Stem Cell Experiment 'Standards'

Experiments
Stem cells can be used to identify drug targets and test potential therapeutics. New drugs may be tested on stem cells for safety and effectiveness, prior to testing new drugs in human trials. Stem cells may be utilized to generate healthy cells to replace diseased cells. iPS cells that are derived from patients can be used to examine individualized responses to drugs and therapies. By studying stem cell differentiation we can deepen our understanding of prevention and treatment of birth defects. Stem cells can be used to generate disease-specific tissues. This allows researchers to better understand disease states and aid the drug development process.
Samples
Stem Cell Transformed Somatic Cancer Purchased Biosample Healthy Tissue Diseased Tissue Primary Cell Line Sample
Sample Processing
Sample Processing Stem cell differentiation involves the changing of a cell to a more specialized cell type, involving a switch from proliferation to specialization. This involves a succession of alterations in cell morphology, membrane potential, metabolic activity and responsiveness to certain signals. Differentiation leads to the commitment of a cell to developmental lineages and the acquisition of specific functions of committed cells depending upon the tissue in which they will finally reside. Stem cell differentiation is tightly regulated by signaling pathways and modifications in gene expression. Mesoderm Heart Muscle Red Blood Cell Ectoderm Skin Neuron Endoderm Lung Pancreas Gene Modification Organoids grown from stem cells - cells that can divide indefinitely and produce different types of cells as part of their progeny. Scientists have learned how to create the right environment for the stem cells so they can follow their own genetic instructions to self-organize, forming tiny structures that resemble miniature organs composed of many cell types. Organoids can range in size from less than the width of a hair to five millimeters. Organoids derived from ESCs and iPSCs undergo directed differentiation towards the desired germ lineage, eventually generating floating spheroids that are subsequently embedded in ECM to initiate organoid culture. Organoids can be generated from primary tissue that is dissociated into functional sub-tissue units containing stem cells. These are further digested into single cells and FACS sorted to enrich for stem cells. Both the functional units and single stem cells can give rise to organoids under the appropriate culture conditions. IPSC Reprogramming Overall, integrating viral vectors efficiently and reliably generate iPSCs, but they have several safety concerns. First, they require the use of potentially harmful viral particles that express oncogenes, such as Myc. Viral vectors also have the largest genomic footprint of iPSC generating methods due to the risk of insertional mutagenesis. Random integration also creates heterogeneous iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing of transgenes is a concern as well, and reactivation of Myc or other oncogenes after differentiation has been linked to tumor formation in iPSC-derived and iPSC-transplanted mice. Cre-deletable or Tet-inducible lentiviruses address some of these concerns, but overall integrating viral systems currently lack the safety required for translational use. Doxycycline-inducible lentivirus are capable of expressing transcription factors (Oct4, Klf4, Sox2 or c-Myc) under the control of the doxycycline (Dox) inducible tetO operator when transduced into cells. Integrating viral vectors that allow delivery of genes into the genome of divinding cells and are usually silenced in mature cells to prevent tumor formation caused by continuous upregulation of transduced oncogenes. Ex. Retrovirus, Lentivirus The integrated transgene is removed from the donor genome following completion of the reprogramming process. Ex: LoxP, piggyBac Non-integrating methods have a smaller genetic footprint compared to integrating approaches. These methods eliminate the risk of insertional mutagenesis, the presence of a genetic scar, and incomplete silencing of transgenes. Overall, non-integrating approaches are safer than integrating methods, with RNA and protein delivery considered the safest since there's minimal risk of lingering expression of reprogramming factors. DNA Minicircles are like mini-plasmids. They contain only a eukaryotic promoter and the cDNA(s) to be expressed and they don't integrate or replicate. Their small size leads to higher transfection efficiencies and they tend to express for longer periods of time than traditional plasmids due to lower activation of DNA-silencing mechanisms. Non-integrating reprogramming method that generates transgene-free and vector-free cells. Generating iPSCs with plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of interest. The advantages of this method are that it's relatively simple to implement and doesn't require time-consuming production of virus. In theory this is a non-integrating approach; however, in practice integration can occur. Non-integrating reprogramming method that generates transgene-free and vector-free cells. Sendai virus is a single stranded, negative sense RNA virus. It's a member of the Paramyxoviridae family of viruses, which also includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm independent of the cell cycle. A challenge of using Sendai is that since it's replication competent, it's difficult to eliminate the virus from all cells, even after many passages. Non-integrating reprogramming method that generates transgene-free and vector-free cells. Adenoviral vectors infect dividing and nondividing cells and have an ~8kb packaging capacity. With this packaging capacity, reprogramming factors can be delivered either as a single polycistronic transgene or with four different adenoviruses, each expressing one factor. These vectors don't integrate into the genome and are instead lost by dilution via cell division. A drawback to this approach is it has lower levels of efficiency at generating iPSCs, usually several orders of magnitude lower than retroviruses; however, because they are less likely to cause insertional mutagenesis, adenoviral vectors are considered a safer way to express reprogramming factors for therapeutic applications. Non-DNA This protocol requires four serial transfections of the miRNA reprogramming cocktail every 48 hr. The efficiency of miRNA-mediated reprogramming is approximately ~0.002-0.01%, depending on the cell type. Similar to other non-DNA-mediated reprogramming strategies, the miRNA technique requires no screening to exclude exogenous DNA sequences that integrate into iPSC clones, but the requirement of multiple miRNAs and multiple transfections renders the strategy more complicated for reprogramming. Chemical approaches can be widely applied to manipulate cell fates and states, including pluripotent reprogramming, directed differentiation, and lineage reprogramming. This method uses in vitro transcription (IVT) reactions utilizing PCR-amplified templates that encode the OSKM or OSKM plus Lin28 reprogramming factors. The delivery of reprogramming factors as proteins represents an alternative way to avoid the introduction of exogenous transgene into donor cells.
Assays
Transcriptomics RNA-seq Bulk RNA Single Cell RNA Fluidigm/C1 10X Drop-seq sc-ATAC-seq Smart-seq Frac-seq CaptureSeq is a targeted RNA sequencing method that is able to provide higher sequencing coverage for selected regions of the genome. This method follows the TruSeq RNA sample preparation protocol, in which mRNA is first isolated from total RNA by poly(A) selection and then fragmented. Double-stranded cDNA copies of the fragments are generated using reverse transcriptase and then ligated to p5 and p7 adapters. Next, these cDNA library fragments are amplified by the polymerase chain reaction (PCR). To increase specificity, custom capture probes are hybridized to the cDNA and bound to an array while other transcripts are washed away prior to PCR amplification. This process leaves the targeted fragments that are ready for sequencing. Genomics WGS Amplicon-seq Other GUIDE-seq Epigenomics Methylome WGBS RRBS Chromatin Modeling ATAC-seq Hi-C Histone Modification Chromatin immunoprecipitation with expectation of narrow peaks as most transcription factors. Chromatin immunoprecipitation with expectation of broad peaks as most modified histones. Assays

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