RESUMO
Recently, interactions between thrombopoietin (TPO) and its receptor, the myeloproliferative leukemia (MPL) virus oncogene, have been shown to play a role in the development and progression of myeloproliferative neoplasms including myelofibrosis (MF). These observations have led to the development of strategies to disrupt the association of TPO with its receptor as a means of targeting MF hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). In this report, we show that although both splenic and peripheral blood MF CD34(+) cells expressed lower levels of MPL than normal CD34(+) cells, TPO promoted the proliferation of MF CD34(+) cells and HPCs in a dose-dependent fashion. Furthermore, the treatment of MF but not normal CD34(+) cells with a synthesized MPL antagonist, LCP4, decreased the number of CD34(+)Lin(-) cells and all classes of assayable HPCs (colony-forming unit-megakaryocyte [CFU-MK], CFU-granulocyte/macrophage, burst-forming unit-erythroid/CFU-erythroid, and CFU-granulocyte/erythroid/macrophage/MK) irrespective of their mutational status. In addition, LCP4 treatment resulted in the depletion of the number of MF HPCs that were JAK2V617F(+) Moreover, the degree of human cell chimerism and the proportion of malignant donor cells were significantly reduced in immunodeficient mice transplanted with MF CD34(+) cell grafts treated with LCP4. These effects of LCP4 on MF HSCs/HPCs were associated with inhibition of JAK-STAT activity, leading to the induction of apoptosis. These findings demonstrate that such specific anti-cytokine receptor antagonists represent a new class of drugs that are capable of targeting MF HSCs.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Mielofibrose Primária/tratamento farmacológico , Receptores de Trombopoetina/antagonistas & inibidores , Idoso , Substituição de Aminoácidos , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Feminino , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Xenoenxertos , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismoRESUMO
A 54-member library of boronated octapeptides, with all but the boronated residue being proteinogenic, was tested for affinity to a set of saccharides commonly found on the terminus of mammalian glycans. After experimentation with a high-throughput dye-displacement assay, attention was focused on isothermal titration calorimetry as a tool to provide reliable affinity data, including enthalpy and entropy of binding. A small number of boronated peptides showed higher affinity and significant selectivity for N-acetylneuraminic acid over methyl-α-d-galactopyranoside, methyl-α/ß-l-fucopyranoside and N-acetyl-d-glucosamine. Thermodynamic data showed that for most of the boronated peptides studied, saccharide binding was associated with a significant increase in entropy, presumably resulting from the displacement of semiordered water molecules from around the sugar and/or peptide.
RESUMO
Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.
Assuntos
Plaquetas/citologia , Técnicas de Cultura de Células , Megacariócitos/citologia , Transfusão de Plaquetas/normas , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/imunologia , Plaquetas/imunologia , Desdiferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Citocinas/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Inativação Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/imunologia , Microfluídica/instrumentação , Microfluídica/métodos , Transfusão de Plaquetas/estatística & dados numéricosRESUMO
Factor V (FV) and factor X (FX) activate and complex to form prothrombinase which subsequently cleaves prothrombin (PT), converting it to active thrombin. Thrombin cleaved osteopontin (tcOPN) contains a cryptic binding site for α4 ß1 and α9 ß1 integrins. We have previously shown that hematopoietic stem cells (HSC) bind to tcOPN via this site resulting in a decrease in their proliferation and differentiation. Therefore, tcOPN and the factors required for its generation are important components of the HSC niche. Herein we show mature megakaryocytes (MM, ≥8N) contain FV, FX, and PT mRNA and protein. Furthermore, we show 8N, 16N, 32N, and 64N MM all release the required factors to enable thrombin cleavage of OPN. Importantly, mice devoid of the myeloproliferative leukemia protein (Mpl), c-Mpl(-/-) mice, contain only approximately 10% of normal megakaryocyte numbers, showed significantly reduced FX and tcOPN protein levels in endosteal bone marrow (BM). In addition, WT hematopoietic progenitors and HSC showed reduced homing to the BM of c-Mpl(-/-) mice. This is the first report identifying MM as a key cellular component in the production of tcOPN in situ, allowing the BM microenvironment to self regulate HSC biology via tcOPN.
Assuntos
Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Megacariócitos/metabolismo , Osteopontina/metabolismo , Trombina/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Megacariócitos/citologia , Camundongos , Nicho de Células-Tronco , Microambiente TumoralRESUMO
The α9ß1 and α4ß1 integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α4ß1 integrin have been thoroughly investigated with respect to HSC function, the role of α9ß1 integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α9ß1 integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α9ß1 integrin with potent cross-reactivity against α4ß1 integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α9ß1 and α4ß1 integrins, which showed faster binding to α4ß1 integrin relative to α9ß1, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α9ß1 and α4ß1 integrins; (2) investigating the biochemical properties of α9ß1 and α4ß1 integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.
Assuntos
Células da Medula Óssea/citologia , Dipeptídeos/farmacologia , Desenho de Fármacos , Corantes Fluorescentes/farmacologia , Integrina alfa4beta1/antagonistas & inibidores , Integrinas/antagonistas & inibidores , Prolina/farmacologia , Rodaminas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Dipeptídeos/síntese química , Dipeptídeos/química , Relação Dose-Resposta a Droga , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Conformação Molecular , Prolina/análogos & derivados , Prolina/química , Rodaminas/síntese química , Rodaminas/química , Relação Estrutura-AtividadeRESUMO
A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels. The preferential homing of HSPCs to the metaphysis occurs rapidly after transplantation, suggesting that blood vessels within this region may express a unique repertoire of endothelial adhesive molecules. One candidate is hyaluronan (HA), which is highly expressed on the blood vessel endothelium in the metaphysis. Analysis of the early stages of homing and the spatial dis-tribution of transplanted HSPCs at the single-cell level in mice devoid of Has3-synthesized HA, provides evidence for a previously undescribed role for HA expressed on endothelial cells in directing the homing of HSPCs to the metaphysis.
Assuntos
Vasos Sanguíneos/citologia , Medula Óssea/irrigação sanguínea , Osso e Ossos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/ultraestrutura , Osso e Ossos/metabolismo , Moléculas de Adesão Celular/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Fêmur/citologia , Fêmur/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nicho de Células-Tronco/irrigação sanguínea , Nicho de Células-Tronco/citologia , Migração Transendotelial e Transepitelial , Microtomografia por Raio-XRESUMO
Hemopoietic stem cells (HSCs) reside within a specified area of the bone marrow (BM) cavity called a "niche" that modulates HSC quiescence, proliferation, differentiation, and migration. Our previous studies have identified the endosteal BM region as the site for the HSC niche and demonstrated that hemopoietic stem and progenitor populations (HSPCs, LSK) isolated from different BM regions exhibit significantly different hemopoietic potential. In this study, we have analyzed subpopulations of LSK cells isolated from different regions of the BM and showed that CD150(+)CD48(-)LSK HSCs within the endosteal BM region have superior proliferative capacity and homing efficiency compared with CD150(+)CD48(-)LSK HSCs isolated from the central BM. Furthermore, we show, for the first time, that a subset of CD150(+)CD48(+)LSK progenitor cells, previously defined as B-lymphoid primed hemopoietic cells, are capable of multilineage reconstitution, however, only when isolated from the endosteal region. In addition, we provide evidence for an unrecognized role of CD48 in HSC homing. Together, our data provide strong evidence that highly purified HSCs show functional differences depending on their origin within the BM and that the most primitive HSCs reside within the endosteal BM region.
Assuntos
Antígenos CD/metabolismo , Medula Óssea/anatomia & histologia , Proliferação de Células , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos Ly/metabolismo , Antígeno CD48 , Ciclo Celular , Transplante de Células-Tronco Hematopoéticas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
Granulocyte colony stimulating factor (G-CSF) is clinically well established for the mobilization of hematopoietic stem cells (HSC). Extensive data on the underlying mechanism of G-CSF induced mobilization is available; however, little is known regarding the functional effect of G-CSF on HSC within the bone marrow (BM). In this study we analyzed the proportion and number of murine HSC in the endosteal and central bone marrow regions after 4 days of G-CSF administration. We demonstrate that the number of HSC, defined as CD150(+)CD48(-)LSK cells (LSKSLAM cells), increased within the central BM region in response to G-CSF, but not within the endosteal BM region. In addition the level of CD150 and CD48 expression also increased on cells isolated from both regions. We further showed that G-CSF mobilized proportionally fewer LSKSLAM compared to LSK cells, mobilized LSKSLAM had colony forming potential and the presence of these cells can be used as a measure for mobilization efficiency. Together we provide evidence that HSC in the BM respond differently to G-CSF and this is dependent on their location. These findings will be valuable in developing new agents which specifically mobilize HSC from the endosteal BM region, which we have previously demonstrated to have significantly greater hematopoietic potential compared to their phenotypically identical counterparts located in other regions of the BM.
Assuntos
Medula Óssea/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD/imunologia , Antígeno CD48 , Ciclo Celular , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/imunologiaRESUMO
Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express alpha(9)beta(1) integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the alpha(9)beta(1) and alpha(4)beta(1) integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with alpha(9)beta(1) and alpha(4)beta(1) integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Osteopontina/fisiologia , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Cricetinae , Cricetulus , Primers do DNA/genética , Sangue Fetal/citologia , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/farmacologia , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Técnicas In Vitro , Integrina alfa4beta1/genética , Integrinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteopontina/deficiência , Osteopontina/genética , Osteopontina/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/metabolismoRESUMO
Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the betac subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene betac Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34(+)CD38(-)CD123(+) leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.
Assuntos
Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide/genética , Proteínas de Neoplasias/fisiologia , Osteopontina/fisiologia , Adulto , Idoso , Sobrevivência Celular , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Feminino , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/mortalidade , Leucemia Mieloide/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Osteopontina/biossíntese , Osteopontina/genética , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosfosserina/metabolismo , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
OBJECTIVES: Objective: To undertake a pilot, feasibility RCT of umbilical cord blood derived cell therapy for treatment of adult patients infected with SARS-CoV-2 virus related moderate-to-severe pneumonia to prevent progression to severe ARDS. HYPOTHESIS: Expanded cord blood derived cell therapy will be feasible, well tolerated and show potential efficacy in the treatment of acute COVID-19 related moderate to severe pneumonia in adult patients because of their powerful anti-inflammatory and immunomodulatory properties. TRIAL DESIGN: Pilot, parallel design randomised controlled trial. PARTICIPANTS: The trial will recruit 24 hospitalised patients with confirmed SARS-CoV-2 infection and pneumonia from July to December 2020 at Monash Medical Centre in Melbourne, Australia. INTERVENTION AND COMPARATOR: Intervention: Intravenous injection of expanded umbilical cord blood cells at a dose of 5 million cells/kg (maximum dose - 500 million cells). Cell infusion will occur over 30-60 minutes through a peripheral intravenous cannula. Standard supportive care will continue as needed. Comparator: Standard supportive care. MAIN OUTCOMES: Safety and tolerability of cell administration within first 24 hours of administration; clinical improvement on a seven-category clinical improvement ordinal scale. RANDOMISATION: Randomisation will be done using computer generated allocation to intervention/ control groups in a 1:1 ratio (in blocks of 6) using sealed opaque envelopes. BLINDING (MASKING): This will be an unblinded study, given that it is the first study using expanded cord blood cells in COVID-19 patients. There will be no placebo infusion. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): Twelve participants in each group. Total n=24. TRIAL STATUS: CBC-19 protocol v2, dated 23rd April 2020. Recruitment has not started yet. Estimated recruitment timeline is between 1st July - 31st December 2020. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12620000478910, registered 16th April 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.
Assuntos
Betacoronavirus/patogenicidade , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Infecções por Coronavirus/cirurgia , Pneumonia Viral/cirurgia , COVID-19 , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Progressão da Doença , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Projetos Piloto , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Ensaios Clínicos Controlados Aleatórios como Assunto , SARS-CoV-2 , Fatores de Tempo , Resultado do Tratamento , VitóriaRESUMO
The recent explosion in the understanding of the cellular and molecular mechanisms underlying hematopoietic stem and progenitor cell (HSPC) mobilization has facilitated development of novel therapeutic agents, targeted at improving mobilization kinetics as well as HSPC yield. With the development of new agents comes the challenge of choosing efficient and relevant preclinical studies for the testing of the HSPC mobilization efficacy of these agents. This article reviews the use of the mouse as a convenient small animal model of HSPC mobilization and transplantation, and outlines the range of murine assays that can be applied to assess novel HSPC mobilizing agents. Techniques to demonstrate murine HSPC mobilization are discussed, as well as the role of murine assays to confirm human HSPC mobilization, and techniques to investigate the biologic phenotype of HSPC mobilized by these novel agents. Technical aspects regarding mobilization regimens and control arms, and choice of experimental animals are also discussed.
Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Modelos Animais , Animais , Animais Congênicos , Técnicas de Cultura de Células/métodos , Linhagem da Célula , Células Cultivadas/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Sobrevivência de Enxerto , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Endogâmicos , Camundongos SCID , Agonistas Mieloablativos/farmacologia , Quimera por RadiaçãoRESUMO
OBJECTIVE: Hematopoietic progenitor cells (HPC) as well as tissue committed stem cells expressing mRNA specific to various somatic tissues are thought to be part of the CD34+ bone marrow compartment. In this study, we explore and quantify their mobilization in patients with multiple myeloma undergoing chemotherapy upon administration of granulocyte colony-stimulating factor (G-CSF) plus/minus erythropoietin (EPO). PATIENTS AND METHODS: HPC were quantified by flow cytometry and functional assays within the blood of healthy donors and myeloma patients before and after chemotherapy followed by G-CSF or G-CSF + EPO given subcutaneously. The mRNA expression was studied by quantitative polymerase chain reaction (PCR). Cytokines and peripheral blood protease levels were measured by an enzyme-linked immunosorbent assay. RESULTS: EPO did not significantly alter the number of HPC mobilized by G-CSF alone, and mRNA specific for liver, brain, muscle and kidney was detected in both treatment groups. Quantitative PCR analysis revealed a 2.7-fold increased expression of glial fibrillary acidic protein after G-CSF + EPO administration compared to G-CSF alone (P = 0.003). The concentration of G-CSF rose from 62 +/- 22 pg/mL and 48 +/- 10 pg/mL to 28 +/- 9 ng/mL and 85 +/- 10 ng/mL after 10 d of treatment with G-CSF and G-CSF + EPO, respectively. The concentration of neutrophil elastase (NE) rose only in the G-CSF group by a factor 1.5. CONCLUSION: The alteration of G-CSF and NE levels as well as the expression of tissue committed RNA after the administration of EPO in addition to G-CSF indicate that different growth factors mobilize different stem cells that might potentially be used for the support of tissue repair in future treatment protocols.
Assuntos
Eritropoetina/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Mieloma Múltiplo/terapia , RNA Mensageiro/análise , Antígenos CD34 , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Células Sanguíneas , Estudos de Casos e Controles , Contagem de Células , Citocinas/sangue , Eritropoetina/farmacologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Elastase de Leucócito/sangue , Especificidade de Órgãos , RNA Mensageiro/efeitos dos fármacosRESUMO
BACKGROUND: The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. RESULTS: We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling) to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. CONCLUSION: These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo.
Assuntos
Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Linhagem da Célula , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Humanos , Camundongos , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/genéticaRESUMO
Mesenchymal stem cells (MSCs) have the potential to revolutionize medicine due to their ability to differentiate into specific lineages for targeted tissue repair. Development of materials and cell culture platforms that improve differentiation of either autologous or allogenic stem cell sources into specific lineages would enhance clinical utilization of MCSs. In this study, nanoscale amyloid fibrils were evaluated as substrate materials to encourage viability, proliferation, multipotency, and differentiation of MSCs. Fibrils assembled from the proteins lysozyme or ß-lactoglobulin, with and without chitosan coatings, were deposited on planar mica surfaces. MSCs were cultured and differentiated on fibril-covered surfaces, as well as on unstructured controls and tissue culture plastic. Expression of CD44 and CD90 proteins indicated that multipotency was maintained for all fibrils, and osteogenic differentiation was similarly comparable among all tested materials. MSCs grown for 7days on fibril-covered surfaces favored multicellular spheroid formation and demonstrated a >75% increase in adipogenesis compared to tissue culture plastic controls, although this benefit could only be achieved if MSCs were transferred to TCP for the final differentiation step. The largest spheroids and greatest tendency to undergo adipogenesis was evidenced among MSCs grown on fibrils coated with the positively-charged polysaccharide chitosan, suggesting that spheroid formation is prompted by both topography and cell-surface interactivity and that there is a connection between multicellular spheroid formation and adipogenesis.
Assuntos
Células-Tronco Mesenquimais , Adipogenia , Amiloide , Diferenciação Celular , Células Cultivadas , Quitosana , Humanos , OsteogêneseRESUMO
Tissue engineering technology platforms constitute a unique opportunity to integrate cells and extracellular matrix (ECM) proteins into scaffolds and matrices that mimic the natural microenvironment in vitro. The development of tissue-engineered 3D models that mimic the endosteal microenvironment enables researchers to discover the causes and improve treatments for blood and immune-related diseases. The aim of this study was to establish a physiologically relevant in vitro model using 3D printed scaffolds to assess the contribution of human cells to the formation of a construct that mimics human endosteum. Melt electrospun written scaffolds were used to compare the suitability of primary human osteoblasts (hOBs) and placenta-derived mesenchymal stem cells (plMSCs) in (non-)osteogenic conditions and with different surface treatments. Using osteogenic conditions, hOBs secreted a dense ECM with enhanced deposition of endosteal proteins, such as fibronectin and vitronectin, and osteogenic markers, such as osteopontin and alkaline phosphatase, compared to plMSCs. The expression patterns of these proteins were reproducibly identified in hOBs derived from three individual donors. Calcium phosphate-coated scaffolds induced the expression of osteocalcin by hOBs when maintained in osteogenic conditions. The tissue-engineered endosteal microenvironment supported the growth and migration of primary human haematopoietic stem cells (HSCs) when compared to HSCs maintained using tissue culture plastic. This 3D testing platform represents an endosteal bone-like tissue and warrants future investigation for the maintenance and expansion of human HSCs. STATEMENT OF SIGNIFICANCE: This work is motivated by the recent interest in melt electrospinning writing, a 3D printing technique used to produce porous scaffolds for biomedical applications in regenerative medicine. Our team has been among the pioneers in building a new class of melt electrospinning devices for scaffold-based tissue engineering. These scaffolds allow structural support for various cell types to invade and deposit their own ECM, mimicking a characteristic 3D microenvironment for experimental studies. We used melt electrospun written polycaprolactone scaffolds to develop an endosteal bone-like tissue that promotes the growth of HSCs. We combine tissue engineering concepts with cell biology and stem cell research to design a physiologically relevant niche that is of prime interest to the scientific community.
Assuntos
Materiais Biomiméticos/síntese química , Substitutos Ósseos/síntese química , Galvanoplastia/métodos , Matriz Extracelular/química , Células-Tronco Hematopoéticas/citologia , Alicerces Teciduais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Desenho de Equipamento , Feminino , Células-Tronco Hematopoéticas/fisiologia , Temperatura Alta , Humanos , Masculino , Impressão Tridimensional , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
Current stem cell protocols for ischemic heart disease are limited by the small numbers of cells that can be obtained by bone marrow aspirate. To increase myocardial delivery of bone marrow stem cells in patients with chronic ischemic heart disease (CIHD), we used granulocyte colony stimulating factor (G-CSF) for bone marrow mobilization of CD34+ cells, enabling intracoronary infusion of large numbers of CD34+ stem cells. Patients with CIHD (n = 5) demonstrated significantly reduced numbers of CD34+ cells mobilized by G-CSF in comparison to age-matched controls. Sustained reduction in anginal symptoms and improvement in quality of life scores was seen in all patients following infusion of cells. Moreover, mean collateral flow grade at 12-month follow-up angiography significantly improved, indicating sustained myocardial neovascularization. No proliferative retinopathy was induced and no in-stent restenosis seen. However, in two patients with documented increase in collateral flow, complications arose, one developing an acute coronary syndrome and the other a lentigo maligna. These results demonstrate the feasibility of G-CSF mobilization, leukapheresis and intracoronary transfer of CD34+ stem cells in patients with CIHD, but longer-term studies are required to ensure that this protocol is safe and effective.
Assuntos
Antígenos CD34/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Mobilização de Células-Tronco Hematopoéticas , Isquemia Miocárdica/terapia , Idoso , Angioplastia Coronária com Balão , Circulação Colateral , Angiografia Coronária , Feminino , Seguimentos , Humanos , Leucaférese , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Tomografia Computadorizada de Emissão de Fóton Único , Função Ventricular Esquerda , Remodelação VentricularRESUMO
The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Mesonefro/citologia , Mesonefro/embriologia , Neovascularização Fisiológica/fisiologia , Aorta/citologia , Aorta/embriologia , Aorta/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Gônadas/citologia , Gônadas/embriologia , Gônadas/crescimento & desenvolvimento , Células-Tronco Hematopoéticas/fisiologia , Humanos , Mesonefro/crescimento & desenvolvimentoRESUMO
INTRODUCTION: Recent studies in the literature have highlighted the critical role played by cell signalling in determining haemopoietic stem cell (HSC) fate within ex vivo culture systems. Stimulatory signals can enhance proliferation and promote differentiation, whilst inhibitory signals can significantly limit culture output. METHODS: Numerical models of various mitigation strategies are presented and applied to determine effectiveness of these strategies toward mitigation of paracrine inhibitory signalling inherent in these culture systems. The strategies assessed include mixing, media-exchange, fed-batch and perfusion. RESULTS: The models predict that significant spatial concentration gradients exist in typical cell cultures, with important consequences for subsequent cell expansion. Media exchange is shown to be the most effective mitigation strategy, but remains labour intensive and difficult to scale-up for large culture systems. The fed-batch strategy is only effective at very small Peclet number, and its effect is diminished as the cell culture volume grows. Conversely, mixing is effective at high Peclet number, and ineffective at low Peclet number. The models predict that cell expansion in fed-batch cultures becomes independent of increasing dilution rate, consistent with experimental results previously reported in the literature. In contrast, the models predict that increasing the flow rate in perfused cultures will lead to increased cell expansion, indicating the suitability of perfusion for use as an automated, tunable strategy. The effect of initial cell seeding density is also investigated, with the model showing that perfusion outperforms dilution for all densities considered. CONCLUSIONS: The models predict that the impact of inhibitory signalling in HSC cultures can be mitigated against using media manipulation strategies, with the optimal strategy dependent upon the protein diffusion time-scale relative to the media manipulation time-scale. The key messages from this study can be applied to any complex cell culture scenario where cell-cell interactions and paracrine signalling networks impact upon cell fate and cell expansion.
Assuntos
Células-Tronco Hematopoéticas/citologia , Modelos Biológicos , Comunicação Parácrina , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Comunicação Parácrina/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
A facile method for the synthesis of cell supportive, highly macro-porous hyaluronic acid (HA) hydrogels via cryogelation is presented. Unmodified HA was chemically cross-linked via EDC/NHS zero-length cross-linking at sub-zero temperatures to yield cryogels with high porosity and high pore interconnectivity. The physical properties of the HA cryogels including porosity, average pore size, elasticity and swelling properties were characterised as a function of cryogelation conditions and composition of the precursor solution. The HA cryogels swell extensively in water, with the average porosities observed being ~90% under all conditions explored. The morphology of the cryogels can be controlled, allowing scaffolds with an average pore size ranging from 18 ± 2 to 87 ± 5 µm to be formed. By varying the cross-linking degree and HA concentration, a wide range of bulk elastic properties can be achieved, ranging from ~1 kPa to above 10 kPa. Preliminary cell culture experiments, with NIH 3T3 and HEK 293 cell lines, performed on biochemically modified and unmodified gels show the cryogels support cell proliferation and cell interactions, illustrating the biomedical potential of the platform.