搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Human pluripotent stem cells (hPSCs) - including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) - are very promising candidates for cell therapies,tissue engineering,high throughput pharmacology screens,and toxicity testing. These applications require large numbers of high quality cells; however,scalable production of human pluripotent stem cells and their derivatives at a high density and under well-defined conditions has been a challenge. We recently reported a simple,efficient,fully defined,scalable,and good manufacturing practice (GMP) compatible 3D culture system based on a thermoreversible hydrogel for hPSC expansion and differentiation. Here,we describe additional design rationale and characterization of this system. For instance,we have determined that culturing hPSCs as a suspension in a liquid medium can exhibit lower volumetric yields due to cell agglomeration and possible shear force-induced cell loss. By contrast,using hydrogels as 3D scaffolds for culturing hPSCs reduces aggregation and may insulate from shear forces. Additionally,hydrogel-based 3D culture systems can support efficient hPSC expansion and differentiation at a high density if compatible with hPSC biology. Finally,there are considerable opportunities for future development to further enhance hydrogel-based 3D culture systems for producing hPSCs and their progeny. View Publication

A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover,the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end,a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly,this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide,which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage,over 85% viability,and maintained a normal karyotype and the expression of pluripotency markers such as Nanog,Oct4,and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers,the cells adopted respective fates displaying relevant markers. Lastly,engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence,we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable,xeno-free propagation and differentiation of human stem cells intended for therapies. View Publication

The development of novel platforms for large scale production of human embryonic stem cells (hESC) derived cardiomyocytes (CM) becomes more crucial as the demand for CMs in preclinical trials,high throughput cardio toxicity assays and future regenerative therapeutics rises. To this end,we have designed a microcarrier (MC) suspension agitated platform that integrates pluripotent hESC expansion followed by CM differentiation in a continuous,homogenous process.Hydrodynamic shear stresses applied during the hESC expansion and CM differentiation steps drastically reduced the capability of the cells to differentiate into CMs. Applying vigorous stirring during pluripotent hESC expansion on Cytodex 1 MC in spinner cultures resulted in low CM yields in the following differentiation step (cardiac troponin-T (cTnT): 22.83. ??. 2.56%; myosin heavy chain (MHC): 19.30. ??. 5.31%). Whereas the lower shear experienced in side to side rocker (wave type) platform resulted in higher CM yields (cTNT: 47.50. ??. 7.35%; MHC: 42.85. ??. 2.64%). The efficiency of CM differentiation is also affected by the hydrodynamic shear stress applied during the first 3. days of the differentiation stage. Even low shear applied continuously by side to side rocker agitation resulted in very low CM differentiation efficiency (cTnT. textless. 5%; MHC. textless. 2%). Simply by applying intermittent agitation during these 3. days followed by continuous agitation for the subsequent 9. days,CM differentiation efficiency can be substantially increased (cTNT: 65.73. ??. 10.73%; MHC: 59.73. ??. 9.17%). These yields are 38.3% and 39.3% higher (for cTnT and MHC respectively) than static culture control.During the hESC expansion phase,cells grew on continuously agitated rocker platform as pluripotent cell/MC aggregates (166??88??105??m2) achieving a cell concentration of 3.74??0.55??106cells/mL (18.89??2.82 fold expansion) in 7days. These aggregates were further differentiated into CMs using a WNT modulation differentiation protocol for the subsequent 12days on a rocking platform with an intermittent agitation regime during the first 3days. Collectively,the integrated MC rocker platform produced 190.5??58.8??106 CMs per run (31.75??9.74 CM/hESC seeded). The robustness of the system was demonstrated by using 2 cells lines,hESC (HES-3) and human induced pluripotent stem cell (hiPSC) IMR-90. The CM/MC aggregates formed extensive sarcomeres that exhibited cross-striations confirming cardiac ontogeny. Functionality of the CMs was demonstrated by monitoring the effect of inotropic drug,Isoproterenol on beating frequency.In conclusion,we have developed a simple robust and scalable platform that integrates both hESC expansion and CM differentiation in one unit process which is capable of meeting the need for large amounts of CMs. ?? 2014. View Publication

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of textgreater10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb,and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells. View Publication

During drug development,measurement of suitable pharmacodynamic biomarkers is key to establishing in vivo drug activity. Binding of monoclonal antibody (mAb) therapeutics to soluble target proteins often results in elevated serum levels of their target antigen,and measuring total (free and bound) concentration of the target antigen can be an important means of demonstrating that the mAb has reached its specific target. However,accurately measuring soluble circulating antigen in preclinical or clinical samples in the presence of a therapeutic mAb presents a bioanalytical challenge. Particularly in the case of low molecular weight and/or multimeric targets,epitopes for capture and detection of the target by reagent antibodies can be obscured by bound therapeutic mAb. Lymphotoxin-alpha (LTα) is a cytokine in the TNF superfamily that has been implicated in the pathophysiology of autoimmune disease,and is a therapeutic target for neutralizing mAb. During preclinical safety studies in cynomolgus macaques,we encountered difficulties in measuring total LTα in serum of dosed animals. When serum LTα trimer was saturated with the anti-LTα mAb,binding of two reagent antibodies,as required for a classic sandwich ELISA,was not feasible,and dissociation methods were also found to be unsuitable. We therefore developed an approach in which excess anti-LTα mAb was added to the in vitro assay system to fully saturate all binding sites,and an anti-idiotypic antibody was used to detect bound therapeutic antibody. Using this method,total LTα could be accurately measured in cynomolgus macaque serum,and was observed to increase with increasing anti-LTα therapeutic mAb dose. Additional in vitro studies demonstrated that the method worked equally well in human serum. This assay strategy will be useful for quantifying total concentrations of other small and/or multimeric target proteins in the presence of a therapeutic antibody. View Publication

OBJECTIVE: The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study,human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. METHODS: Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). RESULTS: Both transcription factors were expressed efficiently in hES cells expressing either PAX8,NKX2-1,or in combination in the hES cells,which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes,including thyroglobulin (TG),thyroid peroxidase (TPO),the sodium/iodide symporter (NIS),and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably,the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH,these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake,indicating functional thyroid epithelial cells. CONCLUSION: The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be committed to thyroid cell speciation under appropriate conditions. View Publication

Non-viral gene delivery into human embryonic stem cells (hESCs)is an important tool for controlling cell fate. However,the delivery efficiency remains low due in part to the tight colony structure of the cells which prevents effective exposure towards delivery vectors. We herein report a novel approach to enhance non-viral gene delivery to hESCs by transiently altering the cell and colony structure. (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632),a small molecule that inhibits the rho-associated protein kinase pathway,is utilized to induce transient colony spreading which leads to increased transfection efficiency by 1.5 to 2 folds in a spectrum of non-viral transfection reagents including Lipofectamine 2000 and Fugene HD. After removal of Y-27632 post-transfection,cells can revert back to its normal state and do not show alteration of pluripotency. This approach provides a simple,effective tool to enhance non-viral gene delivery into adherent hESCs for genetic manipulation. View Publication

Improvement of methods to produce endoderm-derived cells from pluripotent stem cells is important to realize high-efficient induction of endodermal tissues such as pancreas and hepatocyte. Difficulties hampering such efforts include the low efficiency of definitive endoderm cell induction and establishing appropriate defined culture conditions to ensure a safe cell source for human transplantation. Based on previous studies,we revised the experimental condition of definitive endoderm induction in feeder- and serum-free culture. Our results suggested that CHIR99021 is more effective than Wnt3A ligand in feeder- and serum-free conditions. In addition,keeping cell density low during endoderm induction is important for the efficiency. On the other hand,we showed that overtreatment with CHIR99021 converted the cells into BRACHYURY-expressing posterior mesoderm cells rather than endoderm,indicating strict CHIR99021 treatment requirements for endoderm differentiation. Nevertheless,these results should enable better control in the production of definitive endoderm-derived cells. View Publication

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugate-based vaccines,the prevalence of antibiotic-resistant pneumococcal strains,serotype (ST) replacement in nonconjugate vaccine strains,and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study,we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs,including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC,were produced,and their nucleotide sequences,epitope and fine specificity,and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V(H)) and light (V(L)) chain genes,with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V(H) gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy. View Publication

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development,we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm,then into replicating Nkx2.1+ lung endoderm,and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP,FGF,and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs),creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice. View Publication

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive,low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics. The combination of BD FACS Aria Cell Sorter,CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation,production host cells are first transfected with an expression vector carrying the gene of interest (1),followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody,and are subsequently subject to flow cytometry analysis (2-4). Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed,these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup. This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes,the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure,this automated platform demonstrated advantages of significantly increased capacity,ensured clonality,traceability in cell line history with electronic documentation and much reduced opportunity in operator error. View Publication

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease,White Nose Syndrome,while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study,we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence,the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most,if not all,secreted immunoglobulins utilized lambda light chains. Finally,mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence,this reagent may have both basic and practical applications for studying immune process. View Publication