L. Truszkowski et al. (Sep 2025)
Open Research Europe 4 2
Refined and benchmarked homemade media for cost-effective, weekend-free human pluripotent stem cell culture
Cost-effective,practical,and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However,the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover,we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly,the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation. We formulated B8 with commercially available,growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made,weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry,and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability,genome editing efficiency,single-cell cloning,and differentiation in both monolayer and organoids. Finally,we validated key findings using male (H1) and female (H9) human embryonic stem cells. hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast,cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time,B8+ led to gene expression changes indicative of marked lineage priming,reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state,consistent with intermittent media starvation. Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8,integrating some features of B8 while resembling cE8,emerges as a robust and practical option with limited compromises. B8+,though advantageous in some contexts,warrants caution due to lineage priming effects that may impact differentiation outcomes.
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产品类型：
产品号#：
05230
产品名：
STEMdiff™ 三胚层分化试剂盒
Sammar M et al. (JUL 1994)
International immunology 6 7 1027--36
Heat-stable antigen (CD24) as ligand for mouse P-selectin.
Heat-stable antigen (HSA)/CD24 is a cell surface molecule expressed by many cell types in the mouse. The molecule has an unusual structure because of its small protein core and extensive glycosylation. In order to study the functional role of the HSA-associated glycoconjugates we have isolated different forms of HSA. Using lectin analysis we provide evidence for extensive heterogeneity in carbohydrate composition and sialic acid linkage. Several HSA forms were recognized by mouse P-selectin-IgG but not E-selectin-IgG in ELISA. As expected,P-selectin-IgG also bound to L2/HNK-1-positive neural glycoproteins (L2-glycoproteins) and sulfatides but not to gangliosides and other control glycoproteins. The binding of P-selectin-IgG to L2-glycoproteins and HSA required bivalent cations. The reactivity to HSA was sensitive to sialidase treatment whereas the binding to L2-glycoproteins was not. Studies with alpha 2-6 sialytransferase indicated that alpha 2-6 linked sialic acid was not involved in the P-selectin binding to HSA. Surprisingly,an L2/HNK-1 specific antibody was found to cross-react with some HSA glycoforms and its binding correlated with P-selectin-IgG reactivity. L2/HNK-1-positive or L2/HNK-1-negative HSA glycoforms were also analyzed after coating to polystyrene beads. Only the L2/HNK-1-positive HSA coated beads were reactive with P-selectin-IgG and could bind to activated bend3 endothelioma cells expressing P-selectin whereas the L2/HNK-1-negative HSA beads did not. It is suggested that in its L2/HNK-1 modified form the HSA molecule on leukocytes could represent a ligand for P-selectin on endothelial cells or platelets.
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产品类型：
产品号#：
01434
产品名：
Wognum AW et al. (OCT 1990)
Blood 76 7 1323--9
A specific in vitro bioassay for measuring erythropoietin levels in human serum and plasma.
The accurate measurement of biologically active erythropoietin (Ep) in human serum and plasma using present in vivo and in vitro bioassays is difficult because of the presence of both inhibitors and non-Ep stimulators of erythropoiesis. We have developed a simple procedure to quantitatively purify Ep from serum and plasma for subsequent testing in the phenylhydrazine-treated mouse spleen cell assay. The method involves absorption of Ep to an immobilized high-affinity anti-Ep monoclonal antibody and acid elution of the antibody-bound material. After neutralization,the eluted EP is then tested directly in the in vitro bioassay without interference by other serum proteins. By using magnetic beads as a solid support for the antibody,washing and elution steps can be performed rapidly and efficiently. Recoveries of Ep after this procedure show very little sample-to-sample variation and are consistently between 45% and 55%,which is close to the maximum binding expected for the anti-Ep antibody. Coupled with the 7.4-fold concentration that this procedure affords,there is an overall increase in sensitivity of three- to fourfold,which makes this assay suitable for accurately measuring Ep levels in patients with below-average titers. Results with this magnetic bead assay indicate that accurate and reproducible estimates for Ep levels in the serum and plasma from healthy donors as well as from patients with hematologic disorders can be obtained. Titers of biologically active Ep in the sera from a group of patients with either leukemia or lymphoma were found to be elevated,and the values correlated well with titers of immunoreactive Ep measured in the Ep radioimmunoassay. Because of its specificity and high sensitivity,the magnetic bead assay is a valuable alternative to immunoassays for the measurement of elevated,normal,and even subnormal Ep levels in human serum and plasma.
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产品类型：
产品号#：
01630
产品名：
促红细胞生成素（EPO） ELISA试剂盒
Bruserud O et al. (DEC 2000)
Journal of hematotherapy & stem cell research 9 6 923--32
In vitro culture of human acute myelogenous leukemia (AML) cells in serum-free media: studies of native AML blasts and AML cell lines.
The functional characteristics were compared for acute myelogenous leukemia (AML) cells (native blasts and AML cell lines) cultured in three serum-free media (X-vivo 10,X-vivo 15,[Bio-Whitacker,Walkersville,MD] and StemSpan [Stem Cell Technologies,Vancouver,BC,Canada]) and in medium containing 10% inactivated fetal calf serum (FCS). For native AML blasts the following functions were compared: (1) autonomous and cytokine-dependent proliferation; (2) frequency of clonogenic cell; and (3) constitutive cytokine secretion. AML blast proliferation differed between patients independent of the culture medium used,and clonogenic cells were maintained after in vitro culture in all media. In contrast,constitutive cytokine secretion was higher for cells cultured in StemSpan and FCS-containing medium than for cells cultured in the X-vivo media. Native AML blasts incubated in StemSpan also showed a low frequency of apoptotic cells. The three serum-free media could also be used for long-term expansion of well-characterized AML cell lines,but the optimal medium for cell expansion and cytokine secretion differed between cell lines. We conclude that standardized serum-free culture conditions can be used for in vitro studies of native AML blasts and AML cell lines.
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Bryja V et al. ( 2006)
Nature protocols 1 4 2082--2087
Derivation of mouse embryonic stem cells.
Here we describe a simple and efficient protocol for derivation of germline chimera-competent mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) blastocysts. The protocol involves the use of early-passage mouse embryonic fibroblast feeders (MEF) and the alternation of fetal bovine serum- and serum replacement (SR)-containing media. As compared to other available protocols for mESCs derivation,our protocol differs in the combination of commercial availability of all reagents,technical simplicity and high efficiency. mESC lines are derived with approximately 50% efficiency (50 independent mESC lines derived from 96 blastocysts). We believe that this protocol could be a good starting point for (i) setting up the derivation of mESC lines in a laboratory and (ii) incorporating further steps to improve efficiency or adapt the protocol to other applications. The whole process (from blastocyst extraction to the freezing of mESC line) usually takes between 15 and 20 d.
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产品类型：
产品号#：
73272
73274
100-1048
产品名：
丝裂霉素C
丝裂霉素C
Kang M and Han Y-M (APR 2014)
PloS one 9 4 e94888
Differentiation of human pluripotent stem cells into nephron progenitor cells in a serum and feeder free system.
OBJECTIVES Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney,stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources,pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However,little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study,we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system. MATERIALS AND METHODS We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak,intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR,real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility. RESULTS After modification of culture period and concentration of exogenous factors,hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2,GDNF,HOXD11,WT1 and CITED1 in addition to OSR1,PAX2,SALL1 and EYA1. Moreover,NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular,approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems. CONCLUSIONS Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases.
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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
Chang M-YY et al. (NOV 2015)
Stem cell research 15 3 608--613
Doxycycline supplementation allows for the culture of human ESCs/iPSCs with media changes at 3-day intervals.
Culturing human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) is one of the most costly and labor-intensive tissue cultures,as media containing expensive factors/cytokines should be changed every day to maintain and propagate undifferentiated hESCs/iPSCs in vitro. We recently reported that doxycycline,an anti-bacterial agent,had dramatic effects on hESC/iPSC survival and promoted self-renewal. In this study,we extended the effects of doxycycline to a more practical issue to save cost and labor in hESC/iPSC cultures. Regardless of cultured cell conditions,hESCs/iPSCs in doxycycline-supplemented media were viable and proliferating for at least 3 days without media change,while none or few viable cells were detected in the absence of doxycycline in the same conditions. Thus,hESCs/iPSCs supplemented with doxycycline can be cultured for a long period of time with media changes at 3-day intervals without altering their self-renewal and pluripotent properties,indicating that doxycycline supplementation can reduce the frequency of media changes and the amount of media required by 1/3. These findings strongly encourage the use of doxycycline to save cost and labor in culturing hESCs/iPSCs.
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产品类型：
产品号#：
05850
05857
05870
05875
07920
85850
85857
85870
85875
07922
产品名：
ACCUTASE™
mTeSR™1
mTeSR™1
ACCUTASE™
Oeda S et al. (JAN 2013)
The International journal of developmental biology 57 5 383--9
Induction of intermediate mesoderm by retinoic acid receptor signaling from differentiating mouse embryonic stem cells.
Renal lineages including kidney are derived from intermediate mesoderm,which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined,serum-free,adherent,monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm,odd-skipped related 1 (Osr1) and Wilms Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist,but not by retinoid X receptor (RXR) agonists. Furthermore,the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney.
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产品类型：
产品号#：
72892
产品名：
TTNPB
Ishimoto T et al. ( 2014)
PloS one 9 2 e89434
Organic cation transporter-mediated ergothioneine uptake in mouse neural progenitor cells suppresses proliferation and promotes differentiation into neurons.
The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4,which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [(3)H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [(3)H]ERGO uptake. On the other hand,exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker βIII-tubulin,but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP),with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly,edaravone and ascorbic acid did not affect such differentiation of NPCs,in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP,but decreased the number immunoreactive for βIII-tubulin,with concomitant down-regulation of Math1 in P19-NPCs. Thus,OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress,and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action.
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产品类型：
产品号#：
05707
产品名：
NeuroCult™化学解离试剂盒（小鼠）
Zhang Z et al. (SEP 2003)
The EMBO journal 22 18 4759--69
Enforced expression of EBF in hematopoietic stem cells restricts lymphopoiesis to the B cell lineage.
Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment,which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted,multipotential progenitor cells. To test this hypothesis,we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were textgreater95 and 90% GFP+EBF+ mature B cells,respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways.
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产品类型：
产品号#：
03434
03444
产品名：
MethoCult™ GF M3434
MethoCult™ GF M3434
Hannoun Z et al. (APR 2010)
Cellular reprogramming 12 2 133--140
The comparison between conditioned media and serum-free media in human embryonic stem cell culture and differentiation.
Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such,hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However,for their full potential to be realized,both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however,they suffer from some major limitations including lack of definition,xenobiotic nature,batch-to-batch variation,and labor-intensive production. Therefore,hESC culture definition is essential if hESC lines,and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state,for over 30 passages using MT and SP. Additionally,we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture,contributing to the standardization of hESC in vitro models and ultimately their application.
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EasySep™小鼠TIL（CD45）正选试剂盒
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