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OBJECTIVES: Cerebral palsy (CP) remains the world's most common childhood physical disability with total annual costs of care and lost well-being of $A3.87b. The PREDICT-CP (NHMRC 1077257 Partnership Project: Comprehensive surveillance to PREDICT outcomes for school age children with CP) study will investigate the influence of brain structure, body composition, dietary intake, oropharyngeal function, habitual physical activity, musculoskeletal development (hip status, bone health) and muscle performance on motor attainment, cognition, executive function, communication, participation, quality of life and related health resource use costs. The PREDICT-CP cohort provides further follow-up at 8-12 years of two overlapping preschool-age cohorts examined from 1.5 to 5 years (NHMRC 465128 motor and brain development; NHMRC 569605 growth, nutrition and physical activity). METHODS AND ANALYSES: This population-based cohort study undertakes state-wide surveillance of 245 children with CP born in Queensland (birth years 2006-2009). Children will be classified for Gross Motor Function Classification System; Manual Ability Classification System, Communication Function Classification System and Eating and Drinking Ability Classification System. Outcomes include gross motor function, musculoskeletal development (hip displacement, spasticity, muscle contracture), upper limb function, communication difficulties, oropharyngeal dysphagia, dietary intake and body composition, participation, parent-reported and child-reported quality of life and medical and allied health resource use. These detailed phenotypical data will be compared with brain macrostructure and microstructure using 3 Tesla MRI (3T MRI). Relationships between brain lesion severity and outcomes will be analysed using multilevel mixed-effects models. ETHICS AND DISSEMINATION: The PREDICT-CP protocol is a prospectively registered and ethically accepted study protocol. The study combines data at 1.5-5 then 8-12 years of direct clinical assessment to enable prediction of outcomes and healthcare needs essential for tailoring interventions (eg, rehabilitation, orthopaedic surgery and nutritional supplements) and the projected healthcare utilisation. TRIAL REGISTRATION NUMBER: ACTRN: 12616001488493.
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Parálisis Cerebral , Vigilancia de la Población , Parálisis Cerebral/diagnóstico , Niño , Estudios de Cohortes , Humanos , Pronóstico , Proyectos de InvestigaciónRESUMEN
Human placenta is rich in mesenchymal stem/stromal cells (MSC), with their origin widely presumed fetal. Cultured placental MSCs are confounded by a high frequency of maternal cell contamination. Our recent systematic review concluded that only a small minority of placental MSC publications report fetal/maternal origin, and failed to discern a specific methodology for isolation of fetal MSC from term villi. We determined isolation conditions to yield fetal and separately maternal MSC during ex vivo expansion from human term placenta. MSCs were isolated via a range of methods in combination; selection from various chorionic regions, different commercial media, mononuclear cell digest and/or explant culture. Fetal and maternal cell identities were quantitated in gender-discordant pregnancies by XY chromosome fluorescence in situ hybridization. We first demonstrated reproducible maternal cell contamination in MSC cultures from all chorionic anatomical locations tested. Cultures in standard media rapidly became composed entirely of maternal cells despite isolation from fetal villi. To isolate pure fetal cells, we validated a novel isolation procedure comprising focal dissection from the cotyledonary core, collagenase/dispase digestion and explant culture in endothelial growth media that selected, and provided a proliferative environment, for fetal MSC. Comparison of MSC populations within the same placenta confirmed fetal to be smaller, more osteogenic and proliferative than maternal MSC. We conclude that in standard media, fetal chorionic villi-derived MSC (CV-MSC) do not grow readily, whereas maternal MSC proliferate to result in maternal overgrowth during culture. Instead, fetal CV-MSCs require isolation under specific conditions, which has implications for clinical trials using placental MSC. Stem Cells Translational Medicine 2017;6:1070-1084.
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Vellosidades Coriónicas , Células Madre Fetales/citología , Células Madre Mesenquimatosas/citología , Placenta/citología , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Humanos , EmbarazoRESUMEN
Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.
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Células Madre Mesenquimatosas , Placenta , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Femenino , Feto , Humanos , EmbarazoRESUMEN
Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues.
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Analysis of archival samples from cohorts of pregnant women may be key to discovering prognosticators of stillbirth and pregnancy/perinatal complications. Growth hormone (GH) and its receptor (GHR) are pivotal in feto-placental development and pregnancy maintenance. We report a rapid, optimized method for genotyping the GHR full-length versus exon 3-deleted isoform (GHRd3). TaqMan single nucleotide polymorphism (SNP) genotyping proved superior to standard multiplex polymerase chain reaction (PCR) in allele detection and GHR genotyping from archived samples, including those with poor genomic deoxyribonucleic acid quality/quantity such as formalin fixed, paraffin embedded, blood, and serum. Furthermore, this assay is suitable for high through put 96 or 384-well plate quantitative PCR machines with automated genotype calling software. The TaqMan genotyping assay can increase the data obtained from precious archival human samples.
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Proteínas Portadoras/genética , Placenta/química , Adulto , Australia/epidemiología , Células Cultivadas , Estudios de Cohortes , Exones , Femenino , Sangre Fetal/química , Eliminación de Gen , Frecuencia de los Genes , Genotipo , Humanos , Desequilibrio de Ligamiento , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple/genética , Embarazo , Fijación del TejidoRESUMEN
BACKGROUND: Fetal mesenchymal stem/stromal cells (MSC) represent a developmentally-advantageous cell type with translational potential.To enhance adult MSC migration, studies have focussed on the role of the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12), but more recent work implicates an intricate system of CXCR4 receptor dimerization, intracellular localization, multiple ligands, splice variants and nuclear accumulation. We investigated the intracellular localization of CXCR4 in fetal bone marrow-derived MSC and role of intracellular trafficking in CXCR4 surface expression and function. RESULTS: We found that up to 4% of human fetal MSC have detectable surface-localized CXCR4. In the majority of cells, CXCR4 is located not at the cell surface, as would be required for 'sensing' migratory cues, but intracellularly. CXCR4 was identified in early endosomes, recycling endosomes, and lysosomes, indicating only a small percentage of CXCR4 travelling to the plasma membrane. Notably CXCR4 was also found in and around the nucleus, as detected with an anti-CXCR4 antibody directed specifically against CXCR4 isoform 2 differing only in N-terminal sequence. After demonstrating that endocytosis of CXCR4 is largely independent of endogenously-produced SDF-1, we next applied the cytoskeletal inhibitors blebbistatin and dynasore to inhibit endocytotic recycling. These increased the number of cells expressing surface CXCR4 by 10 and 5 fold respectively, and enhanced the number of cells migrating to SDF1 in vitro (up to 2.6 fold). These molecules had a transient effect on cell morphology and adhesion, which abated after the removal of the inhibitors, and did not alter functional stem cell properties. CONCLUSIONS: We conclude that constitutive endocytosis is implicated in the regulation of CXCR4 membrane expression, and suggest a novel pharmacological strategy to enhance migration of systemically-transplanted cells.
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Endocitosis , Feto/citología , Células Madre Mesenquimatosas/citología , Receptores CXCR4/análisis , Receptores CXCR4/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Movimiento Celular , Núcleo Celular/metabolismo , Células Cultivadas , Endosomas/metabolismo , Femenino , Humanos , Lisosomas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Transporte de ProteínasRESUMEN
Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors.
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Janus Quinasa 2/metabolismo , Receptores de Somatotropina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cisteína/química , Activación Enzimática , Células HEK293 , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Somatotropina/química , Receptores de Somatotropina/genéticaRESUMEN
Early fetal and placental MSCs have translationally-advantageous characteristics compared to later pregnancy MSCs. During the first trimester, the fetus and placenta grow rapidly with divergent developmental requirements, but studies comparing mesenchymal stem cells (MSCs) from different origins have paid little attention to the effect of gestational age over this temporal window. Here we present the transcriptome through first trimester development of MSC isolated from fetal bone marrow (BM) or placental structures. Samples were collected weekly from 8 to 12 weeks. The raw microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-1224. Additionally, the data have been integrated into the stem cell collaboration platform www.Stemformatics.org. These data provide a valuable resource for developmental biology and stem cell investigation.
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The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-ß pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.
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Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Antígenos CD/genética , Antígenos CD/metabolismo , Benzamidas/farmacología , Biomarcadores/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Proliferación Celular , Forma de la Célula , Células Cultivadas , Medio de Cultivo Libre de Suero , Dioxoles/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Transición Epitelial-Mesenquimal , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunofenotipificación , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Medicina Regenerativa/métodos , Factores de Tiempo , Vimentina/genética , Vimentina/metabolismoRESUMEN
Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.
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Células de la Médula Ósea/citología , Inmunofenotipificación/métodos , Riñón/citología , Células Madre Mesenquimatosas/citología , Miocardio/citología , Transcriptoma/genética , Animales , Células de la Médula Ósea/metabolismo , Forma de la Célula , Ensayo de Unidades Formadoras de Colonias , Epítopos/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes/genética , Terapia de Inmunosupresión , Células Madre Mesenquimatosas/metabolismo , Ratones , Especificidad de Órganos/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Clinical grade human mesenchymal stem cells (MSC) are manufactured and used in clinical trials for a range of regenerative and inflammatory diseases. Human MSC have now been derived from tissues other than bone marrow, such as placenta, as described in this laboratory protocol. It provides instructions for clinical grade MSC manufacturing according to the Code of Good Manufacturing Practice (cGMP) principles and according to policies and procedures of our internal Quality Management System (QMS), which is based on the International Organization for Standardization (ISO) standard requirements. Relevant organizational structure and QMS elements are presented.
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Materiales Manufacturados/normas , Células Madre Mesenquimatosas/citología , Células Madre Multipotentes/citología , Placenta/citología , Animales , Técnicas de Cultivo de Célula , Núcleo Celular/metabolismo , Separación Celular , Criopreservación , Femenino , Citometría de Flujo , Humanos , Fenotipo , Embarazo , Etiquetado de Productos , EsterilizaciónRESUMEN
Mesenchymal stem cells (MSC) are being used increasingly in clinical trials for a range of regenerative and inflammatory diseases. Bone marrow is the traditional source but is relatively inaccessible in large volume. MSC have now been derived from tissues other than bone marrow including placenta and adipose tissue. We have used placenta obtained after delivery as a source of MSC and have been unable to detect any marked differences from marrow-derived MSC in terms of cell surface phenotype, chemokine receptor display, mesodermal differentiation capacity or immunosuppressive ability. This report described our manufacturing process for isolating and expanding placenta-derived human MSC and their safe infusion into the first patient in a clinical trial program of human placenta-derived MSC.
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Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Placenta/citología , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Separación Celular/métodos , Criopreservación/métodos , Resultado Fatal , Femenino , Citometría de Flujo/métodos , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/terapia , Masculino , Células Madre Mesenquimatosas/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
Bone marrow is the traditional source of human multipotent mesenchymal stem cells (MSCs), but placenta appears to be an alternative and more readily available source. This study comprehensively compared human placenta-derived MSC (hpMSC) and human bone marrow-derived MSC (hbmMSC) in terms of cell characteristics, optimal growth conditions and in vivo safety specifically to determine if hpMSC could represent a source of human MSC for clinical trial. MSC were isolated from human placenta (hpMSC) and human bone marrow (hbmMSC) and expanded ex vivo using good manufacturing practice-compliant reagents. hpMSC and hbmMSC showed similar proliferation characteristics in different basal culture media types, fetal calf serum (FCS) concentrations, FCS heat-inactivation experiments, flask types and media replacement responsiveness. However, hpMSC and hbmMSC differed with respect to their proliferation capabilities at different seeding densities, with hbmMSC proliferating more slowly than hpMSC in every experiment. hpMSC had greater long-term growth ability than hbmMSC. MSC from both sources exhibited similar light microscopy morphology, size, cell surface phenotype, and mesodermal differentiation ability with the exception that hpMSC consistently appeared less able to differentiate to the adipogenic lineage. A comparison of both hbmMSC and hpMSC from early and medium passage cultures using single-nucleotide polymorphism (SNP) GeneChip analysis confirmed GTG-banding data that no copy number changes had been acquired during sequential passaging. In three of three informative cases (in which the gender of the delivered baby was male), hpMSC were of maternal origin. Neither hpMSC nor hbmMSC caused any acute toxicity in normal mice when injected intravenously at the same, or higher, doses than those currently used in clinical trials of hbmMSC. This study suggests that human placenta is an acceptable alternative source for human MSC and their use is currently being evaluated in clinical trials.
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Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Proliferación Celular , Células Madre Mesenquimatosas/citología , Células Madre Multipotentes/citología , Placenta/citología , Adulto , Animales , Células de la Médula Ósea/metabolismo , Separación Celular/métodos , Células Cultivadas , Femenino , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones , Células Madre Multipotentes/metabolismo , Placenta/metabolismo , Factores de TiempoRESUMEN
SUMMARY: Therapeutic applications of cells are likely to increase greatly in the future. Cell and cell-based gene therapy manufacturing facilities need to be purpose-designed and accredited by their national medicinal regulatory body. Production scientists need to work in close tandem with quality assurance and ethics committees to absolutely ensure the safety of new cellular products. In this review, we consider the need for preclinical safety and efficacy data, tissue source for manufacture of clinical grade human mesenchymal stem cells, aseptic tissue processing, indemnification, and the role of the national medicinal regulatory body in appropriate clinical trial design.
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Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. They have been demonstrated to play a role in tissue repair and regeneration in both preclinical and clinical studies. They also have remarkable immunosuppressive properties. We describe their application in settings that include the cardiovascular, central nervous, gastrointestinal, renal, orthopaedic and haematopoietic systems. Manufacturing of MSC for clinical trials is also discussed. Since tissue matching between MSC donor and recipient does not appear to be required, MSC may be the first cell type able to be used as an "off-the-shelf" therapeutic product.
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Trasplante de Células Madre Mesenquimatosas/métodos , Células del Estroma/citología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Células Madre Multipotentes/citología , Células Madre Multipotentes/fisiología , Regeneración , Células del Estroma/fisiología , Células del Estroma/trasplanteRESUMEN
Growth hormone (GH) is a major regulator of postnatal growth and metabolism. There are extensive clinical applications for GH or its antagonists, including treatments for dwarfism, cancer and metabolic wasting. Owing to this, there is considerable interest in the mechanisms of GH receptor (GHR) activation. It is conventionally thought that GH induces dimerization of two GHR monomers, which initiates intracellular signaling cascades. However, recent studies have provided evidence for a ligand-induced conformational change within constitutively dimerized GHRs being responsible for activating signaling pathways. This review will relate the new model of GHR activation to the activation of related cytokine receptors and discuss the implication of this new model for the design of small GH mimetics and antagonists for therapeutic use.
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Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.