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Models for Regenerative Medicine

Full SOP with Academic or a plan

Source Takahashi et al., 2007 · Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University · 10.1016/j.cell.2007.11.019

Authors: Kazutoshi Takahashi, Koji Tanabe, Mari Ohnuki, Megumi Narita, Tomoko Ichisaka, Kiichiro Tomoda, Shinya Yamanaka Duration: 30 days 9 phases Cell source: Adult human dermal fibroblasts (HDF) from facial dermis

Abstract

This protocol describes the generation of induced pluripotent stem (iPS) cells from adult human dermal fibroblasts using retroviral transduction of four defined transcription factors: Oct3/4, Sox2, Klf4, and c-Myc. The resulting human iPS cells are comparable to human embryonic stem cells in morphology, gene expression, telomerase activity, and pluripotency, with capacity to differentiate into cell types from all three germ layers in vitro and form teratomas in vivo.

Cell source
Adult human dermal fibroblasts (HDF) from facial dermis
Application
Disease modeling and regenerative medicine

Protocol overview

49 steps across 9 phases

Phase 1 · Lentiviral Transduction of Slc7a1 Receptor Day -2 to Day 0
  1. a Plate 293FT cells for lentivirus production
  2. b Transfect 293FT cells with lentiviral plasmids
  3. c Harvest lentiviral supernatant
  4. d Transduce human fibroblasts with lentivirus
Phase 2 · Retroviral Transduction of Reprogramming Factors Day 0 to Day 6
  1. a Plate PLAT-E packaging cells
  2. b Transfect PLAT-E cells with reprogramming factor constructs
  3. c Collect first retroviral supernatant
  4. d Collect second retroviral supernatant
  5. e Mix and transduce HDF-Slc7a1 with four retroviruses
  6. f Replace medium with second viral supernatant
  7. g Culture transduced cells
Phase 3 · Plating on Feeder Cells and Initial Selection Day 6 to Day 30
  1. a Harvest transduced fibroblasts by trypsinization
  2. b Plate harvested cells onto mitomycin C-treated SNL feeder cells
  3. c Change medium to primate ES cell medium with bFGF
  4. d Monitor colony morphology and continue culture
  5. e Pick hES-like colonies at day 30
  6. f Mechanically disaggregate colonies into small clumps
Phase 4 · Expansion of iPS Cell Lines Day 30 onwards (Passage 1+)
  1. a Transfer cell clumps to SNL feeder cells in 24-well plates
  2. b Culture iPS cells on SNL feeders
  3. c Passage iPS cells by mechanical dissociation
  4. d Re-plate iPS cell clumps on fresh SNL feeders
  5. e Monitor cell morphology and pluripotency markers
Phase 5 · Feeder-Free Culture (Alternative) Optional, any passage
  1. a Coat plates with Matrigel
  2. b Warm Matrigel-coated plates
  3. c Seed iPS cells on Matrigel-coated plates in MEF-conditioned medium
  4. d Monitor pluripotency in feeder-free conditions
Phase 6 · Embryoid Body (EB)-Mediated Differentiation Day 0 to Day 16
  1. a Harvest iPS cells for EB formation
  2. b Suspend cells in EB differentiation medium
  3. c Plate cells in poly(2-hydroxyethyl methacrylate)-coated dishes
  4. d Culture cells in floating suspension for 8 days
  5. e Transfer EBs to gelatin-coated plates for attachment culture
  6. f Continue differentiation on gelatin-coated plates for 8 days
  7. g Verify differentiation by immunocytochemistry and RT-PCR
Phase 7 · Directed Neural Differentiation (PA6 Coculture) Day 0 to Day 18
  1. a Prepare PA6 stromal feeder layer
  2. b Seed iPS cells on PA6 feeders
  3. c Culture on PA6 for 2 weeks under differentiation conditions
  4. d Harvest and analyze differentiated neural cells
Phase 8 · Directed Cardiac Differentiation (Activin A / BMP4 Protocol) Day 0 to Day 20+
  1. a Maintain iPS cells on Matrigel-coated plates in feeder-free conditions
  2. b Induce differentiation with activin A
  3. c Add BMP4 for cardiac specification
  4. d Continue culture without cytokines
  5. e Monitor and verify cardiac differentiation
Phase 9 · Teratoma Formation and In Vivo Pluripotency Assessment Day 0 to Week 9
  1. a Harvest iPS cells for transplantation
  2. b Prepare immunodeficient mice
  3. c Inject iPS cells subcutaneously
  4. d Monitor tumor formation
  5. e Harvest tumors at week 9
  6. f Prepare histological samples
  7. g Stain and analyze teratoma tissue

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Attribution

This SOP was written by Organthis based on the published method in Takahashi et al., 2007. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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