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|Cultivo de células madre|
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|Componentes||Napthol AS-BI phosphate solution (4mg/mL) in AMPD buffer (2mol/L), pH 9.5 (Part No. 90234). One 15mL bottle. Fast Red Violet solution (0.8g/L stock) (Part No. 90239). Two 15mL bottles.|
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|Tamaño material||100 assays|
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Ficha datos de seguridad (MSDS)
Referencias bibliográficas | 18 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Pub Med ID|
|Establishment of LIF-dependent human iPS cells closely related to basic FGF-dependent authentic iPS cells. |
Hiroyuki Hirai,Meri Firpo,Nobuaki Kikyo
PloS one 7 2012
Human induced pluripotent stem cells (iPSCs) can be divided into a leukemia inhibitory factor (LIF)-dependent naïve type and a basic fibroblast growth factor (bFGF)-dependent primed type. Although the former are more undifferentiated than the latter, they require signal transduction inhibitors and sustained expression of the transgenes used for iPSC production. We used a transcriptionally enhanced version of OCT4 to establish LIF-dependent human iPSCs without the use of inhibitors and sustained transgene expression. These cells belong to the primed type of pluripotent stem cell, similar to bFGF-dependent iPSCs. Thus, the particular cytokine required for iPSC production does not necessarily define stem cell phenotypes as previously thought. It is likely that the bFGF and LIF signaling pathways converge on unidentified OCT4 target genes. These findings suggest that our LIF-dependent human iPSCs could provide a novel model to investigate the role of cytokine signaling in cellular reprogramming.
|BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells. |
Zhongwei Li,Teng Fei,Jianping Zhang,Gaoyang Zhu,Lu Wang,Danyu Lu,Xiaochun Chi,Yan Teng,Ning Hou,Xiao Yang,Hongquan Zhang,Jing-Dong J Han,Ye-Guang Chen
Cell stem cell 10 2012
Extrinsic BMP and LIF signaling collaboratively maintain mouse embryonic stem cell (ESC) pluripotency, whereas appropriate ERK activity is essential for ESC fate commitment. However, how the extrinsic signals restrain appropriate ERK activity remains elusive. Here, we show that, whereas LIF sustains relatively high ERK activity, BMP4 can steadily attenuate ERK activity by upregulating ERK-specific dual-specificity phosphatase 9 (DUSP9). This upregulation requires Smad1/5 and Smad4 and specifically occurs to DUSP9, but not other DUSPs, and only in ESCs. Through DUSP9-mediated inhibition of ERK activity, BMP signaling reinforces the self-renewal status of mouse ESCs together with LIF. Upon LIF withdrawal, ESCs spontaneously undergo neural differentiation, during which process DUSP9 can partially mediate BMP inhibition on neural commitment. Collectively, our findings identify DUSP9 as a critical mediator of BMP signaling to control appropriate ERK activity critical for ESC fate determination.
|Efficient iPS cell production with the MyoD transactivation domain in serum-free culture. |
Hiroyuki Hirai,Nobuko Katoku-Kikyo,Peter Karian,Meri Firpo,Nobuaki Kikyo
PloS one 7 2012
A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O), along with Sox2, Klf4 and c-Myc (SKM). In addition, M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs, including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast, the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs, M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition, in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.
|Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD. |
Hirai, Hiroyuki, et al.
Stem Cells, 29: 1349-61 (2011) 2011
Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c-Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M(3)O). Transduction of M(3) O as well as Sox2, Klf4, and c-Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50-fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far-reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology.
|Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells. |
Derk ten Berge,Dorota Kurek,Tim Blauwkamp,Wouter Koole,Alex Maas,Elif Eroglu,Ronald K Siu,Roel Nusse
Nature cell biology 13 2011
Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.
|Large scale phosphoproteome profiles comprehensive features of mouse embryonic stem cells. |
Li QR, Xing XB, Chen TT, Li RX, Dai J, Sheng QH, Xin SM, Zhu LL, Jin Y, Pei G, Kang JH, Li YX, Zeng R
Molecular & cellular proteomics : MCP 10 M110.001750. Epub 2010 Dec 13. 2011
Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.
|Establishment of induced pluripotent stem cells from aged mice using bone marrow-derived myeloid cells. |
Zhao Cheng,Sachiko Ito,Naomi Nishio,Hengyi Xiao,Rong Zhang,Haruhiko Suzuki,Yayoi Okawa,Toyoaki Murohara,Ken-Ichi Isobe
Journal of molecular cell biology 3 2011
If induced pluripotent stem (iPS) cells are to be used to treat damaged tissues or repair organs in elderly patients, it will be necessary to establish iPS cells from their tissues. To determine the feasibility of using this technology with elderly patients, we asked if it was indeed possible to establish iPS cells from the bone marrow (BM) of aged mice. BM cells from aged C57BL/6 mice carrying the green fluorescence protein (GFP) gene were cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for 4 days. Four factors (Oct3/4, Sox2, Klf4 and c-Myc) were introduced into the BM-derived myeloid (BM-M) cells. The efficiency of generating iPS cells from aged BM cultured in GM-CSF was low. However, we succeeded in obtaining BM-M-iPS cells from aged C57BL/6 mice, which carried GFP. Our BM-M-iPS cells expressed SSEA-1 and Pou5f1 and were positive for alkaline phosphatase staining. The iPS cells did make teratoma with three germ layers following injection into syngeneic C57BL/6 mice, and can be differentiated to three germ layers in vitro. By co-culturing with OP9, the BM-M-iPS cells can be differentiated to the myeloid lineage. The differentiated BM-M-iPS cells proliferated well in the presence of GM-CSF, and lost expression of Nanog and Pou5f1, at least in part, due to methylation of their promoters. On the contrary, Tnf and Il1b gene expression was upregulated and their promoters were hypomethylated.
|Efficient feeder-free episomal reprogramming with small molecules. |
Junying Yu,Kevin Fongching Chau,Maxim A Vodyanik,Jinlan Jiang,Yong Jiang
PloS one 6 2011
Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3? inhibitor CHIR99021, TGF-?/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.Artículo Texto completo
|Ethanol, acetaldehyde, and estradiol affect growth and differentiation of rhesus monkey embryonic stem cells. |
Catherine A VandeVoort,Dana L Hill,Charles L Chaffin,Alan J Conley
Alcoholism, clinical and experimental research 35 2011
The timing of the origins of fetal alcohol syndrome has been difficult to determine, in part because of the challenge associated with in vivo studies of the peri-implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development.
|Epigenetic regulation of Nanog expression by Ezh2 in pluripotent stem cells. |
Aranzazu Villasante,Daniela Piazzolla,Han Li,Gonzalo Gomez-Lopez,Malek Djabali,Manuel Serrano
Cell cycle (Georgetown, Tex.) 10 2011
Nanog levels in pluripotent stem cells are heterogeneous and this is thought to reflect two different and interchangeable cell states, respectively poised to self-renew (Nanog-high subpopulation) or to differentiate (Nanog-low subpopulation). However, little is known about the mechanisms responsible for this pattern of Nanog expression. Here, we have examined the impact of the histone methyltransferase Ezh2 on pluripotent stem cells and on Nanog expression. Interestingly, induced pluripotent stem (iPS) cells lacking Ezh2 presented higher levels of Nanog due to a relative expansion of the Nanog-high subpopulation, and this was associated to severe defects in differentiation. Moreover, we found that the Nanog promoter in embryonic stem (ES) cells and iPS cells coexists in two alternative univalent chromatin configurations, either H3K4me3 or H3K27me3, the latter being dependent on the presence of functional Ezh2. Finally, the levels of expression of Ezh2, as well as the amount of H3K27me3 present at the Nanog promoter, were higher in the Nanog-low subpopulation of ES/iPS cells. Together, these data indicate that Ezh2 directly regulates the epigenetic status of the Nanog promoter affecting the balance of Nanog expression in pluripotent stem cells and, therefore, the equilibrium between self-renewal and differentiation.
|Using small molecules to improve generation of induced pluripotent stem cells from somatic cells |
Desponts C. & Ding S.
Methods Mol. Biol. 636 207-218 2010
Induction of pluripotent stem cells from somatic cells by defined factors was shown to be possible only recently, but already several laboratories have made tremendous strive toward improving and understanding the process. Originally, Oct4, Sox2, Klf4, and cMyc were identified as being the combination of genes necessary to induce reprogramming. It was later shown that cMyc was dispensable; however, in its absence the process was less efficient and took a considerably longer period of time to occur. Furthermore, others have shown that the combination of Oct4, Sox2, Nanog, and Lin28 could also induce reprogramming. One major caveat associated with these techniques remains the need for overexpression of several genes using viral systems. Until very recently, most studies were done using integrating viruses such as retroviruses and lentiviruses. This method ensured that the protein of interested would be expressed at a high concentration and for an adequate period of time necessary to induce reprogramming. Up to date, others have now been able to use different nonintegrative method such as adenovirus and plasmid transfection to induce reprogramming. Furthermore, piggyBac transposition was successfully used to induce reprogramming of murine cells. Most importantly, it was recently published that reprogramming can be induced in the absence of virus, with proteins and small molecules. All of the later methods are appealing since they do not require the integration of the virus or plasmid to exert its effect. However, one avenue that would be all the more therapeutically appealing would be to induce reprogramming in the absence of gene overexpression systems, using small molecules to modulate signaling pathways in the somatic cells. A few molecules have already been identified with the ability to either improve the process or replace one or two of the genes deemed necessary for reprogramming. We have screened successfully for compounds that can replace some of these factors, and share the methods developed following these screens.
|The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. |
Alessandro Prigione,Beatrix Fauler,Rudi Lurz,Hans Lehrach,James Adjaye
Stem cells (Dayton, Ohio) 28 2010
The ability of stem cells to propagate indefinitely is believed to occur via the fine modulation of pathways commonly involved in cellular senescence, including the telomerase, the p53, and the mitochondrial/oxidative stress pathways. Induced pluripotent stem cells (iPSCs) are a novel stem cell population obtained from somatic cells through forced expression of a set of genes normally expressed in embryonic stem cells (ESCs). These reprogrammed cells acquire self-renewal properties and appear almost undistinguishable from ESCs in terms of morphology, gene expression, and differentiation potential. Accordingly, iPSCs exhibit alterations of the senescence-related telomerase and p53 signaling pathways. However, although treatments with antioxidants have been recently shown to enhance cellular reprogramming, detailed information regarding the state of the mitochondrial/oxidative stress pathway in iPSCs is still lacking. Mitochondria undergo specific changes during organismal development and aging. Thus, addressing whether somatic mitochondria within iPSCs acquire ESC-like features or retain the phenotype of the parental cell is an unanswered but relevant question. Herein, we demonstrate that somatic mitochondria within human iPSCs revert to an immature ESC-like state with respect to organelle morphology and distribution, expression of nuclear factors involved in mitochondrial biogenesis, content of mitochondrial DNA, intracellular ATP level, oxidative damage, and lactate generation. Upon differentiation, mitochondria within iPSCs and ESCs exhibited analogous maturation and anaerobic-to-aerobic metabolic modifications. Overall, the data highlight that human iPSCs and ESCs, although not identical, share similar mitochondrial properties and suggest that cellular reprogramming can modulate the mitochondrial/oxidative stress pathway, thus inducing a rejuvenated state capable of escaping cellular senescence.
|Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development. |
Ichi S, Costa FF, Bischof JM, Nakazaki H, Shen YW, Boshnjaku V, Sharma S, Mania-Farnell B, McLone DG, Tomita T, Soares MB, Mayanil CS
J Biol Chem 285 36922-32. Epub 2010 Sep 10. 2010
The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.
|Feeder-independent culture of mouse embryonic stem cells using vitamin A/retinol. |
Jaspal S Khillan,Liguo Chen
Methods in molecular biology (Clifton, N.J.) 652 2010
Embryonic stem (ES) cells derived from the inner cell mass of a mammalian blastocyst represent unlimited source of all types of cells for regenerative medicine and for drug discovery. Mouse and human ES cells require mouse embryonic fibroblast feeder cells to maintain their undifferentiated state which involve additional time-consuming and labor-intensive steps. Recently we reported a novel function of retinol, the alcohol form of vitamin A, in preventing the differentiation of mouse ES cells. Retinol/vitamin A induces the overexpression of Nanog, a key transcription factor that is important for maintaining the pluripotency of mouse and human ES cells. Further, retinol/vitamin A also supports feeder-independent culture of ES cells in long-term cultures. The cells continue to maintain the expression of pluripotent cell-specific markers such as Nanog, Oct4, and Sox2 and form chimeric animals after injection into blastocysts. In this chapter, we describe feeder-independent cultures of mouse ES cells in the medium supplemented with retinol. The ES cells are cultured over plates coated with gelatin in ES medium with leukemia inhibitory factor (LIF) which is supplemented with 0.5 muM retinol/vitamin A. The cells are passaged every 3-5 days by trypsinization. The pluripotency of the cells is tested by different undifferentiated ES cell-specific markers.
|The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. |
Xiangyun Li, Guoliang Meng, Roman Krawetz, Shiying Liu, Derrick E Rancourt
Stem cells and development 17 1079-85 2008
After slow freezing, the survival rate of human embryonic stem (hES) cells is poor and inconsistent. The aim of this study was to increase the freeze-thaw survival rate of hES cells by utilizing the ROCK inhibitor Y-27632. hES cell colonies were first treated with Y-27632, followed by collagenase IV and TrypLE Select dissociation whereupon small clumps were slow frozen using 90% Knockout serum replacement and 10% dimethyl sulfoxide. After thawing at 37 C, the clumps were cultured in medium supplemented with 10 microM Y-27632 for 1 day. Our results show that the use of Y-27632 significantly increases the survival of hES cells after thawing compared with that of the control group. Y-27632-treated freeze-thawed hES cells retain morphology, stable karyotype, expression of cell surface markers, and the potential to differentiate into derivatives of all three germ layers after long-term culture. We have concluded that conventional slow freezing with Y-27632 treatment is efficient and convenient for the cryopreservation of hES cells.
|A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture. |
Guoliang Meng, Shiying Liu, Roman Krawetz, Michael Chan, Judy Chernos, Derrick E Rancourt
Stem cells and development 17 413-22 2008
Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.
|Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method. |
Lin, Han-Tso, et al.
World J. Gastroenterol., 13: 1672-9 (2007) 2007
|Derivation of human embryonic stem cells in xeno-free conditions. |
Mohan C Vemuri,Tim Schimmel,Pere Colls,Santiago Munne,Jacques Cohen
Methods in molecular biology (Clifton, N.J.) 407 2007
Human embryonic stem cells (hESC) have the potential to treat a wide range of diseases. Currently, the use of existing hESC lines in human clinical applications is limited, as they are derived from blastocysts subjected to immunosurgery with animal derived antibodies, and are maintained on mouse embryonic feeder (MEF) cells, in the presence of either fetal calf serum (FCS) or on Matrigel or with conditioned media from MEFs. Successful derivation of hESCs in xeno-free conditions is crucial in advancing stem cell therapy applications. Two hESC lines, one from chromosomally abnormal embryos and another cell line from normal embryos from the inner cell mass of human blastocysts are derived using a culture media that had 20% serum replacement (SR) and human FGF2 on human foreskin fibroblasts as feeder cells. Derivation and characterization of such xenofree hESCs suitable for clinical studies is described in this chapter.
|Human Stem Cell Systems|
|Murine Embryonic Stem Cell Culture Procedures & Protocols|
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|STEMCCA Lentivirus Reprogramming Kits|
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|Alkaline Phosphatase Detection Kit|