 and examined its effect on growth factor dependence. Materials and Methods ETK2 cDNAs encoding full-length or kinase domain-deleted receptor were retrovirally transduced into murine FDC-P1 cells. Survival, cell cycle status, and proliferative responses of ETK2 expressing clones were studied at normal and reduced growth factor concentrations. Results ETK2 was expressed as a functional tyrosine kinase of 110 and 150 kDa. This proto-oncogene altered the growth of FDC-P1 cells, allowing survival at reduced growth factor concentrations and delaying apoptosis after IL-3 withdrawal. ETK2-expressing clones contained a higher fraction of cells in the S/G2/M phases of the cell cycle, both after cytokine withdrawal and in the presence of IL-3. Furthermore, these cells had a modestly enhanced proliferative response to IL-3 and granulocyte-macrophage colony-stimulating factor, suggesting that ETK2 intracellular signaling may converge with that of hematopoietic growth factors. The effects of ETK2 expression on viability and proliferation were largely dependent on a functional intracellular tyrosine kinase domain. Conclusion These results support a role for ETK2 in the survival and/or expansion of primitive hematopoietic cells and suggest that this tyrosine kinase may be implicated in myeloid leukemogenesis as well.
- Published
- 2000
30. Lymphoid Hyperplasia, Autoimmunity, and Compromised Intestinal Intraepithelial Lymphocyte Development in Colitis-Free Gnotobiotic IL-2-Deficient Mice
- Author
-
Nikhat V. Contractor, Hamid Bassiri, Tannishtha Reya, Audrey Y. Park, Daniel C. Baumgart, Mariusz A. Wasik, Stephen G. Emerson, and Simon R. Carding
- Subjects
Immunology ,Immunology and Allergy - Abstract
IL-2-deficient (IL-2−/−) mice develop disorders of the hemopoietic and immune systems characterized by anemia, lymphocytic hyperplasia, and colitis. The mechanisms responsible for these abnormalities remain unclear. To investigate the underlying basis of autoimmunity, the particular role of commensal gut flora in the initiation of colitis, and the role of IL-2 in the development of intestinal intraepithelial lymphocytes (iIEL), we evaluated IL-2−/− mice reared and maintained under gnotobiotic (germfree) conditions. By 8 wk of age, 80% (20 of 25) of germfree IL-2−/− mice show signs of disease, including anemia, disturbances in bone marrow hemopoietic cells, lymphocytic hyperplasia, and generalized autoimmunity, similar to those seen in specific pathogen-free (SPF) IL-2−/− mice. In striking contrast to SPF IL-2−/− mice, germfree IL-2−/− mice do not develop colitis. However, the numbers of γδ+ and TCRαβ+CD8αα+ iIELs are reduced, and in lethally irradiated SPF IL-2+/+ mice, reconstituted with IL-2−/− bone marrow TCRγδ+ iIELs fail to develop, consistent with an important role of IL-2/IL-2R signaling in the development of γδ iIELs. Consequently, our findings demonstrate that the colitis seen in SPF IL-2−/− mice depends upon the presence of intestinal bacterial flora and that environmental Ags are not responsible for the anemia and extraintestinal lymphoid hyperplasia that occur in IL-2−/− mice. Thus, germfree IL-2−/− mice represent a unique system in which the role of IL-2 deficiency in hemopoietic and immune system disorders can be investigated in dissociation from complications that may arise due to colitis.
- Published
- 1998
31. Augmented Production of Interleukin-6 by Normal Human Osteoblasts in Response to CD34+ Hematopoietic Bone Marrow Cells In Vitro
- Author
-
Rama Shanker Verma, Stephen G. Emerson, Russell S. Taichman, and Marcelle J. Reilly
- Subjects
Immunology ,CD34 ,Osteoblast ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Granulocyte colony-stimulating factor ,Cell biology ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Cytokine secretion ,Bone marrow ,Stem cell ,Leukemia inhibitory factor - Abstract
Based on anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, we have begun an analysis of osteoblast/hematopoietic cell interactions. We explore here the functional interdependence between these two cell types from the standpoint of de novo cytokine secretion. We determined that, over a 96-hour period, CD34+ bone marrow cells had no significant effect on osteoblast secretion of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-β1 , but in some experiments minor increases in leukemia inhibitory factor levels were observed. However, when CD34+ bone marrow cells were cocultured in direct contact with osteoblasts, a 222% ± 55% (range, 153% to 288%) augmentation in interleukin-6 (IL-6) synthesis was observed. The accumulation of IL-6 protein was most rapid during the initial 24-hour period, accounting for nearly 55% of the total IL-6 produced by osteoblasts in the absence of blood cells and 77% of the total in the presence of the CD34+ cells. Cell-to-cell contact does not appear to be required for this activity, as determined by coculturing the two cell types separated by porous micromembranes. The identity of the soluble activity produced by the CD34+ cells remains unknown, but is not likely due to IL-1β or tumor necrosis factor-α, as determined with neutralizing antibodies. To our knowledge, these data represent the first demonstration that early hematopoietic cells induce the production of molecules required for the function of normal bone marrow microenvironments, in this case through the induction of hematopoietic cytokine (IL-6) secretion by osteoblasts.
- Published
- 1997
32. Ex vivo expansion of hematopoietic precursors, progenitors, and stem cells: the next generation of cellular therapeutics
- Author
-
Stephen G. Emerson
- Subjects
Haematopoiesis ,Immunology ,Cell Biology ,Hematology ,Ex vivo expansion ,Biology ,Progenitor cell ,Stem cell ,Biochemistry ,Cell biology - Published
- 1996
33. Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures
- Author
-
Stephen G. Emerson, Marcelle J. Reilly, and Russell S. Taichman
- Subjects
Hematopoietic stem cell niche ,Hematopoietic stem cell differentiation ,Immunology ,CD34 ,Clinical uses of mesenchymal stem cells ,Osteoblast ,Stem cell factor ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Cell biology ,Endothelial stem cell ,medicine.anatomical_structure ,medicine ,Bone marrow - Abstract
Hematopoietic stem cell differentiation occurs in direct proximity to osteoblasts within the bone marrow cavity. Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently, we showed that human osteoblasts produce a variety of cytokine mRNAs including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6. We examined here the ability of osteoblasts to support the development of hematopoietic colonies from progenitors as well the ability to maintain long-term culture-initiating cells (LTC-IC) in vitro. Examination of the hematopoietic cells recovered after 2 weeks of culture showed that osteoblasts support the maintenance of immature hematopoietic phenotypes. In methylcellulose assays, osteoblasts stimulate the development of hematopoietic colonies to a level at least 10-fold over controls from progenitor cells. Using limiting dilutional bone marrow cultures, we observed an activity produced by osteoblasts resulting in an threefold to fourfold expansion of human LTC-IC and progenitor cells in vitro. Thus, the presence of hematopoietic stem cells in close proximity to endosteal surfaces in vivo may be due in part to a requirement for osteoblast-derived products.
- Published
- 1996
34. Contents, Vol. 96, 1996
- Author
-
A.S. Warsy, Aliza Zeidman, A.A. Mohamed, M. Erkan, Tapani Ruutu, I. Al-Fawaz, Wolmar Pulcheri, Annamaria Nosari, M.A.F El-Hazmi, Masaki Saito, S. Refai, Stephen G. Emerson, H. Balkan, Silvia Cantoni, Fabíola Passeri Lavrado, Kiyohiko Hatake, Shamsel Rab, Naomi Sasaki, Elisa Piva, Tomohiko Miyata, C. Öner, Ari Aimolahti, Marcio Nucci, A.M. Abdulkade, Satendra Sharma, Meir Djaldetti, J. Peláez, M. Farid, Youichi Amemiya, P.S. Sugathan, Sérgio A. P. Novis, H. Abu Taleb, Kazuo Muroi, Meera Sikka, G. Erdem, Enrica Morra, Usha Rusia, James Pitágoras de Mattos, J. Pinilla, A. Gürgey, Jan Samuelsson, Nelson Spector, Moshe Mittelman, Yuko Ogawa, Jan Palmblad, H.T. Hassan, José Carlos Morais, Osamu Tanabe, Ç. Aliay, F. Gümrük, Yasuo Takimoto, Mukul C. Datta, Preben Johansen, A.R. Zander, Daniele Ben-Dayan, Hideo Tanaka, Aisushi Kuramoto, Kunwar K. Srivastava, Eeva Juvonen, Ghil Busnach, F.H. Navarro, Riitta Jantunen, Luis Felipe Nobre, A.D. Opawoye, N Madan, Alberto Strinchini, H. Özdağ, H.B.M. Ahmed, Giuliana Muti, D. Hernández-Maraver, M. Stockchläder, M. Abulaban, Morten Krogh Jensen, Mario Plebani, Tetsuya Ohtsuki, Stanislaw Pulezynski, R. Öner, Yasusada Miura, Tohru Izumi, Stefania DeToni, A.W. Afy, Warren D. Shlomchik, Hatim G. Abdelrahman, Koji Nanba, Halley Pacheco de Oliveira, C. Löliger, and Z. Howsawi
- Subjects
Hematology ,General Medicine - Published
- 1996
35. The Immunobiology of T Cell Therapies for Leukemias
- Author
-
Stephen G. Emerson and Warren D. Shlomchik
- Subjects
Immunity, Cellular ,Chemotherapy ,Leukemia ,business.industry ,T-Lymphocytes ,medicine.medical_treatment ,T cell ,Alloimmunity ,Graft vs Host Disease ,Myeloid leukemia ,Hematology ,General Medicine ,medicine.disease ,Major Histocompatibility Complex ,Graft-versus-host disease ,medicine.anatomical_structure ,Acute lymphocytic leukemia ,Immunology ,medicine ,Humans ,Bone marrow ,business ,Bone Marrow Transplantation ,Chronic myelogenous leukemia - Abstract
Allogeneic bone marrow transplantation has revolutionized the treatment of chronic myelogenous leukemia, acute myeloid leukemia, and acute lymphocytic leukemia. Some of this success is due to the high doses of chemotherapy and radiotherapy allowable by rescuing fatal hematologic toxicity with the bone marrow allograft. However, it has become increasingly clear that an alloimmune antileukemic effect mediated by mature donor derived T cells, termed the graft-versus-leukemia reaction (GVL), makes a strong contribution to the efficacy of alloBMT. This has been dramatically illustrated by the complete remissions obtained in patients with relapsed leukemias after alloBMT following the infusion of donor mononuclear cells from their original marrow donors. In this review, after summarizing the clinical data in support of this effect, we discuss current paradigms of T cell mediated immunity and how they can be applied to explain the immunobiology of the GVL reaction.
- Published
- 1996
36. Short interfering RNA (siRNA) targeting the Lyn kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+) leukemia cells
- Author
-
Alan M. Gewirtz, Yuji Nakata, Anna Kalota, Andrzej Ptasznik, and Stephen G. Emerson
- Subjects
Small interfering RNA ,Blotting, Western ,Fusion Proteins, bcr-abl ,Tetrazolium Salts ,Apoptosis ,Drug resistance ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Bcr abl1 ,LYN ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,hemic and lymphatic diseases ,In Situ Nick-End Labeling ,Tumor Cells, Cultured ,medicine ,Humans ,RNA, Small Interfering ,hemic and immune systems ,General Medicine ,medicine.disease ,Thiazoles ,Haematopoiesis ,Leukemia ,src-Family Kinases ,Immunology ,Cancer research ,RNA Interference ,Blast Crisis ,Signal Transduction ,Chronic myelogenous leukemia - Abstract
We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80-95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable. This increased dependence on Lyn signaling for BCR-ABL1(+) blast survival provides the basis for rational treatment of drug-resistant CML blast crisis, particularly when lymphoid in nature.
- Published
- 2004
37. Interleukin-1 alpha upregulates tumor necrosis factor receptors expressed by a human bone marrow stromal cell strain: implications for cytokine redundancy and synergy
- Author
-
Jerry Caldwell and Stephen G. Emerson
- Subjects
medicine.medical_specialty ,Stromal cell ,medicine.medical_treatment ,Growth factor ,Immunology ,Alpha (ethology) ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Endocrinology ,Cytokine ,Granulocyte macrophage colony-stimulating factor ,Cell surface receptor ,Internal medicine ,medicine ,Tumor necrosis factor alpha ,Receptor ,medicine.drug - Abstract
To explore the biochemical and physiologic basis of the overlapping effects of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha) on myeloid cytokine production, we have studied the dynamics of granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) production as well as IL-1 receptor and TNF receptor expression in a clonally derived bone marrow stromal cell strain (CDCL). IL-1 alpha and TNF alpha act in a synergistic manner to stimulate G-CSF and GM-CSF production by CDCL, resulting in an increase in CSF secretion that is 250-fold greater than that observed with either cytokine alone. This synergism in protein secretion is paralleled by synergistic increases the steady-state level of GM- and G-CSF mRNA, with supra-additive levels achieved by 24 hours. Coincident with this synergistic induction of myeloid CSFs, treatment of CDCL cells with IL-1 alpha induces a 300% increase in the expression of TNF receptors. IL-1 alpha induction of TNF receptors reaches a peak after 6 hours and gradually returns to baseline level by 24 hours. IL-1 alpha does not affect TNF receptor ligand binding affinity. A kinetic study comparing IL-1/TNF synergistic induction of growth factor secretion with IL-1 alpha induction of TNF receptors shows that these events occur in parallel. In contrast with the induction of TNF receptors by IL-1 alpha, treatment with TNF alpha has no effect on either the number of IL-1 receptors expressed by CDCL cells or IL-1 receptor ligand binding affinity. Brief treatment of IL-1 alpha/TNF alpha-stimulated CDCL cells with cycloheximide before receptor induction reduces the synergistic increase in growth factor mRNA by 40% to 60% compared with cells not treated with CHX. Taken together, these results raise the possibility that IL-1 alpha cross-induction of TNF receptors may contribute to the biochemical mechanisms underlying the synergistic stimulation of G-CSF and GM-CSF production by IL-1 alpha and TNF alpha.
- Published
- 1995
38. Notch signaling directs splenic resident hematopoietic stem cell fate decisions
- Author
-
Ashley Nicole Vanderbeck, Ruben H. Land, Melanie D. Mumau, Elizabeth D. Lynch, Sophia B. Golec, Jennifer A. Punt, and Stephen G. Emerson
- Subjects
Immunology ,Immunology and Allergy - Abstract
Adult hematopoiesis occurs primarily in the bone marrow (BM), generating mature blood cells as well as maintaining a heterogeneous pool of self-renewing hematopoietic stem cells (HSCs). However, studies suggest that the BM may not be the only site of adult hematopoiesis: during times of BM hematopoietic distress stemming from radiation or disease, the spleen can independently reconstitute the blood. Given that the spleen does indeed contain a small, resident population of lineage-sca1+ckit+CD48-CD150+ long-term (LT)-HSCs with potent reconstitution ability, we investigated the peripheral blood cell contribution of these extra-medullary HSCs by transplanting UBC-GFP Tg reporter splenic fragments into a splenectomized syngenic wildtype mouse and tracking the blood cell production over time. While the spleen gives rise to both mature myeloid and lymphoid cells, we find that the proportion is skewed in favor of T cells. We see spleen-derived donor HSCs generating CD4+CD8+ double positive thymocytes for over five months post-transplant, suggesting that this peripheral T cell population derives from a long-lived stem or progenitor cell resident in the spleen. RNA sequencing demonstrates that splenic LT-HSCs are more responsive to Notch signaling than BM LT-HSCS, as indicated by increased levels of Hes1 transcription and decreased levels of the granulocyte/monocyte (GM) lineage priming genes Mpo, Gfi1, and Fcgr2b. Taken together with our finding that splenic HSCs have higher levels of cytoplasmic Notch2, we suggest that the Notch2-Hes1 axis is simultaneously repressing GM priming and enforcing T cell programming even among the most immature HSCs resident in the spleen.
- Published
- 2016
39. Abstract PR01: Precision in Pediatric Sequencing (PIPseq): Clinical implementation of genomic sequencing into pediatric hematology-oncology practice
- Author
-
Darrell J. Yamashiro, Jennifer A. Oberg, Mahesh M. Mansukhani, Carrie Koval, Susan J. Hsiao, Samantha Cano, Stuart J. Andrews, Danielle Pendrick Denney, Andrew L. Kung, Filamon Dela Cruz, Anthony N. Sireci, Stephen G. Emerson, Wendy K. Chung, Julia L. Glade Bender, Maria Luisa Sulis, Peter L. Nagy, and Rebecca J. Zylber
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,Genetic counseling ,Pediatric Hematology/Oncology ,Cancer ,medicine.disease ,Bioinformatics ,Precision medicine ,Pediatric cancer ,Hematologic disease ,Internal medicine ,medicine ,Medical genetics ,Copy-number variation ,business - Abstract
Background: Molecular characterization of tumor and/or host has the potential to advance the management of pediatric cancer and high risk hematologic disease, but the clinical utility of integrating genomic profiling into standard clinical practice has been limited. The PIPseq Program at Columbia University has instituted prospective CLIA-compliant genomic sequencing for newly diagnosed, high risk, relapsed or refractory pediatric cancer patients and patients referred for bone marrow transplantation. Methods: Families are consented for clinical cancer whole-exome sequencing (cWES) or constitutional whole-exome sequencing (WES) with opt out options for return of results, exclusion of results from medical records, receipt of American College of Medical Genetics (ACMG) recommended secondary germline variants, and data/ sample use in research. Molecular characterization utilizes next generation cWES, WES, RNAseq (transcriptome), or targeted sequencing of select cancer genes. Clinical cancer reports include: known tumor type-specific actionable somatic mutations (Tier 1); somatic mutations actionable in other tumor types, in targetable pathways, or in well-established cancer genes (Tier 2); other somatic mutations in cancer genes (Tier 3); and somatic variants of uncertain significance (VUS; Tier 4). Reports for cWES testing also note translocations, significantly over expressed genes, segmental copy number variation, and germline variants. Institutional Review Board approval was obtained to conduct a retrospective review of results to date. Five categories were developed to assess clinical utility and describe significance: 1) diagnostic, 2) prognostic, 3) potentially actionable target, 4) other critical role in decision making, and 5) implications for health maintenance and genetic counseling. Results: Since January 2014, adequate tissue samples were available for 47 patients, including 31 (66%) with solid tumors and 16 (34%) with hematologic conditions. Testing included cWES (n=8), cWES with transcriptome (n=15), transcriptome only (n=1), targeted somatic panel (n=8), constitutional WES only (n=6), and multiple sequencing platforms (n=7). Normal tissue was obtained from buccal swab (n=8), blood (n=18), and unaffected tissue (n=1). Three families opted out of receiving secondary findings. Genomic aberrations were reported in 41/47 patients. Of the 127 cancer alterations found, 70 (55%) were in 15 patients with hematologic disease (median 2, range 1-11) and 57 (45%), were in 26 patients with solid tumors (median 1, range 1-6). Among the hematologic cases, alterations of known or potential clinical relevance were categorized as Tier 1 (n=0), Tier 2 (n=27), Tier 3 (n=2) mutation, or translocation (n=4); whereas in solid tumors these were categorized as Tier 1 (n=1), Tier 2 (n=14), Tier 3 (n=3) mutation, or translocation (n=9). Twenty-four Tier 4 somatic VUS were identified in hematologic specimens and 26 in solid tumor specimens. Genomic interrogation informed diagnosis in 10 patients (3 previously unknown); provided new prognostic information in 4; identified potentially actionable targets in 15; influenced clinical decision making regarding bone marrow transplant in 2; and revealed cancer or other disease predisposition in 7. Secondary germline ACMG findings in BRCA1 and PMS2 were found. Germline APC mutation was confirmed in one patient and germline VUS in SDHC was seen in another. Novel germline findings were also observed in RUNX1, MLL2 and DICER1. Overall, the PIPseq platform provided clinically impactful results in 30/47 cases (64%). Conclusions: Utilizing a CLIA-compliant prospective WES-based platform, more than half of selected patients derived clinically impactful information. The potential clinical utility of genomic sequencing in pediatric hematology-oncology has likely been underestimated. This abstract is also presented as Poster 50. Citation Format: Julia L. Glade Bender, Jennifer A. Oberg, Maria Luisa Sulis, Filamon Dela Cruz, Anthony N. Sireci, Susan J. Hsiao, Darrell J. Yamashiro, Carrie Koval, Wendy K. Chung, Stephen G. Emerson, Rebecca Zylber, Samantha Cano, Danielle P. Denney, Stuart Andrews, Peter L. Nagy, Mahesh M. Mansukhani, Andrew L. Kung. Precision in Pediatric Sequencing (PIPseq): Clinical implementation of genomic sequencing into pediatric hematology-oncology practice. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr PR01.
- Published
- 2016
40. NF-Ya protein delivery as a tool for hematopoietic progenitor cell expansion
- Author
-
Alevtina D, Domashenko, Susan, Wiener, and Stephen G, Emerson
- Subjects
Colony-Forming Units Assay ,Protein Transport ,CCAAT-Binding Factor ,Protein Stability ,Recombinant Fusion Proteins ,Blotting, Western ,Cell Culture Techniques ,Escherichia coli ,Humans ,Amino Acid Sequence ,Hematopoietic Stem Cells ,K562 Cells ,Cell Proliferation - Abstract
The clinical potential of therapeutic quantities of primary hematopoietic cells, either unmodified or altered via genetic modification, has stimulated the search for techniques that allow the production of large numbers of hematopoietic precursors, more primitive progenitors, and perhaps hematopoietic stem cells (HSC) themselves. Modifications of in vitro culture conditions to promote progenitor cell expansion have included combinations of polypeptide cytokines, small molecules, and transcription factors. Here we describe the methods for use of the transcription factor linked to a TAT-based protein transcription domain, in combination with cytokines and serum-free culture condition to stimulate the proliferation of primary cells. Human peripheral blood (PB) CD34(+) cells treated with TAT-NF-Ya fusion protein and grown in vitro for 1 month proliferate four times more than did cells in cultures that contained only cytokines, including increased production of hematopoietic cells of all maturities. These results and techniques should be suitable for multiple applications of ex vivo generation of hematopoietic cells using protein transduction.
- Published
- 2012
41. Long-term functional engraftment of mesenchymal progenitor cells in a mouse model of accelerated aging
- Author
-
Lakshman, Singh, Tracy A, Brennan, Jung-Hoon, Kim, Kevin P, Egan, Emily A, McMillan, Qijun, Chen, Kurt D, Hankenson, Yi, Zhang, Stephen G, Emerson, F Brad, Johnson, and Robert J, Pignolo
- Subjects
Aging ,Disease Models, Animal ,Mice ,Animals ,Osteoporosis ,Cell Differentiation ,Mesenchymal Stem Cells ,Mice, Transgenic ,Telomere ,Mesenchymal Stem Cell Transplantation ,Survival Analysis ,Cellular Senescence ,Article - Abstract
Age-related osteoporosis is characterized by a decrease in bone-forming capacity mediated by defects in the number and function of osteoblasts. An important cellular mechanism that may in part explain osteoblast dysfunction that occurs with aging is senescence of mesenchymal progenitor cells (MPCs). In the telomere-based Wrn(-/-) Terc(-/-) model of accelerated aging, the osteoporotic phenotype of these mice is also associated with a major decline in MPC differentiation into osteoblasts. To investigate the role of MPC aging as a cell-autonomous mechanism in senile bone loss, transplantation of young wild-type whole bone marrow into Wrn(-/-) Terc(-/-) mutants was performed and the ability of engrafted cells to differentiate into cells of the osteoblast lineage was assessed. We found that whole bone marrow transplantation in Wrn(-/-) Terc(-/-) mice resulted in functional engraftment of MPCs up to 42 weeks, which was accompanied by a survival advantage as well as delays in microarchitectural features of skeletal aging.
- Published
- 2012
42. Blockade of lymphocyte chemotaxis in visceral graft-versus-host disease
- Author
-
Noelle V. Frey, Robert H. Vonderheide, Sarah Bailey, David L. Porter, Selina M. Luger, Daniel F. Heitjan, James A. Hoxie, Stephen G. Emerson, Edward A. Stadtmauer, Jacqueline Smith, Sunita D. Nasta, Rosemarie Mick, Steven C. Goldstein, Alison W. Loren, Elizabeth O. Hexner, and Ran Reshef
- Subjects
Adult ,Male ,Chemokine receptor CCR5 ,medicine.medical_treatment ,T-Lymphocytes ,Graft vs Host Disease ,Hematopoietic stem cell transplantation ,CCR5 receptor antagonist ,Kaplan-Meier Estimate ,Maraviroc ,Lymphocyte chemotaxis ,chemistry.chemical_compound ,Young Adult ,immune system diseases ,Cyclohexanes ,Medicine ,Humans ,Transplantation, Homologous ,Cumulative incidence ,Chemokine CCL5 ,Aged ,Chemokine CCL3 ,biology ,business.industry ,Hematopoietic Stem Cell Transplantation ,General Medicine ,Middle Aged ,Triazoles ,medicine.disease ,Transplantation ,Chemotaxis, Leukocyte ,surgical procedures, operative ,Graft-versus-host disease ,chemistry ,Hematologic Neoplasms ,Immunology ,CCR5 Receptor Antagonists ,biology.protein ,Female ,business - Abstract
Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic stem-cell transplantation (HSCT). The chemokine receptor CCR5 appears to play a role in alloreactivity. We tested whether CCR5 blockade would be safe and limit GVHD in humans.We tested the in vitro effect of the CCR5 antagonist maraviroc on lymphocyte function and chemotaxis. We then enrolled 38 high-risk patients in a single-group phase 1 and 2 study of reduced-intensity allogeneic HSCT that combined maraviroc with standard GVHD prophylaxis.Maraviroc inhibited CCR5 internalization and lymphocyte chemotaxis in vitro without impairing T-cell function or formation of hematopoietic-cell colonies. In 35 patients who could be evaluated, the cumulative incidence rate (±SE) of grade II to IV acute GVHD was low at 14.7±6.2% on day 100 and 23.6±7.4% on day 180. Acute liver and gut GVHD were not observed before day 100 and remained uncommon before day 180, resulting in a low cumulative incidence of grade III or IV GVHD on day 180 (5.9±4.1%). The 1-year rate of death that was not preceded by disease relapse was 11.7±5.6% without excessive rates of relapse or infection. Serum from patients receiving maraviroc prevented CCR5 internalization by CCL5 and blocked T-cell chemotaxis in vitro, providing evidence of antichemotactic activity.In this study, inhibition of lymphocyte trafficking was a specific and potentially effective new strategy to prevent visceral acute GVHD. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00948753.).
- Published
- 2012
43. NF-Ya Protein Delivery as a Tool for Hematopoietic Progenitor Cell Expansion
- Author
-
Susan Wiener, Alevtina Domashenko, and Stephen G. Emerson
- Subjects
Haematopoiesis ,Chemistry ,CD34 ,Progenitor cell ,Stem cell ,Transcription factor ,Fusion protein ,In vitro ,Ex vivo ,Cell biology - Abstract
The clinical potential of therapeutic quantities of primary hematopoietic cells, either unmodified or altered via genetic modification, has stimulated the search for techniques that allow the production of large numbers of hematopoietic precursors, more primitive progenitors, and perhaps hematopoietic stem cells (HSC) themselves. Modifications of in vitro culture conditions to promote progenitor cell expansion have included combinations of polypeptide cytokines, small molecules, and transcription factors. Here we describe the methods for use of the transcription factor linked to a TAT-based protein transcription domain, in combination with cytokines and serum-free culture condition to stimulate the proliferation of primary cells. Human peripheral blood (PB) CD34(+) cells treated with TAT-NF-Ya fusion protein and grown in vitro for 1 month proliferate four times more than did cells in cultures that contained only cytokines, including increased production of hematopoietic cells of all maturities. These results and techniques should be suitable for multiple applications of ex vivo generation of hematopoietic cells using protein transduction.
- Published
- 2012
44. IL-1? and TNF? act synergistically to stimulate production of myeloid colony-stimulating factors by cultured human bone marrow stromal cells and cloned stromal cell strains
- Author
-
Jerry Caldwell and Stephen G. Emerson
- Subjects
Myeloid ,Stromal cell ,Physiology ,Clinical Biochemistry ,Alpha (ethology) ,Bone Marrow Cells ,Biology ,Bone Marrow ,Granulocyte Colony-Stimulating Factor ,medicine ,Humans ,Cells, Cultured ,Tumor Necrosis Factor-alpha ,Granulocyte-Macrophage Colony-Stimulating Factor ,Drug Synergism ,Cell Biology ,Colony-stimulating factor ,Recombinant Proteins ,Clone Cells ,Kinetics ,Granulocyte macrophage colony-stimulating factor ,medicine.anatomical_structure ,Cell culture ,Immunology ,Cancer research ,Tumor necrosis factor alpha ,Bone marrow ,Mitogens ,Stromal Cells ,Interleukin-1 ,medicine.drug - Abstract
Human bone marrow stromal cells respond to stimulation by the monokines IL-1 and TNF by producing colony-stimulating factors such as GM-CSF and G-CSF. In this study we show that IL-1 alpha and TNF alpha act synergistically to stimulate GM-CSF and G-CSF production by cultured marrow stromal cells. We further show that IL-1 alpha and TNF alpha synergistically stimulate production of GM-CSF and G-CSF by a clonal stroma-derived cell strain. Although IL-1 and TNF share many of the same biological activities, we show that IL-1 alpha and TNF alpha have an unequal ability to induce myeloid-CSF production by both cultures, with IL-1 alpha being the more potent inducer. We found that induction by IL-1 alpha and TNF alpha was independent of cell proliferation. The effect of IL-1 alpha and TNF alpha on production of the two myeloid-CSFs by the clonal cells was significantly greater than the unfractionated passaged stromal cultures, having the greater effect on G-CSF production. The clonally derived stromal cells constitutively produced colony-stimulating activity, in particular GM-CSF, at levels easily detected by ELISA. These findings show that, in addition to the overlapping and additive activities of IL-1 alpha and TNF alpha, they can interact synergistically. Our findings further suggest that a small subpopulation of stroma cells may be the major producer of G-CSF in the marrow microenvironment during immune response.
- Published
- 1994
45. Granulocyte colony-stimulating factor (G-CSF) production and G-CSF receptor structure in patients with congenital neutropenia
- Author
-
Laurence A. Boxer, Susan C. Guba, Stephen G. Emerson, Raymond Hutchinson, and Carolyn A. Sartor
- Subjects
medicine.medical_specialty ,Stromal cell ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Biology ,Granulocyte ,Neutropenia ,medicine.disease ,Biochemistry ,Granulocyte colony-stimulating factor ,medicine.anatomical_structure ,Endocrinology ,Cytokine ,Internal medicine ,medicine ,Bone marrow ,Granulocyte colony-stimulating factor receptor ,Congenital Neutropenia - Abstract
Congenital neutropenia (Kostmann's syndrome [KS]) is an autosomal recessive syndrome that is characterized by profound neutropenia, resulting in major clinical infections and death. Since the neutropenia and symptoms in KS improve in response to exogenous administration of granulocyte colony-stimulating factor (G-CSF), we studied bone marrow cytokine (G-CSF, granulocyte-macrophage CSF [GM-CSF], and interleukin- 6) production under both basal and stimulated conditions. No differences in G-CSF, GM-CSF, or IL-6 gene expression were found in bone marrow stromal cells between normal controls and KS patients, and all three cytokines were detected by enzyme-linked immunosorbent assay (ELISA) in medium conditioned by bone marrow stromal cells from normal donors and patients with KS. Each KS patient tested had detectable, functional G-CSF in their own serum before exogenous G-CSF administration. Since G-CSF production appeared normal in KS patients, we then asked whether we could detect structural defects in the signaling portion of G-CSF receptor genes. Polymerase chain reaction (PCR) amplification of the G-CSF receptor transmembrane region alone, and of the transmembrane plus cytosolic portions of the receptor, yielded the size products predicted from the sequences of the normal G- CSF receptor. Single-strand conformational polymorphism (SSCP) analysis of G-CSF receptor PCR products demonstrated no variance in structural conformation between KS patients and normal subjects. These results demonstrate that bone marrow stromal cells in patients with KS secrete normal concentrations of functional G-CSF and suggest that the neutropenia in KS patients is caused by an inability of neutrophilic progenitor and precursor cells to respond to normal, physiologic levels of G-CSF. Such a defect, clinically responsive to pharmacologic doses of G-CSF, might be caused by defects in the post-G-CSF receptor signal transduction pathway.
- Published
- 1994
46. Human osteoblasts support hematopoiesis through the production of granulocyte colony-stimulating factor
- Author
-
Russell S. Taichman and Stephen G. Emerson
- Subjects
Hematopoietic stem cell niche ,Immunology ,Molecular Sequence Data ,Stem cell factor ,Biology ,Granulocyte ,In Vitro Techniques ,Polymerase Chain Reaction ,Granulocyte Colony-Stimulating Factor ,medicine ,Immunology and Allergy ,Humans ,DNA Primers ,Osteoblasts ,Base Sequence ,Osteoblast ,Articles ,Immunohistochemistry ,Granulocyte colony-stimulating factor ,Cell biology ,Hematopoiesis ,Haematopoiesis ,medicine.anatomical_structure ,Myelopoiesis ,Stem cell - Abstract
Previous attempts at identifying the constitutive source(s) of granulocyte colony-stimulating factor (G-CSF) in human bone marrow have been unsuccessful despite the fact that normal bone marrow supports abundant myelopoiesis in vivo. We hypothesized that the intimate physical association between bone and hematopoietic cells facilitates interactions between osteoblasts and hematopoietic stem cells. Here we provide the first direct evidence that human osteoblasts participate in hematopoiesis by constitutively producing G-CSF and present the protein in a membrane-associated fashion to human hematopoietic progenitors. These results suggest a direct and central role for osteoblasts in normal myelopoiesis.
- Published
- 1994
47. NF-Y is necessary for hematopoietic stem cell proliferation and survival
- Author
-
Gerd Bungartz, David T. Scadden, Stephen G. Emerson, and Hannah S. Land
- Subjects
Male ,Cell Survival ,Hematopoiesis and Stem Cells ,Immunology ,Blotting, Western ,Down-Regulation ,Apoptosis ,Bone Marrow Cells ,Cell Cycle Proteins ,Biology ,Biochemistry ,Mice ,Proto-Oncogene Proteins ,Conditional gene knockout ,Animals ,Receptor, Notch1 ,Cyclin B1 ,Transcription factor ,Cells, Cultured ,Cyclin ,Cell Proliferation ,Homeodomain Proteins ,Mice, Knockout ,Polycomb Repressive Complex 1 ,Thymocytes ,Cell growth ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Nuclear Proteins ,Cell Biology ,Hematology ,Cell Cycle Checkpoints ,Cell cycle ,Hematopoietic Stem Cells ,Hematopoietic stem cell proliferation ,Cell biology ,Hematopoiesis ,Repressor Proteins ,Haematopoiesis ,CCAAT-Binding Factor ,Female ,Tumor Suppressor Protein p53 ,Spleen ,Transcription Factors - Abstract
HSC function depends on the tight control of proliferation and the balance between self-renewal and differentiation. Here, we report that the trimeric transcription factor NF-Y is critical for the survival of cycling, but not quiescent HSCs. With the use of a conditional knockout mouse model, we demonstrate that NF-Ya deletion creates an accumulation of HSCs in G2/M and prompts apoptosis, causing hematopoietic failure and death of the animal. These defects are accompanied by the dysregulation of multiple genes that influence cell cycle control (cyclin b1 and p21), apoptosis (Bcl-2), and self-renewal (HoxB4, Notch1, Bmi-1) and are independent of p53. Our results identify NF-Y as a pivotal upstream participant in a regu-latory network necessary for the pre-servation of cycling HSCs.
- Published
- 2011
48. Identification of four murine cDNAs encoding putative protein kinases from primitive embryonic stem cells differentiated in vitro
- Author
-
Leslie G. Biesecker, Stephen G. Emerson, and Lisa R. Gottschalk
- Subjects
Cellular differentiation ,Molecular Sequence Data ,Gene Expression ,In Vitro Techniques ,Biology ,Sequence Homology, Nucleic Acid ,Complementary DNA ,Tumor Cells, Cultured ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Protein kinase A ,Multidisciplinary ,Base Sequence ,Sequence Homology, Amino Acid ,Kinase ,Stem Cells ,Teratoma ,Cell Differentiation ,Embryonic stem cell ,Molecular biology ,Cell biology ,Phosphorylation ,Stem cell ,Signal transduction ,Protein Kinases ,Sequence Alignment ,Research Article - Abstract
Protein kinases transduce signals from extracellular ligands in the hematopoietic and other systems through direct phosphorylation of tyrosine, serine, or threonine residues. Little is known about the ligands and receptors that are important in the earliest stages of development--i.e., stem cell self-renewal and lineage commitment. We have made use of the lineage differentiation potential of the murine embryonic stem cell system to clone partial cDNAs encoding four putative protein kinases. Three of the four genes contain the highly conserved residues Asp-Phe-Gly in domain VII of the protein kinase family. These genes are candidates for receptors or downstream effectors of cytokines that regulate self-renewal and lineage commitment in embryogenesis.
- Published
- 1993
49. Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures
- Author
-
Bernhard O. Palsson, Stephen G. Emerson, and Manfred R. Koller
- Subjects
Pathology ,medicine.medical_specialty ,Stromal cell ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Trypsinization ,Transplantation ,medicine.anatomical_structure ,medicine ,Autologous transplantation ,Bone marrow ,Progenitor cell ,Stem cell ,Ex vivo - Abstract
There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells. We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment. In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation. At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed. The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal. Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14. The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8. In contrast to CFU- GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations. Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay. The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8- fold) expansion. In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate. These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.
- Published
- 1993
50. Expansion of Human Bone Marrow Progenitor Cells in a High Cell Density Continuous Perfusion System
- Author
-
Mahshid A. Palsson, Samuel M. Silver, Bernhard O. Palsson, Stephen G. Emerson, Richard M. Schwartz, Gyun Min Lee, and Se-Hwan Paek
- Subjects
Time Factors ,Population ,Bone Marrow Cells ,Cell Count ,Peripheral blood mononuclear cell ,medicine ,Humans ,Progenitor cell ,education ,Cells, Cultured ,Erythroid Precursor Cells ,education.field_of_study ,Cell growth ,Chemistry ,Macrophages ,Hematopoietic Stem Cells ,Culture Media ,Cell biology ,Oxygen ,Perfusion ,Blood ,medicine.anatomical_structure ,Cell culture ,Immunology ,Bone marrow ,Stem cell ,Cell Division ,Granulocytes ,Biotechnology - Abstract
We describe here a continuous perfusion bioreactor system that enables a population of unselected human mononuclear bone marrow cells obtained from adult donors to expand up to 20 to 25-fold over a two-week period. Colony-forming units of granulocyte-macrophage (CFU-GM) progenitor cells expand 10 to 30-fold. These expansions depend on the gas phase oxygen concentration, the seeding density and time of cell harvest. Under operating conditions that allow for good cell proliferation, 3 to 4 million mononuclear cells can be obtained per square centimeter, with 0.5 to 0.8% being progenitor cells. Autologous human sera supported cell expansion as efficiently as animal sera. Increasing the size of the perfusion system to produce a clinically meaningful number of CFU-GMs could have important applications in bone marrow transplantation therapies.
- Published
- 1993
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