RESUMEN
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.
Asunto(s)
Plaquetas/citología , Técnicas de Cultivo de Célula , Megacariocitos/citología , Transfusión de Plaquetas/normas , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/inmunología , Plaquetas/inmunología , Desdiferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Citocinas/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Silenciador del Gen , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/inmunología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Megacariocitos/efectos de los fármacos , Megacariocitos/inmunología , Microfluídica/instrumentación , Microfluídica/métodos , Transfusión de Plaquetas/estadística & datos numéricosRESUMEN
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.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Osteopontina/fisiología , Animales , Secuencia de Bases , Células CHO , Línea Celular , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Cricetinae , Cricetulus , Cartilla de ADN/genética , Sangre Fetal/citología , Expresión Génica , Factor Estimulante de Colonias de Granulocitos/farmacología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Técnicas In Vitro , Integrina alfa4beta1/genética , Integrinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Osteopontina/deficiencia , Osteopontina/genética , Osteopontina/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/metabolismoRESUMEN
Ventilator-associated pneumonia (VAP) is a highly common hospital-acquired infection affecting people that require mechanical ventilation. The endotracheal tube (ETT) used during the ventilation process provides a surface that can allow bacterial colonization and biofilm formation, which can lead to VAP. Although various approaches, including ETT design and material selection, as well as antimicrobial coatings have been employed to minimize adverse events, VAP remains a significant unresolved clinical issue. In this study, we have utilized a novel styrylbenzene-based antimicrobial (BCP3) in a simple and robust coating that allows its long-term release at an effective level. BCP3 was applied onto PVC ETT segments blended together with poly(lactic-co-glycolic acid) via a facile dip-coating process with controlled loadings. In vitro studies demonstrated concentration-dependent release of BCP3 from the coatings for at least 31 days. Bacterial assays using major VAP culprits, Staphylococcus aureus and Pseudomonas aeruginosa, demonstrated significant growth inhibition, with a stronger effect on S. aureus. Despite its ability to inhibit bacterial growth, BCP3 showed no cytotoxicity toward mammalian (L929) fibroblasts, which makes it attractive from a clinical perspective. The coating procedure was successfully translated to coat the entire ETTs, making it highly amenable for large-scale manufacturing.
RESUMEN
Compared to soluble cytokines, surface-tethered ligands can deliver biological signalling with precise control of spatial positioning and concentration. A strategy that immobilises ligand molecules on a surface in a uniform orientation using non-cleavable linkages under physiological conditions would enhance the specific and systemic delivery of signalling in the local environment. We used mixed self-assembled monolayers (SAMs) of oxyamine- and oligo(ethylene glycol)-terminated thiols on gold to covalently install aldehyde- or ketone-functionalised ligands via oxime conjugation. Characterisation by electrochemistry and X-ray photoelectron spectroscopy showed quantitative immobilisation of the ligands on SAM surfaces. The thrombopoietin mimetic peptide, RILL, was immobilised on SAMs and the bioactivity of the substrate was demonstrated by culturing factor-dependent cells. We also optimised the immobilisation and wash conditions so that the peptide was not released into the culture medium and the immobilised RILL could be re-used for consecutive cell cultures. The surface also supported the growth of haematopoietic CD34+ cells comparable to the standard thrombopoietin-supplemented culture. Furthermore, the RILL-immobilised SAM surface was as effective in expanding uncommitted CD34+ cells as standard culture. The stimulatory effect of surface-tethered ligands in haematopoietic stem cell expansion supports the use of ligand immobilisation strategies to replicate the haematopoietic stem cell niche.
Asunto(s)
Antígenos CD34/metabolismo , Proteínas Inmovilizadas/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas Electroquímicas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Ligandos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Espectroscopía de Fotoelectrones , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
Several growth factors feature prominently in the control of hematopoiesis. Thrombopoietin, a classâ I hematopoietic cytokine, plays critical roles in regulating hematopoietic stem cell numbers and also stimulates the production and differentiation of megakaryocytes, the bone marrow cells that ultimately produce platelets. Thrombopoietin interacts with the c-Mpl cell-surface receptor. Recently, several peptide and small-molecule agonists and antagonists of c-Mpl have been reported. We conducted a bioinformatics and molecular modeling study aimed at understanding the agonist activities of peptides that bind to c-Mpl, and developed new potent peptide agonists with low nanomolar activity. These agonists also show very high activity in human CD34(+) primary cell cultures, and doubled the mean blood platelet counts when injected into mice.