We would like to acknowledge the financial support from the Monash Larkins Program as well as from a NHMRC CDF and a NHMRC project grant. Furthermore, we would like to thank Sue Mei Lim for her constructive suggestions, Edwina McGlinn for her support and the Monash Flowcore team (in particular Adam Dinsdale) for their help in producing the video.

1. Instrument Settings/Reagent Preparation/Genotyping
<ol>
	<li>Prepare iPS cell medium: Supplement 500 ml Knockout DMEM media with 75 ml fetal bovine serum (FBS), 5 ml L-glutamine, 5 ml Non-essential amino acids, 500 &#181;l &#946;-Mercaptoethanol, 5 x 105 units Leukaemia inhibitory factor (LIF). Please refer to Materials List for product and purchasing information for reagents used in the context of this manuscript.</li>
	<li>Prepare MEF medium: Supplement 500 ml DMEM media with 50 ml FBS, 5 ml L-glutamine, 5 ml Non-essential amino acids, 5 ml Sodium pyruvate, 500 &#181;l &#946;-Mercaptoethanol.</li>
	<li>Prepare freezing medium: For 10 ml, combine 9 ml of FBS with 1 ml of DMSO.</li>
	<li>Prepare Gelatin coated dishes Add 3-5 ml 0.1% porcine gelatin solution to a T75 flask, incubate at RT for at least 10 min and aspirate right before use.</li>
	<li>Prepare FACS labeling medium: For 10 ml, combine 9.9 ml of PBS with 0.1 ml of FBS.</li>
	<li>Perform a genotyping PCR for the Rosa26 m2rtTA locus: Perform reactions with Taq DNA polymerase according to manufacturer&#8217;s instructions. Use a modified MgCl2-concentration of 3.5 mM and the following 3 primers: oIMR8052: 5&#8217; GCG AAG AGT TTG TCC TCA ACC 3&#8217;, oIMR8545: 5&#8217;AAA GTC GCT CTG AGT TGT TAT 3&#8217;, oIMR8546: 5&#8217; GGA GCG GGA GAA ATG GAT ATG 3&#8217;. Cycling conditions: 94 &#176;C for 3 min; 35 cycles of (94 &#176;C for 30 sec, 65 &#176;C for 1 min, 72 &#176;C for 1 min); 72 &#176;C for 2 min; hold at 12 &#176;C. Expected product size: 650 bp for wild type allele and 340 bp for the mutant allele.</li>
	<li>Perform a genotyping PCR for the Collagen-StemCa/OKSM locus: Perform reactions with Taq DNA polymerase as per instructions. Use a modified MgCl2-concentration of 2.5 mM and the following 3 primers: col/frt-B: 5&#8217; CCCTCCATGTGTGACCAAGG 3&#8217;, col/frtA1: 5&#8217; GCACAGCATTGCGGACATGC 3&#8217;, col/frtC1: 5&#8217; GCAGAAGCGCGGCCGTCTGG 3&#8217;. Cycling conditions: 95 &#176;C for 1 min: 2 cycles of (94 &#176;C for 30 sec, 70 &#176;C for 45 sec), 6 cycles of (94 &#176;C for 30 sec, 68 &#176;C for 45 sec) and 30 cycles of (94 &#176;C for 20 sec, 60 &#176;C for 1 min); 72 &#176;C for 5 min; hold at 12 &#176;C. Expected product size: 331 bp for wild type allele and 551 bp for mutant allele.</li>
	<li>Carry out all centrifugation steps at 400 x g for 3 min at 4 &#176;C.</li>
</ol>
2. Generation of Mouse Embryonic Fibroblasts
<ol>
	<li>Perform a timed mating between an animal of the OKSM strain with a member of the opposite gender of the m2rtTA strain. Harvest embryos at embryonic day 13.5 by culling the mother and removing the uterine horn. Prior to removal of the uterine horn, spray the abdominal region with an 80% ethanol solution, to avoid microbial contamination.</li>
	<li>Transfer the uterine horn into a 10 cm tissue culture dish containing 10 ml of sterile Phosphate Buffered Saline (PBS). Use sterilized surgical grade scissors to cut the uterine horn into pieces containing one embryo (see Figure 3A,B).</li>
	<li>Use a dissection microscope (ideally placed within a tissue culture hood) and sterilized forceps to carefully remove the uterine envelope and the extra-embryonic membranes surrounding each embryo. Transfer each embryo to a separate 10 cm dish with 10 ml of PBS.</li>
	<li>Proceed by removing the embryo&#8217;s head, limbs, tail and internal organs (heart, liver, intestine etc.) with the forceps (see Figure 3C). Transfer the embryo&#8217;s head into a 1.5 ml tube and freeze down so it can be used for genotyping if necessary.</li>
	<li>Transfer the embryo&#8217;s torso into an empty 10cm plate and use two surgical blades to mince the embryo for 2 min. Add 200 &#181;l of trypsin/EDTA solution on top of the minced embryo and incubate for 3-5 min at RT. Continue mincing for an additional 2 min, add 2 ml of MEF media to inactivate trypsin, and then transfer into a 15 ml tube.</li>
	<li>Use a 1,000 &#181;l pipette to further mechanically dissociate the tissue by gentle pipetting. Transfer to a gelatin coated 10 cm dish and add an additional 10 ml of MEF media. Note: Both normoxic and hypoxic incubators can be used for culture, refer to discussion for more information.</li>
	<li>After 24-48 hr the plate should be densely covered with MEFs.</li>
	<li>Alternatively, the cells can then be further propagated or frozen down. For the freezing of cells, remove the culture media, rinse the dish with 10 ml PBS to remove traces of media and overlay with 3 ml Trypsin/EDTA solution. Incubate for 3-5 min at 37 &#176;C, inactivate trypsin digestion by adding 5 ml of MEF media and transfer to a 15 ml centrifugation tube. Spin down and resuspend the pellet in 3 ml freezing media. Transfer to 3 cryovials and freeze down in a cell freezing container.</li>
</ol>
3. Reprogramming of MEFs
NOTE: Pellet cells by centrifugation at 200 x g for 5 min at 4 &#176;C and use normoxic tissue culture incubators for reprogramming. It can be beneficial to derive and expand MEFs under hypoxic conditions (5% oxygen, see discussion for more information).
<ol>
	<li>Quickly thaw a low passage cryovial (P0-P1, 2-3 Mio cells) in a water bath (37 &#176;C) and transfer the contents into a tube with 10 ml of pre-warmed MEF media. Pellet cells, resuspend in 12 ml fresh MEF media, transfer into a gelatin coated T75 culture vessel, and allow cells to recover for 24-48 hr before proceeding. Note: MEFs can also be used directly after derivation.</li>
	<li>Following the removal of culture media, wash MEFs with PBS and cellularize by overlaying with a Trypsin/EDTA solution (3 ml for 3-5 min at 37 &#176;C). Quench trypsin by adding 5 ml of MEF media and further dissociate cells by gentle mixing with a 10 ml pipette. Determine cell numbers using a hemocytometer.</li>
	<li>For reprogramming experiments, seed MEFs onto gelatin coated T75 flasks at densities of 6.7 x 103 cells/cm2 (~0.5 Mio cells per flask) in iPSC media containing 2 &#181;g/ml doxycycline. Refer to Table 1 regarding seeding recommendations to obtain around 2 Mio reprogramming intermediates for each time point. Note: Reprogramming commences with the addition of doxycycline (2 &#956;g/ml) supplemented media</li>
	<li>For the first 6 days replace media every other day with fresh doxycycline supplemented iPSC media (12 ml of media per T75 flask). Beyond this point renew media on a daily basis if there are high cell numbers. Double the culture volume if media changes can only be performed every alternate day.</li>
	<li>Harvest reprogramming intermediates at the required time points (suggestion: Days 3, 6, 9, 12) by removing media, rinsing with appropriate volume of PBS (10 ml for a T75 flask) and overlaying with Trypsin-EDTA solution (3 ml for a T75 flask). Incubate for 3-5 min at 37 &#176;C and quench by adding 5 ml of iPSC media followed by gentle pipetting.</li>
	<li>Count cell suspension with a hemocytometer (see above), pellet by centrifugation, aspirate the supernatant and resuspend cells in 10 ml of PBS to remove any traces of trypsin. Pellet cells once again, aspirate supernatant and proceed with step 4.1 to isolate the reprogramming intermediates. Note: Cells undergoing reprogramming can be cryopreserved and thawed for FACS isolation at a later stage.</li>
	<li>To establish fully reprogrammed iPS cultures, grow cells in dox-free iPSC media for a further 4-7 days. Note: Successfully reprogramming cultures will contain a large number of colonies by day 12 (see Figure 4). During this time, aberrant iPS cells that still require OKSM expression will disappear.</li>
	<li>Alternatively, to propagate the remaining fully reprogrammed iPS cultures: initially, seed irradiated MEFs at a density of 15 x 103 cell/cm2 in 12 ml of iPSC media onto a gelatin coated T75 flask one day or 6 hr prior to experiments.</li>
	<li>Next, cellularize iPS cell cultures by removing media, rinsing the T75 flask with 10 ml of PBS, overlaying with 3 ml of Trypsin-EDTA solution and incubating for 3-5 min at 37 &#176;C. Quench trypsin by adding 5 ml of iPSC media followed by gentle mixing, transfer to a 15 ml conical tube, spin down and then resuspended in 10 ml iPS media.</li>
	<li>Transfer 500 &#181;l of this cell suspension onto the irradiated MEFs (T75 flask) and allow the cultures to grow for 4-5 days. Note: Established iPS cultures can be cryopreserved or used for experiments. Clonal cell lines can be derived by picking individual iPSC colonies under a dissecting microscope 22.</li>
	<li>Cryo preserve confluent iPSC cultures as described in 2.8 for MEFs.</li>
</ol>
4. Antibody Labeling
<ol>
	<li>Resuspend cell pellets of reprogramming cultures, as prepared in Section 3, in FACS labeling media supplemented with antibodies (anti-Thy-1.2 pacific blue 1:400, anti-Ssea-1 biotin 1:400 and anti-Epcam Fitc 1:400). Use 200 &#181;l of supplemented labeling mix per 5 million cells and incubate on ice.</li>
	<li>After 10 min gently tap the tube to resuspend the cells and incubate for an additional 10 min on ice. Add 10 ml of cold PBS per tube and spin down.</li>
	<li>For each tube to be stained prepare 200 &#181;l of labeling media supplemented with 1 &#181;l Streptavidin-PeCy7 (1:200) per 5 million cells. When cells have pelleted, carefully aspirate the supernatant and resuspend in an appropriate volume of labeling media supplemented with Streptavidin-PeCy7.</li>
	<li>Keep cells on ice for 10 min, tap to resuspend, incubate for another 10 min on ice, add 10 ml of cold PBS per tube, spin down and aspirate supernatant.</li>
	<li>Resuspend cell pellets in labeling media supplemented with propidium iodide (PI) (2 &#181;g/ml) at approximately 1 x 107 cells/ml. Remove cell clumps by passing the suspension through a 70 &#181;m strainer. Transfer cells to an appropriate FACS tube and keep them on ice.</li>
	<li>Prepare separate samples with individual antibodies (anti-Thy-1.2, anti-Ssea-1 and anti-Epcam). NOTE: These are required to perform color compensation in the three channels used in the analysis. In addition, unlabeled cells are needed to set voltages and gates for sorting. Ideally iPS cells are used for compensation: stocks of 0.5-1 x 106 iPS cells maintained on irradiated MEFs are stored in cryovials in liquid nitrogen. The presence of MEFs is essential to get a signal in the Thy-1.2 channel.</li>
	<li>Thaw a cryovial of iPS cells as described for MEFs (section 3). Following centrifugation, resuspend cells in 800 &#181;l of labeling buffer and 200 &#181;l of this sample is set aside as the unlabeled control.</li>
	<li>Use the remaining cells as compensation controls. Divide cells between three 15 ml tubes and add antibodies as follows: Pacific Blue compensation control: add 0.5 &#181;l of the anti-Thy-1.2 Pacific Blue antibody. Fitc compensation tube: add 0.5 &#181;l of the anti-Epcam-Fitc antibody. Pe-Cy7 compensation control: 0.5 &#181;l of the anti Ssea-1-Biotin antibody.</li>
	<li>Keep cell-antibody samples on ice for 10 min, mix by tapping and incubate for 10 min on ice.</li>
	<li>Wash samples with 10 ml cold PBS to remove unbound antibody, spin cells down and aspirate the supernatant. Resuspend cell pellets in 200 &#181;l of FACS labeling media.</li>
	<li>For anti-Ssea-1-Biotin labeled samples, add a Streptavidin-PeCy7 conjugate (1 &#181;l per 200 &#181;l cells). Label cells as described for primary antibody (Ssea-1-Biotin).</li>
	<li>Pass all samples including the unlabeled control through a 70 &#181;m strainer and add PI (2 &#181;g/ml). Transfer cells to FACS tubes and keep on ice until required.</li>
</ol>
5. FACS Isolation of Intermediates
NOTE: Cells are sorted using a Fluorescence Activated Cell Sorter with 405 nm, 488 nm and 560 nm excitation lasers and a 100 &#181;m nozzle.
<ol>
	<li>Set up voltages for forward and side scatter so that the cell population is properly visualized. Draw a gate around cells as indicated in Figure 5A to exclude debris. Note: The correct set up of voltages on the cell sorter can be complex and requires an experienced FACS operator.</li>
	<li>Use Forward scatter (FSC) height versus FSC area to exclude aggregates (Figure 5B).</li>
	<li>Visualize the PI channel versus FSC to gate in on the PI negative live cells (Figure 5C).</li>
	<li>Use the unlabeled cells to adjust the voltages for the fluorescent channels of PB, Fitc/GFP, Pe-Cy7. Ideally position the cell population at the lower end of the respective channel.</li>
	<li>Set gates with the unlabeled control cells as shown in Figure 6A,B to sub fraction the reprogramming cultures into day 3, 6 and 9 populations: Ssea-1-/Thy-1.2+ cells; Ssea-1-/Thy-1.2- cells; and the reprogramming intermediates Ssea-1+/Thy-1.2- cells.</li>
	<li>Further subdivide the Day 9 Ssea-1+/Thy-1.2- fraction using the Pe-Cy7 versus Fitc blot; draw gates around the Sse-a1+/Epcam+ cells and the Ssea-1+/Epcam- cells. Set gates with the unlabeled control cells as shown in Figure 6C,D.</li>
	<li>Prefill appropriate collection tubes with iPS cell media (1-2 ml) before sorting the desired subpopulations (see Figure 7 for representative FACS profiles for MEFs, Day 3/Day 6/Day 9/Day 12 cultures and iPS cells).</li>
	<li>Perform FACS sorting according to manufactures protocol.</li>
	<li>After FACS isolation submit cells to molecular profiling (RNA/ protein extraction, chromatin immunoprecipitation, DNA methylome analysis etc.). Alternatively, the various cell fractions may be cultured.</li>
</ol>

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The authors have nothing to disclose.

In order to successfully reprogram MEFs into iPS cells and to purify reprogramming intermediates at high quantity it is essential to be aware of factors that have an impact on the overall efficiency. In particular the batch of FBS used to supplement iPS media can have a detrimental effect. In general positive experiences were made with Embryonic stem (ES) cell qualified FBS but batch testing of sera from a variety of vendors might identify a cheaper alternative.
Another factor that impacts on reprogramming efficiency is the genotype of MEFs 15,20. Although MEFs derived from mice heterozygous (het) for both the OKSM and the m2rtTA locus do reprogram, homozygousity at one or both loci results in higher reprogramming efficiencies. Indeed, MEFs derived from the offspring of OKSM and m2rtTA crosses show an increase in reprogramming efficiency in the following order (OKSM/m2rtTA): het/het &#60; homo/het &#60; het/homo &#60; homo/homo (please note that the numbers given in Table 1 are for het/het animals). On the rare occasion a male homo/homo animal is identified, a harem mating with multiple m2rtTA females can be set up. All offspring from these crosses will be het/homo for the OKSM/m2rtTA loci, respectively. In addition, the rapid genotyping of litters is recommended as larger litters are obtained when using younger mice for breeding (6-15 weeks of age).
Moreover, the use of low passage MEFs for reprogramming is emphasized 23. If MEFs were derived and expanded in a normoxic tissue culture incubator we strongly recommend to only use these cells up to passage 2. However, if the experimenter has access to a low oxygen incubator (5% oxygen) for the derivation and expansion of MEFs, passage numbers as high as 3 will still yield good results 23.
In case the experimenter encounters problems associated with reprogramming, as outlined, the most likely explanations are high passage numbers at the time of dox addition, incorrect genotype of the mice or the use of FBS that is not conducive for iPS cell generation. However, in rare cases we observed that MEFs were not able to reprogram even under ideal conditions. In these cases a spontaneous de novo deletion within the m2rtTA&#8217;s open reading frame was identified as the underlying reason.
This methodology was successfully adapted to extract Ssea-1+ intermediates via magnetic cell isolation (MACS). There is a clear time advantage in using MACS, however, the Ssea-1+ cell populations&#8217; purity is reduced to 80-90%, rather than more than 95%, which depending on the type of experiment the cells are destined for might pose a problem. Thus, an option is to use MACS to enrich for Ssea-1+ cells followed by FACS to increase purity and/or fraction them into Ssea-1+/Epcam+ and Ssea-1+/Epcam- cells.
While the iPS cells produced by the outlined protocol and mouse model have been shown to be pluripotent and effectively contribute to all tissues of chimeric animals 15,20, a reduced capacity to produce embryos entirely composed of these iPS cells via tetraploid complementation has been demonstrated 24. Aberrant methylation patterns at the Dlk-Dio3 gene cluster have been identified as the underlying cause 24. However, addition of ascorbic acid at a concentration of 50 &#181;g/ml to the media during reprogramming will produce tetraploid complementation competent iPS cells 24. Please be advised that an alternative reprogrammable mouse model engineered to express the reprogramming factors at a different stoichiometry can produce tetraploid competent iPS cells even in the absence of ascorbic acid treatment 25. However, in our hands cells from this strain reprogram at considerably lower frequency compared to the herein used mouse model.
The methodology described in this manuscript allows the separation of reprogramming intermediates from the refractory bulk of the population and should be seen as valuable tool to dissect and understand the reprogramming process, removing the previous limitations of having to use an unfractioned population for profiling (e.g., high signal noise from the refractory cells). The antibody combination described herein allows isolation of intermediates from mouse cells at high purity, however it is possible that additional cell surface markers will be uncovered in the future that will allow to obtain intermediates at even higher purity. Please note that Ssea-1 expression is not a hallmark of human induced pluripotent stem cells and as such this protocol cannot be used on reprogramming cells of human origin. In conclusion, reprogramming intermediates once isolated with the described method can be used for molecular analyses including expression profiling, chromatin immunoprecipitation, methylome analysis, and protein assays.

Embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos1. Under appropriate culture conditions they self-renew and remain pluripotent. In 2006 Yamanaka and colleagues demonstrated that mature cells can be reprogrammed into so called induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct-4, Klf-4, Sox-2, c-Myc (OKSM) 2. iPS cells, like ES cells, can give rise to all cell types of the body, however, they are free of the ethical constraints surrounding the generation of ES cells3. Furthermore, iPS cells carry the promise of personalized regenerative medicine and hold tremendous potential for applications like disease modeling and in vitro drug screening4,5. In order for reprogramming technology to fulfill this potential, the basic mechanism of nuclear reprogramming needs to be fully understood. However, efforts to dissect the reprogramming pathway have been hampered by the fact that only a very small number of cells reprogram (0.1-1%). Successfully reprogramming fibroblasts have been reported to undergo a distinct series of events including a mesenchymal to epithelial transition 6-10 and, in the final stages of reprogramming, activation of the endogenous core pluripotency network 11-14. We and others 12,13,15-17 have recently identified a set of cell surface markers that allows for the separation of rare intermediates from the refractory bulk population. Reprogramming mouse embryonic fibroblasts (MEFs) undergo changes in the expression of Thy-1.2, Ssea1 and Epcam (among others) during the 2-week-long reprogramming process15. Early during reprogramming a subset of MEFs down-regulate expression of fibroblast identity marker (Thy-1.2) and then start expressing the pluripotency-associated marker Ssea-112. During the final stages of reprogramming Ssea1-positive cells reactivate endogenous pluripotency genes such as Oct-410-13,15. This last transition is marked on the cell surface by detectable expression of Epcam (see Figure 1) or in a later stage Pecam 15. Recently, O&#8217;Malley et al. reported the use of CD44 and iCAM1 as alternatives or complementary to Thy-1.2 and Ssea-1 for the identification of reprogramming intermediates.&#160;We have previously FACS extracted reprogramming intermediates from Day 0, Day 3, Day 6, Day 9 and Day 12 reprogramming cultures, as well as from established iPS cell lines based on these cell surface markers 15,18. For the below described reprogramming system and conditions we have shown at the single cell level that although the populations are quiet homogenous, there is a certain degree of heterogeneity in the identified intermediate populations. It should be noted that only a subset of cells within these populations are able to progress to the respective next stage of the reprogramming process and give rise to iPS cell colonies at different efficiencies, which have been extensively characterized previously15,19. Moreover, the reprogramming efficiency of these populations will depend as well on the re-plating and culture conditions. To increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and a polycistronic OKSM cassette under control of a doxycycline responsive promoter20,21. Using this mouse model circumvents the unwanted side effects of traditional viral methods of iPS cell generation, i.e., a heterogeneous starting population with cell to cell variability in number and location of integration sites of viral inserts. Two transgenic mouse strains (OKSM, m2rtTA), available as homozygous founder animals at the Jackson Laboratory, have to be crossed in order to establish the reprogrammable mouse model (see Figure 2). In this manuscript we describe in detail how to derive MEFs, generate iPS cells, and isolate the reprogramming intermediates at various stages of the conversion process by FACS.

<table><tbody><tr><td>Anti-mouse CD90.2 (Thy1.2) Pacific Blue&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>48-0902-82&amp;nbsp;</td><td></td></tr><tr><td>Anti-Human/Mouse SSEA-1 Biotin&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>13-8813&amp;nbsp;</td><td></td></tr><tr><td>Streptavidin PE-Cy 7&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>25-4317-82&amp;nbsp;</td><td></td></tr><tr><td>Anti-Mouse CD326(Epcam) Fitc&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>48-5791-82&amp;nbsp;</td><td></td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11360-070&amp;nbsp;</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids (NEAA)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11140-050&amp;nbsp;</td><td></td></tr><tr><td>Embryonic Stem Cell qualified Foetal Bovine Serum (FBS)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>10439024</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>25200-056&amp;nbsp;</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies&amp;nbsp;</td><td>35050-070&amp;nbsp;</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>21985-023&amp;nbsp;</td><td></td></tr><tr><td>DMSO (Dimethyl Sulfoxide)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>15140</td><td></td></tr><tr><td>Propidium Iodide solution (PI)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P4864-10ML&amp;nbsp;</td><td></td></tr><tr><td>Gelatine from porcine skin&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G1890&amp;nbsp;</td><td></td></tr><tr><td>Doxycyclin hyclate&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>33429-100MG-R&amp;nbsp;</td><td></td></tr><tr><td>Leukemia Inhibitory Factor (LIF)&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>ESG1107&amp;nbsp;</td><td></td></tr><tr><td>DMEM High Glucose&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11960-044&amp;nbsp;</td><td></td></tr><tr><td>DPBS (Dulbecco&#039;s phosphate buffer saline)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>14190-144&amp;nbsp;</td><td></td></tr><tr><td>KnockOut DMEM&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>10829-018&amp;nbsp;</td><td></td></tr><tr><td>Irradiated MEFs&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>S1520-100&amp;nbsp;</td><td></td></tr><tr><td>75-cm&lt;sup&gt;2&lt;/sup&gt; Tissue culture flasks&amp;nbsp;</td><td>Corning/BD Bioscience&amp;nbsp;</td><td>430641</td><td></td></tr><tr><td>15-ml centrifuge tubes&amp;nbsp;</td><td>Corning/BD Bioscience&amp;nbsp;</td><td>430791</td><td></td></tr><tr><td>50-ml centrifuge tubes&amp;nbsp;</td><td>Corning/BD Bioscience&amp;nbsp;</td><td>430829</td><td></td></tr><tr><td>Disposable surgical blades&amp;nbsp;</td><td>Clifford Hallam</td><td>SM0501</td><td></td></tr><tr><td>Cryogenic vials&amp;nbsp;</td><td>Corning/BD Bioscience&amp;nbsp;</td><td>430487</td><td></td></tr><tr><td>70 &amp;micro;m Cell strainer&amp;nbsp;</td><td>BD Bioscience&amp;nbsp;</td><td>352340</td><td></td></tr><tr><td>Taq DNA Polymerase&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>10342-020&amp;nbsp;</td><td></td></tr></tbody></table>

cell surface marker mediated purification of ips cell intermediates from a reprogrammable mouse model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

After the dissection, disaggregation and plating of an E13.5 mouse embryo, a 10 cm dish is expected to reach confluency in approximately 1 to 2 days. At this stage, it is normal for the culture to contain some adherent pieces of tissue that have not been cellularized properly. These will disappear after passaging.
Upon doxycycline induction, reprogramming MEFs undergo distinct morphological changes. Around day 6, early colony-like patches should start to emerge (Figure 4C). These will continue to grow in size upon further culture (see Figure 4D,E). A good reprogramming experiment should result in &#62;500 colonies per T75 that was initially seeded with 5 x 105 cells (Figure 4E). Established iPS cultures possess characteristic dome shaped colonies and should mostly be devoid of differentiated cells (Figure 4F). Occasionally, additional passaging of iPS cultures may be required to remove undifferentiated/partially reprogrammed cells; a confluent flask of cells can be split at a ratio of 1:10 onto a feeder layer of irradiated MEFs.
As mentioned above, morphological and molecular changes during reprogramming are reflected by changes in the FACS profiles for Thy-1.2, Ssea-1 and ultimately Epcam (see Figure 7A-F). As reported previously12,15, while MEFs are predominantly positive for Thy-1.2 and negative for the other markers, Day 3 cultures already look markedly different. A large proportion of cells have started to down-regulate the expression of Thy-1.2 and a very small subset of these Thy-1.2 negative cells have become positive for Ssea-1, the actual reprogramming intermediate for this time point. The number of reprogramming intermediates that can be extracted from Day 3 cultures is very unpredictable as the percentage of Ssea-1+/Thy-1.2- usually lies in a range between 1-10% of viable cells. On Days 6 and 9 an increased percentage of Ssea-1+ cells, usually well above 10%, can be detected. Around day 12 a subset of Ssea-1 positive cells can be detected that also label positive for Epcam. Day 12 cultures, like Day 3 cultures, represents a bottleneck in the purification of intermediates, as the percentage of Ssea-1.2+/Epcam+ cells is variable and usually falls in the range of only 2-4% of all live cells. The expected number of reprogramming intermediates presented in Table 1 only serves as a rough approximation. Established bona fide iPS cell cultures will be strongly positive for Ssea-1 and Epcam.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig1highres.jpg" width="500" /> 
Figure 1. Surface marker changes during the reprogramming pathway: Down-regulation of fibroblast identity marker Thy-1.2 is followed by up-regulation of Ssea-1. The transition of a subset of Ssea-1 positive cells towards a pluripotent state is indicated by acquisition of Epcam around day 12. <a href="https://www.jove.com/files/ftp_upload/51728/51728fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig2highres.jpg" width="500" /> 
Figure 2. Schematic representation of the reprogrammable mouse model: m2rtTA expression is under control of the ubiquitously active Rosa26 locus. In the presence of doxycycline (dox) the m2rtTA protein binds to a tetracycline dependent promoter (tetOP) at the Collagen 1a1 (Col1a1) locus resulting in the expression of the four-factor cassette. Two bicistronic cassettes are linked by an internal ribosome entry site (IRES). Open reading frames for Oct-4/Klf-4 and Sox-2/c-Myc are fused by self-cleaving F2A and E2A sequences, respectively. <a href="https://www.jove.com/files/ftp_upload/51728/51728fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig3highres.jpg" width="500" /> 
Figure 3. Embryo dissection: (A) Transfer uterine horn into a 10 cm dish filled with 10 ml PBS and (B) cut into pieces containing one embryo. (C) Using forceps free the embryos from extra embryonic tissue and remove head, limbs, tail and internal organs. <a href="https://www.jove.com/files/ftp_upload/51728/51728fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig4highres.jpg" width="500" /> 
Figure 4. Morphological changes during reprogramming. Colony-like patches become apparent in the cultures from day 6 onwards. Bona fide iPSCs are characterized by a domed shaped morphology. (A) Day 0/MEFs, (B) Day 3, (C) Day 6, (D) Day 9, (E) Day 12, (F) iPS cell culture. Scale bar: 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/51728/51728fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig5highres.jpg" width="500" /> 
Figure 5. Basic FACS setup. (A) Exclude debris with Side Scatter versus Forward Scatter Area blot. (B) Exclude aggregates from non-debris by gating on single cell population with Forward Scatter Area versus Forward Scatter High blot. (C) Exclude dead cells from single cell population by gating on PI low events with PI channel versus Forward Scatter Area blot. <a href="https://www.jove.com/files/ftp_upload/51728/51728fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig6highres.jpg" width="500" /> 
Figure 6. Gating. (A, B) Using the unlabeled control cells set up gates for Thy-1.2+/Ssea-1- cells, Thy-1.2-/Ssea-1- cells and Thy-1.2-/Ssea-1+ cells. (C) Use the unlabeled control cells to set gates for Epcam positive and Epcam negative cells. (D) Ssea-1+/Thy-1.2- cells can be separated into an Epcam positive and into an Epcam negative population. <a href="https://www.jove.com/files/ftp_upload/51728/51728fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig7highres.jpg" width="500" /> 
Figure 7. Thy-1.2 versus Ssea-1 FACS blots at various stages of the reprogramming process. (A) Day 0/MEFs, (B) Day 3, (C) Day 6, (D) Day 9, (E) Day 12, (F) iPS cell culture.
Table 1. Suggested seeding density, flask number and expected outcome for P1 MEFs of an embryo heterozygous for OKSM and m2rtTA. <a href="https://www.jove.com/files/ftp_upload/51728/51728fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Day </td>
			<td>Number of T75 flasks seeded on day 0</td>
			<td>Total number of Ssea1+ cells after FACS </td>
			<td>Total number of Ssea1+/Epcam+ cells after FACS </td>
		</tr>
		<tr>
			<td>3</td>
			<td>8</td>
			<td>2 million </td>
			<td></td>
		</tr>
		<tr>
			<td>6</td>
			<td>4</td>
			<td>2 million </td>
			<td></td>
		</tr>
		<tr>
			<td>9</td>
			<td>4</td>
			<td>2 million </td>
			<td></td>
		</tr>
		<tr>
			<td>12</td>
			<td>10</td>
			<td>10 million </td>
			<td>2 million </td>
		</tr>
	</tbody>
</table>

Watch this Scientific Journal Video about Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model at JoVE.com

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

cell-surface-marker-mediated-purification-ips-cell-intermediates-from

Research

JoVE Journal

Biology

12.3K Views.  Monash University. Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam. 

Video: Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Immunology and Infection

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Medicine

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Neuroscience

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 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.

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 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, ...

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 ...