In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.
A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue
1Regenerative Medicine Institute, Cedars-Sinai Medical Center
This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension. Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.
Published June 15, 2014. Keywords: Neuroscience, neural progenitor cell, neural precursor cell, neural stem cell, passaging, neurosphere, chopping, stem cell, neuroscience, suspension culture, good manufacturing practice, GMP
1NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health
Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.
Published July 24, 2014. Keywords: Stem Cell Biology, Pluripotent stem cells, human embryonic stem cells, induced pluripotent stem cells, cell culture, non-colony type monolayer, single cell, plating efficiency, Rho-associated kinase, Y-27632, transfection, transduction
Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2
1Materials Science and Engineering, CSIRO
We describe the use of the monoclonal antibodies TG30 (CD9) and GCTM-2 for the combined detection of cell surface antigens via fluorescence activated cell sorting (FACS) for the identification and enrichment of live human embryonic stem cells (hESC) using positive selection and also the use of negative selection to purge hESCs from a mixed cell population.
Published December 6, 2013. Keywords: Stem Cell Biology, Stem cells, cell surface antigens, antibodies, FACS, purging stem cells, differentiation, pluripotency, teratoma, human embryonic stem cells (hESC)
1Developmental Biology, Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, 2St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University
Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.
Published May 22, 2014. Keywords: Neuroscience, Embryonic Stem Cells (ESCs), Pluripotent Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Neural Crest, Peripheral Nervous System (PNS), pluripotent stem cells, neural crest cells, in vitro differentiation, disease modeling, differentiation protocol, human embryonic stem cells, human pluripotent stem cells
Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium
The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.
This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.
1Department of Biology, University of Alabama at Birmingham, 2Nutrition Métabolisme Aquaculture, INRA UR1067, 3Laboratoire de Physiologie et Genomique des Poissons, INRA UR1037
In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.
Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells
1Humanitas Clinical and Research Center, Italy, 2Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR)
Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.
Published June 28, 2013. Keywords: Stem Cell Biology, Developmental Biology, Molecular Biology, Cellular Biology, Medicine, Bioengineering, Biomedical Engineering, Genetics, Cardiology, Stem Cell Research, Cardiovascular Diseases, Human cardiomyocytes, iPS cells, induced pluripotent stem cells, stem cells, cardiac differentiation, disease modeling, embryoid bodies, cell lines, cell culture
Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics
1Division of Virology, Department of Pathology, University of Cambridge
SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.
Published July 6, 2014. Keywords: Biochemistry, mass spectrometry, tissue culture techniques, isotope labeling, SILAC, Stable Isotope Labeling of Amino Acids in Cell Culture, proteomics, Interactomics, immunoprecipitation, pulldown, eIF4A, GFP, nanotrap, orbitrap