By Michal Amit (auth.), Uma Lakshmipathy, Mohan C Vemuri (eds.)
Human pluripotent stem cells equivalent to human embryonic stem cells (hESC) and brought about pluripotent stem cells (iPSC) with their particular developmental plasticity carry significant strength as mobile versions for drug discovery and in regenerative drugs as a resource for cellphone substitute. whereas hESC are derived from a constructing embryo, iPSC are generated with pressured expression of key transcription components to transform grownup somatic cells to ESC-like cells, a procedure termed reprogramming. utilizing iPSC overcomes moral concerns about the use of constructing embryos and it may be generated from patient-specific or disease-specific cells for downstream purposes. Pluripotent Stem Cells: tools and Protocols highlights the simplest tools and platforms for the total paintings move. Divided into 4 handy sections, subject matters comprise a spotlight on generating iPSC from assorted somatic assets, media structures for increasing ESC and iPSC with designated protocols for directed differentiation into particular lineages, well-known mobile and molecular characterization equipment , and the aptitude software of categorized stem cells with particular equipment for cloning, gene supply and phone engineering. Written within the winning Methods in Molecular Biology™ sequence layout, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, effectively reproducible protocols, and notes on troubleshooting and fending off identified pitfalls.
Authoritative and simply available, Pluripotent Stem Cells: tools and Protocols seeks to serve either pros and rookies with its well-honed methodologies with a purpose to extra our wisdom of this crucial mobile function.
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Extra resources for Pluripotent Stem Cells: Methods and Protocols
2. 45 µM filter. 3. Pasteur pipets. 4. 6 well plates. 5. T25 flasks. 6. 10 cm dishes. 7. Ultracentrifuge tubes. 1 293FT Growth Medium 1. 435 mL high-glucose DMEM/F12. 2. 50 mL FBS. 3. 5 mL L-glutamine. 4. 5 mL NEAA. 5. 5 mL 100× pen/strep. 2 293 Cell-Freezing Medium 1. 10 mL growth medium. 2. 80 mL FBS. 3. 10 mL DMSO. 3 Fibroblast Growth Medium (hFib Medium) 1. 450 mL DMEM/F12. 2. 75 mL FBS. 3. 5 mL Nonessential amino acids. 4. 5 mL 100× pen/strep. 4 KOSR Medium 1. 400 mL DMEM/F12. 2. 100 mL Knockout Serum Replacer.
L-Glutamine. 4. GM-CSF recombinant human. 5. IL3 recombinant human. 6. SCF recombinant human. 3 PCR Reagents 1. TRIzol® reagent. 2. SuperScript® VILO™ cDNA Synthesis Kit (Cat. no. 11754-050). 3. AccuPrime™ SuperMix I (Cat. no. 12342-010). 4 Equipment 1. , biosafety cabinet) equipped with a stereomicroscope. 2. Inverted microscope. 3. Incubator set at 37°C, 5% CO2. 4. Water bath set at 37°C. 5. Sterile serological pipettes (5-mL, 10-mL). 6. Centrifuge. 7. 15-mL centrifuge tubes. 8. 60-mm and 100-mm tissue culture-treated dishes.
2. Examine dishes under the microscope for colony formation (see Note 9). 2. Prepare Matrigel dishes according to the protocol supplied with the Matrigel. 3. Using a pulled Pasteur pipet, gently break up and scrape off reprogrammed colonies. 4. Using a 100 μL pipet, pick up colony pieces and transfer to Matrigel plates containing mTeSR with 5 μM Rho-Kinase Inhibitor (Y27632). 5. Feed daily with mTeSR. 42 Jennifer C. Moore Fig. 2 Reprogrammed fibroblast colonies at days 16, 20, and 24 after initial reprogramming.