Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H||ELISA, WB, FC, ICC||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at from date of receipt.|
|Reference overview||Pub Med ID|
|High-content imaging-based screening of microenvironment-induced changes to stem cells. |
Sebasti Vega,Er Liu,Parth J Patel,Anthony B Kulesa,Aaron L Carlson,Yanrui Ma,Matthew L Becker,Prabhas V Moghe
Journal of biomolecular screening 17 2012
Effective screening methodologies for cells are challenged by the divergent and heterogeneous nature of phenotypes inherent to stem cell cultures, particularly on engineered biomaterial surfaces. In this study, we showcase a high-content, confocal imaging-based methodology to parse single-cell phenotypes by quantifying organizational signatures of specific subcellular reporter proteins and applied this profiling approach to three human stem cell types (embryonic-human embryonic stem cell [hESC], induced pluripotent-induced pluripotent stem cell [iPSC], and mesenchymal-human mesenchymal stem cell [hMSC]). We demonstrate that this method could distinguish self-renewing subpopulations of hESCs and iPSCs from heterogeneous populations. This technique can also provide insights into how incremental changes in biomaterial properties, both physiochemical and mechanical, influence stem cell fates by parsing the organization of stem cell proteins. For example, hMSCs cultured on polymeric films with varying degrees of poly(ethylene glycol) to modulate osteogenic differentiation were parsed using high-content organization of the cytoskeletal protein F-actin. In addition, hMSCs cultured on a self-assembled monolayer platform featuring compositional gradients were screened and descriptors obtained to correlate substrate variations with adipogenic lineage commitment. Taken together, high-content imaging of structurally sensitive proteins can be used as a tool to identify stem cell phenotypes at the single-cell level across a diverse range of culture conditions and microenvironments.
|Dynamic profiles of Oct-4, Cdx-2 and acetylated H4K5 in in-vivo-derived rabbit embryos. |
Chien-Hong Chen,Jie Xu,Wei-Fang Chang,Chia-Chia Liu,Hwa-Yun Su,Y Eugene Chen,Fuliang Du,Li-Ying Sung
Reproductive biomedicine online 25 2012
This study documents the spatial and temporal distribution of Oct-4, Cdx-2 and acetylated H4K5 (H4K5ac) by immunocytochemistry staining using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where the signal was similarly weak in both the ICM and TE cells. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts, and became evident in the TE cells exclusively afterwards. A consistently strong H4K5ac signal was observed in the TE cells in all blastocyst stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Double staining of H4K5ac with either Oct-4 or Cdx-2 on embryos at different blastocyst stages confirmed these findings. This work suggests that day 4 is a critical timing for lineage formation in rabbit embryos. A combination of Oct-4, Cdx-2 and H4K5ac can be used as biomarkers to identify different lineage cells in rabbit blastocysts. There are very limited studies on key transcription factors and epigenetic programming events in preimplantation-stage rabbit embryos. Previously we reported the spatial and temporal distribution of Oct-4 and acetylated H4K5 (H4K5ac) in-vitro-cultured rabbit embryos. In the present study, we report the similar distribution patterns of Oct-4 and H4K5ac using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where such signal was similarly weak in both the ICM and the TE cells. A consistent strong H4K5ac signal was observed in the TE cells in all stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Importantly, we also report, as far as is known for the first time, the distribution pattern of Cdx-2 in rabbit embryos. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts. It became evident in the TE cells exclusively afterwards. The present study provides novel information on key transcription factors and epigenetic events during rabbit embryo development, and demonstrates that a combination of Oct-4, Cdx-2 and H4K5ac could be used as biomarkers to identify different lineage cells in rabbit blastocysts.
|Oct-4B isoform is differentially expressed in breast cancer cells: hypermethylation of regulatory elements of Oct-4A suggests an alternative promoter and transcriptional start site for Oct-4B transcription. |
Wang, Yajuan, et al.
Biosci. Rep., 31: 109-15 (2011) 2011
The human Oct-4 gene has three isoforms, Oct-4A, Oct-4B and Oct-4B1, which are thought to be derived from alternative splicing. It remains controversial whether the Oct-4 gene is expressed in cancer cells. Expression of Oct-4A is regulated by two elements, the PE (proximal enhancer) and DE (distal enhancer), but the expression and regulation of Oct-4B are not well known. Here, we firstly report that Oct-4B is expressed at low levels in MCF-7 cells, while the Oct-4A gene is inactivated. By analysing the function of different promoter constructs and the DNA methylation status of three regulatory regions, we demonstrate that the Oct-4A gene in MCF-7 cells is repressed by epigenetic control rather than transcriptional control. In addition, we speculate that the transcription of Oct-4B in MCF-7 cells is differentially regulated by additional regulatory elements. This work will enhance the understanding of Oct-4 gene in differential regulation.
|Characterization of differential properties of rabbit tendon stem cells and tenocytes. |
Jianying Zhang,James H-C Wang
BMC musculoskeletal disorders 11 2010
Tendons are traditionally thought to consist of tenocytes only, the resident cells of tendons; however, a recent study has demonstrated that human and mouse tendons also contain stem cells, referred to as tendon stem/progenitor cells (TSCs). However, the differential properties of TSCs and tenocytes remain largely undefined. This study aims to characterize the properties of these tendon cells derived from rabbits.Full Text Article
|Efficient derivation of embryonic stem cells from nuclear transfer and parthenogenetic embryos derived from cryopreserved oocytes. |
Sung LY, Chang CC, Amano T, Lin CJ, Amano M, Treaster SB, Xu J, Chang WF, Nagy ZP, Yang X, Tian XC
Cell Reprogram 12 203-11. 2010
Deriving histocompatible embryonic stem (ES) cells by somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA) requires fresh oocytes, which prevents their applications in humans. Here, we evaluated the efficiency of deriving ES cells from mature metaphase II (MII) and immature metaphase I (MI) vitrified oocytes, by PA or SCNT, in a mouse model. We successfully generated ES cell lines from PA (MII and MI) and SCNT (MII and MI) blastocysts. These cell lines expressed genes and antigens characteristic of pluripotent ES cells and produced full-term pups upon tetraploid embryo complementation. This study established an animal model for efficient generation of patient-specific ES cell lines using cryopreserved oocytes. This is a major step forward in the application of therapeutic cloning and parthenogenetic technology in human regenerative medicine and will serve as an important alternative to the iPS cell technology in countries/regions where these technologies are permitted.
|Ago2 immunoprecipitation identifies predicted microRNAs in human embryonic stem cells and neural precursors. |
LA Goff, J Davila, MR Swerdel, JC Moore, RI Cohen, H Wu, YE Sun, RP Hart
PloS one 4 e7192 2009
BACKGROUND: MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: SOLiD ultra-deep sequencing identified >10(7) unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. CONCLUSIONS/SIGNIFICANCE: Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.Full Text Article
|The intracellular distribution of the ES cell totipotent markers OCT4 and Sox2 in adult stem cells differs dramatically according to commercial antibody used. |
Patricia A Zuk, Patricia A Zuk
Journal of cellular biochemistry 106 867-77 2009
To characterize ES cells, researchers have at their disposal a list of pluripotent markers, such as OCT4. In their quest to determine if adult stem cell populations, such as MSCs and ASCs, are pluripotent, several groups have begun to report the expression of these markers in these cells. Consistent with this, human ASCs (hASCs) are shown in this study to express a plethora of ES pluripotent markers at the gene and protein level, including OCT4, Sox2, and Nanog. When intracellular distribution is examined in hASCs, both OCT4 and Sox2 are expressed within the nuclei of hASCs, consistent with their expression patterns in ES cells. However, a significant amount of expression can be noted within the hASC cytoplasm and a complete absence of nuclear expression is observed for Nanog. Recent descriptions of OCT4 transcript variants may explain the cytoplasmic expression of OCT4 in hASCs and consistent with this, hASCs do express both the OCT4A and 4B transcript variants at the gene level. However, discrepancies arise when these three pluripotent markers are studied at the protein level. Specifically, distinct differences in intracellular expression patterns were noted for OCT4, Sox2, and Nanog from commercial antibody to commercial antibody. These antibody discrepancies persisted when hMSCs and rat ASCs and MSCs were examined. Therefore, confirming the expression of OCT4, Sox2, and Nanog in adult stem cells with today's commercial antibodies must be carefully considered before the designation of pluripotent can be granted.
|Targeting cancer stem cells through L1CAM suppresses glioma growth. |
Bao, Shideng, et al.
Cancer Res., 68: 6043-8 (2008) 2008
Malignant gliomas are lethal cancers that display striking cellular heterogeneity. A highly tumorigenic glioma tumor subpopulation, termed cancer stem cells or tumor-initiating cells, promotes therapeutic resistance and tumor angiogenesis. Therefore, targeting cancer stem cells may improve patient survival. We interrogated the role of a neuronal cell adhesion molecule, L1CAM, in glioma stem cells as L1CAM regulates brain development and is expressed in gliomas. L1CAM(+) and CD133(+) cells cosegregated in gliomas, and levels of L1CAM were higher in CD133(+) glioma cells than normal neural progenitors. Targeting L1CAM using lentiviral-mediated short hairpin RNA (shRNA) interference in CD133(+) glioma cells potently disrupted neurosphere formation, induced apoptosis, and inhibited growth specifically in glioma stem cells. We identified a novel mechanism for L1CAM regulation of cell survival as L1CAM knockdown decreased expression of the basic helix-loop-helix transcription factor Olig2 and up-regulated the p21(WAF1/CIP1) tumor suppressor in CD133(+) glioma cells. To determine if targeting L1CAM was sufficient to reduce glioma stem cell tumor growth in vivo, we targeted L1CAM in glioma cells before injection into immunocompromised mice or directly in established tumors. In each glioma xenograft model, shRNA targeting of L1CAM expression in vivo suppressed tumor growth and increased the survival of tumor-bearing animals. Together, these data show that L1CAM is required for maintaining the growth and survival of CD133(+) glioma cells both in vitro and in vivo, and L1CAM may represent a cancer stem cell-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors.
|Microporous membrane growth substrates for embryonic stem cell culture and differentiation. |
Steven D Sheridan,Sonia Gil,Matthew Wilgo,Aldo Pitt
Methods in cell biology 86 2008
As the field of embryonic stem cell culture and differentiation advances, many diverse culturing techniques will ultimately be necessary in order to fully reproduce the various environments these cells normally encounter during development. Although most of the work to date has been performed on solid plastic supports, this growth support has several limitations in its representation of the in vivo environment. Impermeable substrates force the cells to exchange their gas and nutrients exclusively through the top side of the cultured cells. In contrast, cells growing in vivo are exposed from several directions to factors from the blood, other cells, soluble factors, and liquid-air interfaces. Additionally, solid plastic presents a smooth two-dimensional surface that is not experienced in vivo. Therefore, the use of traditional plastic presents limitations upon normal cellular morphology, function, and differentiation. An important alternative to growth on solid plastic is the growth of cells on microporous membranes. One of the many advantages to cell growth on porous membrane substrates is their ability to provide a surface that better mimics a three-dimensional in vivo setting. A porous membrane allows multidirectional exposure to nutrients and waste products. In addition, the membrane separation of dual chambers allows for the coculture of cells of different origin to study how cells interact through indirect signaling or through providing a conditioned niche for the proper growth and differentiation of cell types.
|Human Stem Cell Systems|
Technical Info and Required Licenses
|A Comparative Analysis of Human Embryonic Stem Cells Cultured in a Variety of Media Conditions|
|Single transfection of a synthetic polycistronic self-replicative RNA yields high numbers of transgene-free human iPSCs|
|Anti-Oct-4, clone 10H11.2 - Data Sheet|