Support to KKB, MJB, JLH and KLN was provided by the California Institute for Regenerative Medicine, the Pew Charitable Trusts Biomedical Scholars Program, the Esther B. O'Keeffe Family Foundation and the Shapiro Family Foundation. KKB is a Donald E. and Delia B. Baxter Foundation Faculty Scholar.

This method was used in the research reported in Boland et al. Nature. 461, 91-96 (2009).1
1. Preparation of Lentivirus
This protocol employs doxycycline-inducible lentiviral shuttle vectors that encode for Oct4, Sox2, Klf4, and c-Myc under control of a tetO response element. Transgenes are activated by the reverse tetracycline trans-activating protein, rtTAM2.216, which induces reprogramming factor expression in the presence of doxycycline. This system allows for tightly controlled, high expression of reprogramming factors. The lentiviral vectors used here are self-inactivating and thus cannot replicate following genomic integration. However, caution is required when working with lentiviruses and should be performed in laboratories compliant with BSL2 (USA) and S2 (Europe) standards.
<ol>
	<li>Thaw HEK293T cells and passage at least once before transfection. Cells should be maintained at subconfluent density in HEK medium at 37 &deg;C, 5% CO2 in a humidified environment. Routinely passage cells every 2 days with a split ratio of 1:6-1:10.</li>
	<li>Seed ~8 x 106 HEK293T cells/T150 with 25 ml HEK medium. Use one T150 for each lentivirus preparation.</li>
	<li>The following day transfect HEK293T cells by calcium phosphate precipitation. (Note: in our hands calcium phosphate precipitation routinely results in 80-90% transfection efficiency; however, cationic lipid transfection reagents such as Lipofectamine 2,000 may also be used). Prepare two 15 ml conical tubes for each virus to be prepared. Label the tubes "A" and "B". Tube A, 10 &mu;g of each of: the lentiviral shuttle vector encoding reprogramming factors (or rtTAM2.2), the viral packaging vectors, and plasmid encoding the viral envelope protein, VSVg. Add 186 &mu;l 2 M CaCl2 to Tube A, and bring the volume to 1.5 ml with sterile H2O. Tube B: 1.5 ml 2x HBS (pre-warmed to room temperature).</li>
	<li>Pipette the mixture in Tube A until it is a homogenous solution. Add solution A to solution B dropwise and let stand at room temperature for 2-3 min.</li>
	<li>Aspirate growth medium of HEK293T cells and replace with 22 ml pre-warmed HEK medium without penicillin and streptomycin.</li>
	<li>Pipette combined solutions AB (calcium phosphate precipitate) directly to HEK293T cells and distribute evenly by gentle rocking.</li>
	<li>24 hr after transfection of HEK-293T cells with lentivirus, remove growth medium and replace it with 25 ml of fresh, pre-warmed HEK medium. Return transfected HEKs to the incubator.</li>
	<li>48 hr after transfection of HEK-293T cells with lentivirus, collect growth media containing lentiviral particles from the transfected HEKs. Remove particulate debris from the harvested viral solution by centrifugation at 3,000 x g for 5 min at 4 &deg;C.</li>
	<li>Concentrate virus by ultracentrifugation through a 20% sucrose cushion (2 ml sucrose/25 ml viral supernatant) for 2 hr at 112,000 x g at 4 &deg;C. Suspend viral pellet in 0.4 ml MEF medium at 4 &deg;C for 15-30 min with gentle rocking. Store viral particles in single use aliquots (i.e. 50 &mu;l) at -80 &deg;C.</li>
</ol>
2. Preparation of Mouse Embryonic Fibroblasts (MEF) for Reprogramming
Note: The protocol outlined here relates to the derivation of iPSCs from E 13.5 mouse embryonic fibroblasts for use in TEC assays. While other groups have generated all-iPSC mice from adult donor cell sources, we have not tested this method on other cell types and cannot be certain that donor cell type is not a factor.
<ol>
	<li>Set up mouse timed matings. On embryonic day 13.5 (E13.5), euthanize the pregnant female and dissect the embryos from the uterine horns. Place and store embryos in 1x PBS (pre-chilled to 4 &deg;C) on ice.</li>
	<li>Remove the extraembryonic tissues (i.e. chorion, amnion and placenta). Decapitate the embryo and remove the tail (optional - if needed for genotyping) and limbs. Scoop out the internal organs, using forceps or a scoop shaped spatula and mince the remaining carcass with the blade of a scalpel or sharp scissors.</li>
	<li>Wash the minced carcass in 5 ml pre-chilled 1x PBS. Centrifuge at 200 x g for 5 min.</li>
	<li>Aspirate the supernatant. Suspend pellet in 5 ml 0.25% Trypsin-EDTA and incubate at 37 &deg;C with vigorous shaking for 20-30 min.</li>
	<li>Add 5 ml of MEF medium (pre-warmed to 37 &deg;C), mix and centrifuge at 200 x g for 5 min.</li>
	<li>Aspirate supernatant and resuspend pellet in pre-warmed MEF medium.</li>
	<li>Plate dissociated MEFs in 2-3 wells of a 6-well plate pre-coated with 0.1% gelatin. This is considered passage 1.</li>
	<li>Passage MEFs at a dilution of 1:4-1:5 every 48 hr. MEFs at passage 3 are ready for lentiviral transduction.</li>
</ol>
3. Derivation of iPSC Lines
<ol>
	<li>The day before lentiviral transduction; seed ~3 x 105 primary MEFs into one well of a 6-well plate pre-coated with 0.1% gelatin.</li>
	<li>Day 1: Primary MEFS should be 80-90% confluent for lentiviral transduction. Add lentiviral particles directly to MEF media and incubate with MEFs overnight at 37 &deg;C, 5% CO2 in a humidified environment.</li>
	<li>The following day (day 2) aspirate the medium and wash twice with 3 ml of 1x PBS to remove viral particles.</li>
	<li>Add 0.5 ml pre-warmed 0.25% Trypsin-EDTA to the cells and incubate at 37 &deg;C for 3-5 min with occasional rocking.</li>
	<li>Triturate to achieve a single-cell suspension. Observe cells by light microscopy to ensure a single cell suspension.</li>
	<li>Transfer MEFs into a 15 ml conical tube containing 5 ml MEF media. Centrifuge at 200 x g for 5 min. Aspirate the supernatant and gently resuspend cells in MEF medium.</li>
	<li>Evenly split the cell suspension between two wells of a 6 well plate pre-coated with 0.1% gelatin.</li>
	<li>Rock the plate back and forth, side to side, and once in a circular motion to achieve an even distribution of cells throughout the well. Incubate overnight at 37 &deg;C, 5% CO2 in a humidified environment.</li>
	<li>Day 3: Repeat steps 4-9 except split the cells evenly from one well to 3 wells of a 6 well plate pre-coated with 0.1% gelatin. This will yield 6 wells of transduced primary fibroblasts.</li>
	<li>Day 4: Add doxycycline (dox) at a concentration of 10 &mu;g/ml to 5/6 wells. One well should remain untreated to serve as a control.</li>
	<li>Add VPA at 1.9 mM to 3 of the 5 wells treated with dox. VPA reduces the proliferation rate of MEFs. Dense cultures of MEFs tolerate prolonged exposure to VPA whereas subconfluent cultures tend to senesce within 2-5 days. Therefore, MEFS should be 100% confluent when VPA is added. Note: We use VPA in our reprogramming experiments because it is a known epigenetic modifier, and has been shown to increase the efficiency of iPSC generation17 although the effects and mechanisms of VPA action with respect to generating fully pluripotent iPSC lines are not known.</li>
	<li>Day 5: Aspirate the medium, wash and trypsinize the cells as before. Passage the cells treated with dox/VPA to a 15 cm2 tissue culture dish pre-coated with 0.1% gelatin and each of the other conditions to pre-coated 10 cm2 dishes in ES cell medium supplemented with fresh dox and VPA.</li>
	<li>Replenish cells every day with ESC medium supplemented with fresh dox and VPA. ESC-like colonies should begin to appear after ~7 days in the dox/VPA treatment and after ~10 days in the dox alone treatment. No colonies should appear in the absence of dox treatment.</li>
	<li>Once colonies possess a bright refractive, well-defined border and contain 30-50 cells, manually isolate the colonies with a gel loading pipette tip and transfer to a U-bottom 96 well plate containing 20 &mu;l of 0.25% trypsin-EDTA. Trypsinize to single cells and transfer to feeders in a flat bottom 96 well plate in 150 &mu;l ESC medium containing dox/VPA or dox alone.</li>
	<li>Continue to clonally expand the isolated iPSC lines on feeders in ESC medium. Remove dox and VPA on day 19 after their addition (post-transduction day 23). Discard iPSC lines that do not maintain self-renewal or proliferation rates similar to ESC controls.</li>
</ol>
It may be helpful to characterize your iPSC lines in relation to ESCs before attempting to perform TEC. We have characterized our lines by 1) expression of endogenous pluripotency markers (SSEA-1, Oct4, Sox2, Nanog) by immunocytochemistry, 2) karyotype analysis by chromosome counting and 3) embryoid body formation. One may also perform lentiviral-specific RT-qPCR to confirm that the proviral transgenes are not expressed in the iPSCs. However, we have identified fully pluripotent iPSCs using only morphology, immunostaining and karyotyping. In our experiments, selection of iPSC lines based on ESC-like morphology and growth characteristics results in the majority of the lines expressing pluripotency markers while we typically identify several lines with potentially abnormal karyotypes.
4. Preparation of iPSCs for Blastocyst Injection
Passage number of a PSC line has been shown to affect its pluripotency18 although this may be line dependent19. We have used iPSCs of from passages 8-14 to produce adult all-iPSC mice.
<ol>
	<li>Thaw iPSCs and plate on feeders in ESC medium. Passage the cells at least once on feeders before use for injection.</li>
	<li>One well of a 6-well plate containing 70-80% confluent iPSCs will provide more than a sufficient number of cells for injection. Aspirate growth medium and wash the cells with ~3 ml 1x PBS (without Ca2+/Mg2+).</li>
	<li>Add 0.5 ml pre-warmed 0.05% Trypsin-EDTA to the cells and incubate at 37 &deg;C for 10 min with occasional rocking.</li>
	<li>Triturate to achieve a single-cell suspension. Observe cells by light microscopy to ensure a single cell suspension. The iPSCs need to be in single cell suspension as colonies/cell aggregates will clog the injection pipette.</li>
	<li>Once a single cell suspension has ben achieved, add 1.0 ml ESC media to the well and return the plate to the 37 &deg;C incubator. Incubate for ~15 min or until the majority of feeders have begun to adhere.</li>
	<li>Gently remove the medium containing the iPSCs taking care not to dislodge the weakly adherent feeders.</li>
	<li>Place the iPSCs in a 15 ml conical tube containing 5 ml ESC medium. Centrifuge at 200 x g for 5 min. Aspirate the supernatant and remove the remainder of ES cell medium with a micropipette. Tap the tube to dislodge the pellet and gently resuspend cells in 0.2-0.5 ml pre-chilled FHM medium. Store cells on ice until and during injection into tetraploid blastocysts.</li>
</ol>
5. Generation of Tetraploid Blastocysts
Procedures performed in this section have been described in detail elsewhere5,6,20. Here we outline our technique, optimized for the BTX Electro Cell Manipulator ECM 2001.
<ol>
	<li>Set up embryo donor mice by priming 23-28 day old female mice (C57BL/6J-Tyrc-2J /BALB/cByJ F1) with PMS and HCG. Administer 5 IU of PMS at 2PM and 5 IU of HCG 47 hr later. After HCG injection, set up female mice with C57BL/6J-Tyrc-2J /BALB/cByJ F1 stud males. Check the following day for vaginal plugs.</li>
	<li>Euthanize plugged female mice and collect oviducts. Collect 1-cell embryos by placing oviducts in FHM with Hyaluronidase and gently tearing the ampulae. Allow the cumulus masses to sit in FHM/Hyaluronidase for 5-7 min.</li>
	<li>Collect 1-cell embryos using a mouth pipette and wash through drops of FHM media before placing them in KSOM-AA culture. Culture at 37 &deg;C, 5% CO2 under mineral oil overnight and select 2-cell embryos the day of electrofusion, discard all other embryos.</li>
	<li>Place a BTX Microslide in a 10 cm Petri dish. Pour enough room temperature electrofusion media to submerge the slide in the solution, but not so much that the poles of the electrode are completely submerged.</li>
	<li>Switch on ECM 2001 and BTX Enhancer 400. Connect the ECM's cables to the microslide's electrode and fix the cables to the side of the Petri dish to prevent unintended movement of the slide.</li>
	<li>Run one manual pulse to get a reading on the BTX enhancer and note the voltage of the AC/DC currents being applied. AC current will control the speed at which the embryos will align between the electrodes, DC current will fuse the blastomeres, and pulse time will set the length of the DC pulse. A good starting point is AC 3V, DC 100V, and time 0.05 msec. The optimal DC varies in the range of 90-150 volts.</li>
	<li>Using a mouth pipette, take about 30-40 two-cell embryos from KSOM-AA culture and wash them through several drops of electrofusion medium. Draw fresh electrofusion media from the microslide dish into mouth pipette and take embryos from the wash. Place them in the 1 mm gap between the electrodes on the microslide. Be careful that they are aligned down the middle of the gap and that they are not in contact with each other.</li>
	<li>Apply AC current by pressing the manual pulse button. The embryos will rotate in the AC field, until the plane of blastomere contact is parallel to the electrodes. If embryos are not aligned in a few seconds, increase AC setting.</li>
	<li>After embryos have aligned, press the manual pulse button again to apply the DC pulse.</li>
	<li>With electrofusion medium in the pipette, collect the embryos from the microslide. Wash embryos through several drops of KSOM-AA and place them in KSOM-AA culture at 37 &deg;C, 5% CO2. The blastomere fusion should be completed in less than 30 min in culture.</li>
	<li>Repeat steps 7-11 for remaining 2-cell embryos. After subsequent fusion groups, monitor and select embryos with fused blastomeres. Successfully fused embryos will appear to be in 1-cell stage. Discard lysed and 2-cell embryos after 30 min in culture. If fusion rate is below 80%, increase voltage and/or time in increments of 5V and 0.01 msec. If lysis is above 20%, decrease DC voltage and/or time accordingly. The optimal settings in our experiments were AC 4V, DC 146V, and 0.07 msec. These settings consistently yielded 90% or higher fusion rates with little or no lysis.</li>
	<li>Continue to culture fused embryos in microdrops of KSOM-AA under mineral oil at 37 &deg;C, 5% CO2. You should expect 85-95% of fused embryos to form tetraploid (4n) blastocysts after 48 hr of incubation.</li>
</ol>
6. Microinjection of iPSCs into Tetraploid Blastocysts
We use a Nikon TE-2000U inverted microscope equipped with DIC optics and Narishige micromanipulators for blastocyst injection. Each tetraploid blastocyst is injected with 10-12 iPSCs using a standard protocol for ESC injection into mouse blastocysts that has been demonstrated in a previous JoVE publication5,20,21
<ol>
	<li>Place a 20 &mu;l drop of FHM in the center of a concave microscope slide and cover it with 150 &mu;l of mineral oil.</li>
	<li>Lower the holding pipette and microinjection needle into the FHM drop. Allow 2-3 min for both needles to partially fill with FHM.</li>
	<li>Wash 20-30 tetraploid blastocysts through drops of FHM and transfer to the FHM drop on the microscope slide.</li>
	<li>Mouth pipette iPSC mixture into the drop. It may be necessary to dilute the cell mixture in a drop of FHM beforehand if cells are too concentrated or aggregated.</li>
	<li>Pick up 100-200 cells with the injection needle.</li>
	<li>Hold the blastocyst with the inner cell mass in the 9 o'clock position. Inject cells into blastocoel by penetrating the zona pellucida and trophoblast at the 3 o'clock position. Inject 16-18 cells per blastocyst.</li>
	<li>Return iPSC complemented blastocysts to KSOM-AA culture.</li>
</ol>
7. Transfer of Complemented Tetraploid Blastocysts into the Uterine Horns of Recipient Mice
Complemented tetraploid blastocysts are surgically transferred to the uterine horns of female recipient mice according to the guidelines of the researcher's institute, using the standard technique20 which we shall briefly summarize. Select female CD-1 mice at the pro-estrus stage and set them up for mating with vasectomized males. Check for vaginal plugs the next morning. Females are ready for uterine embryo transfer two days after the plug was detected (2.5 dpc).
One day before recipient females are mated with vasectomized males, set up additional CD-1 females with non-vasectomized males to be used as foster mothers for all-iPSC mice retrieved by Caesarian section.
8. Caesarean Section and Fostering of iPSC-derived Pups
The transfer of TC embryos typically results in multiple resorptions after the implantation, even if the iPSC or ESC line has a high developmental potential. As a result, one can expect not more than 4 viable pups (usually 1-2) per recipient. These small litters are usually neglected by recipients. To increase the level of neonatal care and the rate of survival, we perform C-sections and fostering according to the standard protocols20. To perform the Caesarean section, euthanize recipient mice 16 days after embryo transfer at 7-8PM (recipient 18.5 dpc) and dissect pups from the uterine horns. Foster viable pups to CD-1 mothers that delivered litters the same day.

<ol>
	<li>Boland, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mice+generated+from+induced+pluripotent+stem+cells.">Adult mice generated from induced pluripotent stem cells.</a> Nature. 461, 91-94 (2009).</li><li>Robinton, D. A., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+induced+pluripotent+stem+cells+in+research+and+therapy.">The promise of induced pluripotent stem cells in research and therapy.</a> Nature. 481, 295-305 (2012).</li><li>Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+completely+cell+culture-derived+mice+from+early-passage+embryonic+stem+cells.">Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 90, 8424-8428 (1993).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 6209-6214 (2001).</li><li>Eggan, K., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Generation of embryonic stem (ES) cell-derived embryos and mice by tetraploid-embryo complementation. , (2006).</li><li>McLaughlin, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+tetraploid+embryos+by+electrofusion.">Production of tetraploid embryos by electrofusion.</a> Methods Enzymol. 225, 919-930 (1993).</li><li>Humpherys, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+instability+in+ES+cells+and+cloned+mice.">Epigenetic instability in ES cells and cloned mice.</a> Science. 293, 95-97 (2001).</li><li>Kang, L., Wang, J., Zhang, Y., Kou, Z., Gao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cells+can+support+full-term+development+of+tetraploid+blastocyst-complemented+embryos.">iPS cells can support full-term development of tetraploid blastocyst-complemented embryos.</a> Cell Stem Cell. 5 (09), 135-138 (2009).</li><li>Zhao, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cells+produce+viable+mice+through+tetraploid+complementation.">iPS cells produce viable mice through tetraploid complementation.</a> Nature. 461, 86-90 (2009).</li><li>Kang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viable+mice+produced+from+three-factor+induced+pluripotent+stem+(iPS)+cells+through+tetraploid+complementation.">Viable mice produced from three-factor induced pluripotent stem (iPS) cells through tetraploid complementation.</a> Cell Res. 21, 546-549 (2011).</li><li>Zhao, X. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viable+Fertile+Mice+Generated+from+Fully+Pluripotent+iPS+Cells+Derived+from+Adult+Somatic+Cells.">Viable Fertile Mice Generated from Fully Pluripotent iPS Cells Derived from Adult Somatic Cells.</a> Stem Cell Reviews and Reports. 6, 390-397 (2010).</li><li>Han, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tbx3+improves+the+germ-line+competency+of+induced+pluripotent+stem+cells.">Tbx3 improves the germ-line competency of induced pluripotent stem cells.</a> Nature. 463, 1096-1100 (2010).</li><li>Stadtfeld, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbic+acid+prevents+loss+of+Dlk1-Dio3+imprinting+and+facilitates+generation+of+all-iPS+cell+mice+from+terminally+differentiated+B+cells.">Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells.</a> Nat. Genet. 44, 398-405 (2012).</li><li>Stadtfeld, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+silencing+of+imprinted+genes+on+chromosome+12qF1+in+mouse+induced+pluripotent+stem+cells.">Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells.</a> Nature. 465, 175-181 (2010).</li><li>Carey, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+factor+stoichiometry+influences+the+epigenetic+state+and+biological+properties+of+induced+pluripotent+stem+cells.">Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells.</a> Cell Stem Cell. 9, 588-598 (2011).</li><li>Go, W. Y., Ho, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+direct+comparison+of+the+dimerizer+and+reverse+tet+transcriptional+control+systems.">Optimization and direct comparison of the dimerizer and reverse tet transcriptional control systems.</a> The Journal of Gene Medicine. 4, 258-270 (2002).</li><li>Huangfu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+by+defined+factors+is+greatly+improved+by+small-molecule+compounds.">Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.</a> Nature. 26, 795-797 (2008).</li><li>Li, X. -. y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passage+number+affects+the+pluripotency+of+mouse+embryonic+stem+cells+as+judged+by+tetraploid+embryo+aggregation.">Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation.</a> Cell and Tissue Research. 327, 607-614 (2007).</li><li>George, S. H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+adult+phenotyping+directly+from+mutant+embryonic+stem+cells.">Developmental and adult phenotyping directly from mutant embryonic stem cells.</a> Proceedings of the National Academy of Sciences. 104, 4455-4460 (2007).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: A laboratory manual. , (2003).</li><li>Kirak, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transnuclear+Mice+with+Pre-defined+T+Cell+Receptor+Specificities+Against+Toxoplasma+gondii+Obtained+Via+SCNT.">Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT.</a> J. Vis. Exp. (43), e2168 (2010).</li></ol>

No conflicts of interest declared.

Generating mice from iPSC lines using TEC assays provides a stringent functional test for the pluripotency of an iPSC line. This test may be useful to assess the relative efficacy of different reprogramming methods or to identify iPSC lines that may be most useful for generating certain cell types in vitro. Mice generated from iPSCs may be used to stringently test the long-term stability and tumorigenicity of iPSC-derived tissues. This protocol will be useful to investigators wishing to generate fully pluripotent iPSC lines or iPSC mice or to compare the relative utility of different reprogramming methods.
The mechanisms that control the generation and identification of fully pluripotent iPSCs remain poorly understood and it is possible that some iPSC lines produced using this method will not pass the TEC test. Many factors may vary between experiments including genetic backgrounds, lentiviral titer, patterns of lentiviral insertion, cell cycle parameters of the donor population, inter-laboratory differences in various steps of the TEC procedure and variable propensities of iPSCs to harbor genetic or epigenetic aberrations. To best ensure success, we take care to establish appropriate levels of lentiviral gene expression in iPSC derivation experiments by testing viral dilutions on control MEFs to ensure that each virus is sufficiently concentrated to produce detectable gene expression at least 80% and ideally 100% of the MEFs. This allows us to identify lines with multiple copies of different lentiviruses while limiting toxicity to the MEFs and producing colonies without overcrowding the wells. It should be noted that multiple other protocols have been shown to produce iPSCs with full developmental potential, using multiple methods and donor cell sources suggesting that multiple paths to full pluripotency may exist1,8-13,15. At present, however, no definitive biomarker of fully pluripotent iPSC has been identified and therefore the TEC assay remains the gold standard test of whether an iPSC line can generate all cell lineages in an organism.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM (high glucose)</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ES cell qualified FBS</td>
			<td>Invitrogen</td>
			<td>104392-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>16140-071</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>Sigma M7522</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.1% Gelatin</td>
			<td>Millipore</td>
			<td>ES006-B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>Invitrogen</td>
			<td>11140</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Invitrogen</td>
			<td>11150-059</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ESGRO (murine LIF)</td>
			<td>Millipore</td>
			<td>ESG1106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Valproic Acid</td>
			<td>Sigma</td>
			<td>P4543</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher</td>
			<td>BP231-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS Ca2+/Mg2+</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS Ca2+/Mg2+ free</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin, for superovulation, freeze-dried, 2,000 IU</td>
			<td>Harbor-UCLA Research Institute</td>
			<td>n/a</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chorionic gonadotropin, human</td>
			<td>Sigma</td>
			<td>C1063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FHM medium with Hyaluronidase</td>
			<td>Millipore</td>
			<td>MR-056-F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KSOM-1/2 AA medium</td>
			<td>Millipore</td>
			<td>MR-106-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FHM</td>
			<td>Millipore</td>
			<td>MR-024-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water, for embryo transfer, embryo tested</td>
			<td>Sigma</td>
			<td>W1503</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mineral oil, embryo tested</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma</td>
			<td>M4125</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), embryo tested</td>
			<td>Sigma</td>
			<td>A3311</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts, non-irradiated</td>
			<td>Millipore</td>
			<td>PMEF-CFL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			Media and buffers used in this protocol
			HEK293T growth medium. 90% DMEM, 10% FBS, 100 U/ml penicillin and 10 mg/ml streptomycin. Exclude penicillin and streptomycin from HEK media used on day of transfection. HEK medium can be stored at 4 &deg;C for up to 1 month.
			2x HBS. 42 mM Hepes, 274 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4&middot;7H2O, 12 mM Dextrose. pH to 7.1 +/- 0.1. pH is critical! Sterile filter and store at 4 &deg;C.
			Mouse embryonic fibroblast (MEF) growth medium (also for use with feeders). 70% DMEM, 20% Medium 199, 10% FBS, 100 U/ml penicillin and 10 mg/ml streptomycin. Store at 4 &deg;C for up to 1 month.
			ESC growth medium. 85% DMEM,15% ES cell qualified FBS, 1x Glutamax, 0.1 mM non-essential amino acids, 0.1 mM &beta;-mercaptoethanol, 1,000 U/ml ESGRO, 100 U/ml penicillin and 10 mg/ml streptomycin. ESC media can be stored at 4 &deg;C for up to three weeks.
			Electrofusion medium. 0.3 M Mannitol, 0.1 mM MgSO4, 50 mM CaCl2, and 3% BSA in embryo tested water. Store at 4 &deg;C for up to 3 months.</td>
		</tr>
	</tbody>
</table>

generation of mice derived from induced pluripotent stem cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

In step 3, "Derivation of iPSCs from MEFs", one should observe morphological heterogeneity and immature iPSC colony formation starting 4-5 days after doxycycline/VPA addition and mature colonies between 7-10 days (Figure 2). The production of one-cell tetraploid embryos in step 5 is highly efficient (Figure 3). We routinely observe up to 95% of treated two-cell embryos successfully fuse to produce tetraploid one-cell embryos. The protocol followed to inject iPSCs into tetraploid blastocysts (Step 6, Figure 4) is similar to the protocol for injection of ESCs into diploid blastocysts to generate chimeric mice, and can be performed by an experienced microinjectionist. The number of live pups born depends on the cell line (Table 1).
<table border="1">
	<tbody>
		<tr>
			<td colspan="5">MEF preprogramming efficiency 0.01-0.03%</td>
		</tr>
		<tr>
			<td colspan="5">Efficiency of iPSC mouse production by TEC</td>
		</tr>
		<tr valign="top">
			<td>Name</td>
			<td>Description</td>
			<td>Blastocysts injected</td>
			<td>Live Newborn</td>
			<td>Live Adult</td>
		</tr>
		<tr valign="top">
			<td>iMZ-21</td>
			<td>iPSC</td>
			<td>867</td>
			<td>53 (6.1%)</td>
			<td>19 (2.2%)</td>
		</tr>
		<tr valign="top">
			<td>iMZ-9</td>
			<td>iPSC</td>
			<td>195</td>
			<td>7 (3.6%)</td>
			<td>4 (2.1%)</td>
		</tr>
		<tr valign="top">
			<td>iMZ-11</td>
			<td>iPSC</td>
			<td>338</td>
			<td>1(0.3%)</td>
			<td>0 (0%)</td>
		</tr>
	</tbody>
</table>
Table 1. Representative Results.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/4003/4003fig1highres.jpg" src="/files/ftp_upload/4003/4003fig1.jpg" /> 
Figure 1. Schematic of experimental design. Top left: Production of tetraploid blastocysts. Fertilized two-cell embryos from albino mice are electrofused to generate tetraploid one-cell embryos, which are cultured in vitro to the blastocyst stage. Bottom left: Reprogramming. Mouse embryonic fibroblasts are transduced with lentiviral particles encoding Oct4, Sox2, Klf4 and c-Myc and the reverse tetracycline transactivating protein, rtTAM2.2. Addition of doxycycline results in transgene expression and the initiation of reprogramming to iPSCs. Right: Production of iPSC mice. iPSCs derived from pigmented mice are injected into the blastocoel of tetraploid blastocysts and then surgically implanted into pseudo-pregnant recipient mice. Newborn iPSC mice are delivered by Caesarian section and cross-fostered. <a href="https://www.jove.com/files/ftp_upload/4003/4003fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/4003/4003fig2highres.jpg" src="/files/ftp_upload/4003/4003fig2.jpg" /> 
Figure 2. Morphological changes associated with reprogramming. From left to right: Examples of the morphological progression from fibroblasts to iPSC colonies during the course of a reprogramming experiment. <a href="https://www.jove.com/files/ftp_upload/4003/4003fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4003/4003fig3.jpg" /> 
Figure 3. Production of tetraploid embryos. Diploid two-cell embryos are subjected to an electric pulse resulting in blastomere fusion and generation of one-cell tetraploid embryos.
<img alt="Figure 4" src="/files/ftp_upload/4003/4003fig4.jpg" /> 
Figure 4. Production of iPSC mice. Left: iPSCs are injected into the blastocoel of a tetraploid blastocyst. Middle: Newborn iPSC mice are distinguished by pigmented eyes. Right: iPSC mouse at three weeks post-delivery.

Watch this Scientific Journal Video about Generation of Mice Derived from Induced Pluripotent Stem Cells at JoVE.com

Generation of Mice Derived from Induced Pluripotent Stem Cells

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

generation-of-mice-derived-from-induced-pluripotent-stem-cells

Research

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Biology

21.1K ビュー.  The Scripps Research Institute. 彼らは多能性のための標準的なテストを通過した時、誘導多能性幹細胞（IPSC）ラインを生成することはあっても発生能の異なる行が生成されます。ここでは、所有している完全な多能性としてIPSC行を定義性IPSC、から完全に由来するマウスを作製するためのプロトコルを記述する 1。

ビデオ: Generation of Mice Derived from Induced Pluripotent Stem Cells

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

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.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.