Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells.
Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific,robust,and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of textgreateror=30 AP(+) cells that coexpress OCT4,NANOG,SSEA3,SSEA4,TRA-1-60,and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition,including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly,this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple,reliable,broadly applicable,and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.
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产品类型：
产品号#：
07923
36254
85850
85857
产品名：
Dispase (1 U/mL)
DMEM/F-12 with 15 mM HEPES
mTeSR™1
mTeSR™1
文献
Tan JY et al. (JUL 2013)
Stem cells and development 22 13 1893--1906
Efficient derivation of lateral plate and paraxial mesoderm subtypes from human embryonic stem cells through GSKi-mediated differentiation.
The vertebrae mesoderm is a source of cells that forms a variety of tissues,including the heart,vasculature,and blood. Consequently,the derivation of various mesoderm-specific cell types from human embryonic stem cells (hESCs) has attracted the interest of many investigators owing to their therapeutic potential in clinical applications. However,the need for efficient and reliable methods of differentiation into mesoderm lineage cell types remains a significant challenge. Here,we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) is an essential first step toward efficient generation of the mesoderm. Under chemically defined conditions without additional growth factors/cytokines,short-term GSK inhibitor (GSKi) treatment effectively drives differentiation of hESCs into the primitive streak (PS),which can potentially commit toward the mesoderm when further supplemented with bone morphogenetic protein 4. Further analysis confirmed that the PS-like cells derived from GSKi treatment are bipotential,being able to specify toward the endoderm as well. Our findings suggest that the bipotential,PS/mesendoderm-like cell population exists only at the initial stages of GSK-3 inhibition,whereas long-term inhibition results in an endodermal fate. Lastly,we demonstrated that our differentiation approach could efficiently generate lateral plate (CD34(+)KDR(+)) and paraxial (CD34(-)PDGFRα(+)) mesoderm subsets that can be further differentiated along the endothelial and smooth muscle lineages,respectively. In conclusion,our study presents a unique approach for generating early mesoderm progenitors in a chemically directed fashion through the use of small-molecule GSK-3 inhibitor,which may be useful for future applications in regenerative medicine.
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产品类型：
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
05270
05275
产品名：
mTeSR™1
mTeSR™1
STEMdiff™ APEL™2 培养基
STEMdiff™ APEL™2 培养基
文献
Matsumoto Y et al. (DEC 2013)
Orphanet journal of rare diseases 8 1 190
Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show increased mineralization and cartilage formation.
BACKGROUND: Abnormal activation of endochondral bone formation in soft tissues causes significant medical diseases associated with disability and pain. Hyperactive mutations in the bone morphogenetic protein (BMP) type 1 receptor ACVR1 lead to fibrodysplasia ossificans progressiva (FOP),a rare genetic disorder characterized by progressive ossification in soft tissues. However,the specific cellular mechanisms are unclear. In addition,the difficulty obtaining tissue samples from FOP patients and the limitations in mouse models of FOP hamper our ability to dissect the pathogenesis of FOP.backslashnbackslashnMETHODS: To address these challenges and develop a disease model in a dish"�
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产品类型：
产品号#：
05850
05857
05870
05875
07920
85850
85857
85870
85875
产品名：
ACCUTASE™
mTeSR™1
mTeSR™1
文献
Chen G et al. (DEC 2014)
Cell and tissue banking 15 4 513--21
Monitoring the biology stability of human umbilical cord-derived mesenchymal stem cells during long-term culture in serum-free medium.
Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have an immunosuppressive effect. The biological stability of MSCs in serum-free medium during long-term culture in vitro has not been elucidated clearly. The morphology,immunophenotype and multi-lineage potential were analyzed at passages 3,5,10,15,20,and 25 (P3,P5,P10,P15,P20,and P25,respectively). The cell cycle distribution,apoptosis,and karyotype of human umbilical cord-derived (hUC)-MSCs were analyzed at P3,P5,P10,P15,P20,and P25. From P3 to P25,the three defining biological properties of hUC-MSCs [adherence to plastic,specific surface antigen expression,multipotent differentiation potential] met the standards proposed by the International Society for Cellular Therapy for definition of MSCs. The cell cycle distribution analysis at the P25 showed that the percentage of cells at G0/G1 was increased,compared with the cells at P3 (P textless 0.05). Cells at P25 displayed an increase in the apoptosis rate (to 183 %),compared to those at P3 (P textless 0.01). Within subculture generations 3-20 (P3-P20),the differences between the cell apoptotic rates were not statistically significant (P textgreater 0.05). There were no detectable chromosome eliminations,displacements,or chromosomal imbalances,as assessed by the karyotyping guidelines of the International System for Human Cytogenetic Nomenclature (ISCN,2009). Long-term culture affects the biological stability of MSCs in serum-free MesenCult-XF medium. MSCs can be expanded up to the 25th passage without chromosomal changes by G-band. The best biological activity period and stability appeared between the third to 20th generations.
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文献
Yen J et al. (SEP 2014)
Journal of materials chemistry. B,Materials for biology and medicine 2 46 8098--8105
Enhanced Non-Viral Gene Delivery to Human Embryonic Stem Cells via Small Molecule-Mediated Transient Alteration of Cell Structure.
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.
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Human pluripotent stem cell tools for cardiac optogenetics
It is likely that arrhythmias should be avoided for therapies based on human pluripotent stem cell (hPSC)-derived cardiomyocytes (CM) to be effective. Towards achieving this goal,we introduced light-activated channelrhodopsin-2 (ChR2),a cation channel activated with 480 nm light,into human embryonic stem cells (hESC). By using in vitro approaches,hESC-CM are able to be activated with light. ChR2 is stably transduced into undifferentiated hESC via a lentiviral vector. Via directed differentiation,hESCChR2-CM are produced and subjected to optical stimulation. hESCChR2-CM respond to traditional electrical stimulation and produce similar contractility features as their wild-type counterparts but only hESCChR2-CM can be activated by optical stimulation. Here it is shown that a light sensitive protein can enable in vitro optical control of hESC-CM and that this activation occurs optimally above specific light stimulation intensity and pulse width thresholds. For future therapy,in vivo optical stimulation along with optical inhibition could allow for acute synchronization of implanted hPSC-CM with patient cardiac rhythms.
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产品类型：
产品号#：
85850
85857
产品名：
mTeSR™1
mTeSR™1
文献
Fukuta M et al. (DEC 2014)
PLoS ONE 9 12 e112291
Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media
Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton,cornea,peripheral nervous system,and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs),such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),further modifications are required to improve the robustness,efficacy,and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions,the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons,glia,melanocytes,and corneal endothelial cells. In addition,cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.
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产品类型：
产品号#：
85850
85857
产品名：
mTeSR™1
mTeSR™1
文献
Maldonado M et al. (MAY 2015)
Biomaterials 50 1 10--19
The effects of electrospun substrate-mediated cell colony morphology on the self-renewal of human induced pluripotent stem cells
The development of xeno-free,chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard,various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal,i.e.,proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly,when surface chemistry of the substrates was uniformly controlled by collagen conjugation,the stiffness of substrate was inversely related to the sphericity,a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture,implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall,we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal.
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产品类型：
产品号#：
85850
85857
产品名：
mTeSR™1
mTeSR™1
文献
Akoto C et al. (MAR 2017)
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 47 3 351--360
Mast cells are permissive for rhinovirus replication: potential implications for asthma exacerbations.
BACKGROUND Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations,with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity. OBJECTIVE In view of the emerging role of MCs in innate immunity and increased localization to the asthmatic bronchial epithelium,we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection. METHODS The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-β or IFN-λ. After 24 h,innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay. RESULTS HRV infection of LAD2 MCs induced expression of IFN-β,IFN-λ and IFN-stimulated genes. However,LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralization of the type I IFN receptor had minimal effects on viral shedding,suggesting that endogenous type I IFN signalling offered limited protection against HRV. However,augmentation of these responses by exogenous IFN-β,but not IFN-λ,protected MCs against HRV infection. CONCLUSION AND CLINICAL RELEVANCE MCs are permissive for the replication and release of HRV,which is prevented by exogenous IFN-β treatment. Taken together,these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbations.
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EasySep™小鼠TIL（CD45）正选试剂盒
文献
产品手册PneumaCult™ Culture System for Airway Epithelial Cells
产品手册Standardized Solutions for Mouse Pluripotent Stem Cells
技术公告Protocol for Total RNA Isolation from Cells
