HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells
Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However,generation of human AID cell lines is challenging,especially in human embryonic stem cells (hESCs). Here,we develop HiHo-AID2,a streamlined procedure for rapid,one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines,including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03187-w.
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产品类型：
产品号#：
100-0276
100-1130
产品名：
mTeSR™ Plus
mTeSR™ Plus
M. Dastpak et al. (Dec 2025)
PLOS One 20 12
SF3B1K700E mutation in human embryonic stem cells causes aberrant expression of immune-related genes
SF3B1,a component of the U2 snRNP pre-mRNA splicing factor,plays a critical role in splicing and is frequently mutated in cancer,particularly hematologic malignancies. We investigated the effects of the most common SF3B1 mutation,heterozygous substitution of Lysine 700 to Glutamate (K700E),in human embryonic stem cells (hESC),using CRISPR-Cas9 to generate heterozygous SF3B1K700E clones. Interestingly,we observed the upregulation of several key transcription regulators associated with hematopoiesis and a broad range of immune genes in SF3B1K700E hESCs. Despite differences in the transcriptional and splicing profiles between hESC and myelodysplastic syndrome (MDS) cells harboring the SF3B1K700E mutation,several common immune gene programs were identified in both cell types. To elucidate the molecular mechanisms underlying dysregulated gene expression in SF3B1K700E hESCs,we mapped actively engaged RNA polymerase II (RNA Pol II) using Precision Run-On sequencing (PRO-seq). These analyses revealed that the SF3B1K700E mutation alters RNA Pol II elongation properties. Specifically,we observed a general increase in pause release in SF3B1K700E hESCs,consistent with recent work in leukemia cells suggesting that the SF3B1K700E mutation affects early transcription elongation. Taken together,our study identifies several candidate genes that could contribute to the SF3B1 mutated phenotype and clarifies the role for the U2 snRNP and pre-spliceosome assembly on transcription by RNA Pol II. Further,our data suggest that mutations of SF3B1 impact immune gene expression independent of cell type,providing new insights into the role of SF3B1K700E in hematologic malignancies.
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Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.
Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However,upgrading them to pluripotency confers refractoriness toward senescence,higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling,such as Down syndrome or $\$-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing,feeder-dependent culture. Here,we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium,a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4,Nanog,Sox2,SSEA-1,SSEA-4,TRA-1-60,TRA-1-81 in a pattern typical for human primed PSC. Additionally,the cells formed teratomas,and were deemed pluripotent by PluriTest,a global expression microarray-based in-silico pluripotency assay. However,we found that the PluriTest scores were borderline,indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology,non-integrating reprogramming and chemically defined culture are more acceptable.
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05850
05857
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05940
07180
07183
07190
27147
07191
07930
07931
07940
07955
07956
07959
07954
85850
85857
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100-1061
07952
100-0763
产品名：
Vitronectin XF™
CellAdhere™ 稀释缓冲液
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
mTeSR™1
mTeSR™1
CryoStor® CS10
CryoStor® CS10
Vitronectin XF™
Zielske SP et al. (NOV 2003)
The Journal of clinical investigation 112 10 1561--70
In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning.
Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo,selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study,we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment,at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore,99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR,compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice,demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity,and CFUs were stained intensely for AGT protein,indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge,these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.
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产品类型：
产品号#：
04434
04444
产品名：
MethoCult™ H4434 Classic
MethoCult™ H4434 Classic
Gupta R et al. (MAY 2012)
Molecular endocrinology (Baltimore,Md.) 26 5 859--72
Squelching of ETS2 transactivation by POU5F1 silences the human chorionic gonadotropin CGA subunit gene in human choriocarcinoma and embryonic stem cells.
The subunit genes encoding human chorionic gonadotropin,CGA,and CGB,are up-regulated in human trophoblast. However,they are effectively silenced in choriocarcinoma cells by ectopically expressed POU domain class 5 transcription factor 1 (POU5F1). Here we show that POU5F1 represses activity of the CGA promoter through its interactions with ETS2,a transcription factor required for both placental development and human chorionic gonadotropin subunit gene expression,by forming a complex that precludes ETS2 from interacting with the CGA promoter. Mutation of a POU5F1 binding site proximal to the ETS2 binding site does not alter the ability of POU5F1 to act as a repressor but causes a drop in basal promoter activity due to overlap with the binding site for DLX3. DLX3 has only a modest ability to raise basal CGA promoter activity,but its coexpression with ETS2 can up-regulate it 100-fold or more. The two factors form a complex,and both must bind to the promoter for the combination to be transcriptionally effective,a synergy compromised by POU5F1. Similarly,in human embryonic stem cells,which express ETS2 but not CGA,ETS2 does not occupy its binding site on the CGA promoter but is found instead as a soluble complex with POU5F1. When human embryonic stem cells differentiate in response to bone morphogenetic protein-4 and concentrations of POU5F1 fall and hCG and DLX3 rise,ETS2 then occupies its binding site on the CGA promoter. Hence,a squelching mechanism underpins the transcriptional silencing of CGA by POU5F1 and could have general relevance to how pluripotency is maintained and how the trophoblast lineage emerges from pluripotent precursor cells.
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产品类型：
产品号#：
05850
05857
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85850
85857
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85875
产品名：
mTeSR™1
mTeSR™1
E. Yamashita et al. (Sep 2025)
The FASEB Journal 39 17
Red Blood Cell‐Mediated Enhancement of Hematopoietic Stem Cell Functions via a Hes1‐Dependent Pathway
In bone marrow,cell numbers are balanced between production and loss. After chemotherapy,blood cell counts decrease initially but later recover as hematopoietic progenitor cells expand,although the mechanisms underlying this recovery are still unclear. We investigated the influence of red blood cells (RBCs) on hematopoietic stem cell (HSC) function during bone marrow recovery. Following chemotherapy,RBC concentrations in bone marrow peaked on day 5 posttreatment,coinciding with the recovery of hematopoiesis. Coculture of HSCs with RBCs resulted in a significant increase in hematopoiesis. Direct contact between RBCs and HSCs was essential for enhancement of hematopoiesis,and HSCs precultured with RBCs resulted in greater numbers of donor‐derived mature hematopoietic cells after transplantation. RNA‐sequencing analysis showed that Hes1 was the most significantly upregulated transcription factor in RBC coculture,and the response to RBC‐induced hematopoiesis of Hes1‐deficient HSCs was reduced. These findings imply a role of RBCs and Hes1 in the enhancement of hematopoietic recovery following bone marrow stress.
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产品类型：
产品号#：
03436
产品名：
MethoCult™ SF M3436
Diniz B et al. (JUL 2013)
Investigative Ophthalmology and Visual Science 54 7 5087--5096
Subretinal Implantation of Retinal Pigment Epithelial Cells Derived From Human Embryonic Stem Cells: Improved Survival When Implanted as a Monolayer
PURPOSE: To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM).backslashnbackslashnMETHODS: Sixty-nine rats (38 male,31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1,6,and 12 months after surgery. Both ocular tissues and systemic organs (brain,liver,kidneys,spleen,heart,and lungs) were fixed in 4% paraformaldehyde,embedded in paraffin,and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker),anti-TRA-1-85 (human cell marker),anti-Ki67 (proliferation marker),anti-CD68 (macrophage),and anti-cytokeratin (epithelial marker).backslashnbackslashnRESULTS: The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P textless 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group.backslashnbackslashnCONCLUSIONS: hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects.
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产品类型：
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Francipane MG and Lagasse E ( 2013)
Oncotarget 4 11 1948--1962
Selective targeting of human colon cancer stem-like cells by the mTOR inhibitor Torin-1.
Metastatic colorectal cancer (CRC) is incurable for most patients. Since mammalian target of rapamycin (mTOR) has been suggested as a crucial modulator of tumor biology,we aimed at evaluating the effectiveness of mTOR targeting for CRC therapy. To this purpose,we analyzed mTOR expression and the effect of mTOR inhibition in cancer stem-like cells isolated from three human metastatic CRCs (CoCSCs). CoCSCs exhibited a strong mTOR complex 2 (mTORC2) expression,and a rare expression of mTOR complex 1 (mTORC1). This latter correlated with differentiation,being expressed in CoCSC-derived xenografts. We indicate Serum/glucocorticoid-regulated kinase 1 (SGK1) as the possible main mTORC2 effector in CoCSCs,as highlighted by the negative effect on cancer properties following its knockdown. mTOR inhibitors affected CoCSCs differently,resulting in proliferation,autophagy as well as apoptosis induction. The apoptosis-inducing mTOR inhibitor Torin-1 hindered growth,motility,invasion,and survival of CoCSCs in vitro,and suppressed tumor growth in vivo with a concomitant reduction in vessel formation. Torin-1 also affected the expression of markers for cell proliferation,angio-/lympho-genesis,and stemness in vivo,including Ki67,DLL1,DLL4,Notch,Lgr5,and CD44. Importantly,Torin-1 did not affect the survival of normal colon stem cells in vivo,suggesting its selectivity towards cancer cells. Thus,we propose Torin-1 as a powerful drug candidate for metastatic CRC therapy.
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产品类型：
产品号#：
73492
73494
产品名：
Torin 1
Torin 1, 50 mg
Badja C et al. (DEC 2014)
Stem cells translational medicine 3 12 1467--72
Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells.
For years,our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular,much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells,hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction,expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days,neuronal precursors can be expanded,frozen,and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction,cells express typical voltage-gated and ionotrophic receptors for GABA,glycine,and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications,especially for modeling human pathologies.
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产品类型：
产品号#：
05850
05857
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05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Shen S-C et al. (DEC 2014)
PloS one 9 12 e114990
Susceptibility of human embryonic stem cell-derived neural cells to Japanese encephalitis virus infection.
Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells,including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses,such as Japanese encephalitis virus (JEV),provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition,glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast,only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition,functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover,we discover that vimentin intermediate filament,reported as a putative neurovirulent JEV receptor,is highly expressed in NPCs and glial cells,but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally,we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.
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产品类型：
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Tateno H et al. (MAY 2015)
Stem Cell Reports 4 5 811--820
Elimination of tumorigenic human pluripotent stem cells by a recombinant lectin-toxin fusion protein
The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously,we identified a human pluripotent stem-cell-specific lectin probe,called rBC2LCN,by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A,termed rBC2LCN-PE23,which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells,followed by its internalization,allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus,rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies.
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产品类型：
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Chen RJ et al. (NOV 2015)
PloS one 10 11 e0142554
Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.
Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report,we employ electron,immunofluorescence microscopy,and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 $$g/$$g proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 $$g/$$g proteins) reported in human cancer cell lines. Moreover,we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states,similar to those naïve-like hPSCs,with increased glycogen synthesis. Furthermore,we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus,our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.
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