Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
It’s hard to believe it’s been just seven years since Shinya Yamanaka produced the first inducible pluripotent stem cells (iPSCs) [1]. Since then, iPSCs have racked up more than 5,400 citations in PubMed, inched towards their first clinical trial and earned their discoverer the 2012 Nobel Prize in Physiology or Medicine.
iPSCs are like embryonic stem cells without the embryo. Researchers create them from more differentiated cells by the introduction of a handful of transcription factors (most commonly Oct4, Klf4, Sox2 and c-Myc, or OKSM), and with them study the origins of genetic disease, run drug development programs and develop cellular therapies.
First, though, they need to make the cells. A number of companies have developed tools to simplify and facilitate the process.
Delivery tools
Yamanaka made his first iPSCs using retroviral vectors, genome-integrating RNA viruses, and the iPSCs thus produced are genetically modified. For some researchers, that’s a deal-breaker, but not for all, and retroviral reprogramming vectors are commercially available.
EMD Millipore's STEMCCA™ reprogramming kits derive from a subclass of retroviruses called lentiviruses, which also integrate into the host genome. Millipore offers both constitutive and doxycycline-inducible forms, as well as a “floxed,” or Cre-excisable construct, and their efficiency is such that they even can reprogram traditionally refractory targets like peripheral blood cells, says Nick Asbrock, product manager for stem-cell and cell-culture products at EMD Millipore.
“There’s advantages and disadvantages to each [reprogramming] system,” Asbrock says. “Ours is very efficient and reproducible. And it is excisable. You’re guaranteed to have colonies, which is nice. The main disadvantage is that it integrates in the genome.”
Millipore also recently launched a cell-permeable form of Cre recombinase called TAT-Cre, in which the protein can be added directly to the cells rather than needing to be expressed; paired with the floxed STEMCCA kit, users can remove the iPSC transgenes from the host genome without an additional transfection or viral-infection step.
Still, some researchers eschew integration because it can induce unanticipated changes in the host genome, and some reprogramming factors are oncogenic. Integration also is verboten if the cells are to be used in a clinical setting. But new strategies can circumvent that limitation.
Life Technologies’ CytoTune®-iPS reprogramming kits leverage the RNA-based Sendai virus, which “doesn’t leave a footprint in the cells,” says Uma Lakshmipathy, principal scientist for cell biology at Life Technologies. According to Lakshmipathy, the company recently released a new version 2.0 of the CytoTune kit, offering higher efficiency and faster viral clearance. This new kit differs from its predecessor in its construction, with the expression cassettes for Klf4, Oct4 and Sox2 in one vector and c-Myc and Klf4 provided on separate vectors. (The previous version contained different vectors for each factor.)
Life Technologies also offers nonviral, DNA plasmid-delivery vehicles, including the Episomal iPSC Reprogramming Vectors (OKSM+Lin28+Nanog) and Epi5™ Episomal iPSC Reprogramming Kit (OKSM+Lin28).
Stemgent provides reprogramming reagents built around synthetically modified mRNAs. mRNA offers the advantage that it is both non-integrative and non-viral, says Brad Hamilton, director of research and development at Stemgent. But, because the cell has no mechanism to retain the RNAs, they must be reintroduced daily via transfection, sometimes up to 20 days. Now, though, the company has greatly simplified the process, Hamilton says.
Using Stemgent’s new “miRNA Booster Kit,” which complements mRNA delivery with microRNAs associated with up-regulation of pluripotency factors and down-regulation of differentiation factors, researchers can create mRNA-based iPSCs with fewer than a dozen transfection steps, usually eight to 10, Hamilton says. “With a new protocol built around miRNA, the Stemfect™ RNA Transfection Kit, and [BD] Matrigel™ or other defined surfaces, we have reduced the daily workload, the total number of transfections required and ultimately the timeline [approximately two weeks] to generate integration-free human iPS cell colonies.”
Finally, researchers can boost reprogramming efficiency by adding small molecules, which are available from such companies as EMD Millipore, Stemgent and Tocris Bioscience. These molecules serve, among other things, to open chromatin. But they can also eliminate the need for specific reprogramming factors; Stemgent’s Stemolecule™ ALK5 Inhibitor can substitute for Sox2, for instance. Usually, though, at least one transcription factor also is needed. But this summer researchers at Peking University demonstrated for the first time that it’s possible to generate mouse iPSCs (which they call CiPS cells) using only a mixture of seven small molecules, no nucleic acids required [2].
Growth media
Embryonic stem cells (ESCs) require special growth media and so, too, do iPSCs.
STEMCELL Technologies offers several such formulations, including the popular mTeSR1™ (for feeder-free pluripotent stem-cell maintenance), TeSR2™ (a version of mTeSR1 containing recombinant proteins) and the simpler, xeno-free and low-protein TeSR™-E8™.
TeSR™-E7™ is STEMCELL’s optimized medium for iPSC reprogramming. According to Simon Hilcove, the company’s product manager for pluripotent stem cell biology, TeSR-E7 produces “better colonies” than other media—that is, primary colonies that are more compact, have sharper borders and are more easily identified, isolated and expanded. “It reduces the amount of differentiation and fibroblast overgrowth [in the culture] and really streamlines the manual selection process,” he says.
Other media for iPSC work include Life Technologies’ KnockOut™ Serum Replacement and Essential 8™ Medium for feeder-based and feeder-free growth, respectively; EMD Millipore’s PluriSTEM™ growth media; and Stemgent’s NutriStem™ and Pluriton™ for growth and reprogramming, respectively.
According to Hamilton, NutriStem and Pluriton are highly compatible, which translates into efficient colony transition from the primary reprogramming well to expansion. “Transitioning from one medium to the other is really smooth.”
PluriSTEM is a small-molecule-based formulation that is low in growth factors and relatively stable, Asbrock says. As a result, researchers can feed cells less frequently than with other media, and at lower cost. “You can skip some of the routine, labor-intensive protocols a lot of stem-cell researchers complain about,” he says, including weekend feedings.
Characterization tools
According to Hilcove, there are two tests researchers should perform in characterizing their iPSCs. One is karyotyping, to test for chromosomal abnormalities. The second is differentiation, to prove your putative iPSCs can form all three cellular germ layers (ectoderm, endoderm and mesoderm). Traditionally, this is accomplished using teratoma assays in mice, but researchers these days increasingly are turning to in vitro differentiation assays, using, for instance, STEMCELL Technologies’ STEMdiff™ formulations. Once the cells have been differentiated, they can examined for markers for each germ layer, using either antibodies or gene expression analyses.
Many researchers also perform antibody-based assays for pluripotency itself, and a number of companies offer antibodies that target cell-surface and intracellular markers of pluripotency and differentiation, including Cell Signaling Technology (CST), EMD Millipore, Life Technologies, R&D Systems, Rockland Immunochemicals, STEMCELL Technologies and Stemgent. These antibodies usually are available in multiple forms and support a range of applications, including flow cytometry and immunofluorescence.
The StemLight™ iPS Cell Reprogramming Antibody Kit from CST includes unconjugated antibodies for Oct4A, Sox2, Nanog, Lin28A, Klf4 and c-Myc, and it is used to assess expression of reprogramming transcription factors. The company’s StemLight Pluripotency Antibody Kit targets both transcription factors (Nanog, Oct4A, Sox2) and surface markers (SSEA4, TRA-1-60, TRA-1-81). “We go to extraordinarily great lengths to validate the antibodies up-front,” says Susan Kane, antibody development scientist in the stem-cell group at Cell Signaling Technology.
Rockland also stresses validation and has its own catalog of iPSC target antibodies, says R&D director Karin Abarca Heidemann. The company currently is assembling an array of 10 antibodies for January release that will also help users track pluripotency and differentiation of iPSCs via immunocytochemistry and immunofluorescence.
For those interested in flow cytometry, EMD Millipore has packaged its flow-ready antibodies into the FlowCellect™ Human iPS Cell Characterization Kit, including antibodies to TRA-1-60, SSEA4 and Oct4 (as well as a negative control antibody targeting SSEA1). (The company also offers standalone antibodies for flow cytometry under its Milli-Mark™ brand.
Detection tools for live cells are also available. Life Technologies has reagents for live-cell staining of alkaline phosphatase activity, for instance, and R&D Systems and Stemgent offer azide-free formulations of directly conjugated antibodies for imaging of iPSC colonies without secondary antibodies. R&D Systems’ GloLIVE™ antibodies target SSEA1, SSEA4 and TRA-1-60 (a TRA-1-81 antibody is expected shortly, says Joy Aho, who manages the company’s stem-cell group), as do Stemgent’s fluorescently labeled StainAlive™ antibodies.
“They are for use in live cell cultures,” Hamilton explains. “You use them in active culture … take images and mark which [colonies] you want to monitor, and then over 24 to 48 hours, the signal fades away.”
EMD Millipore’s SmartFlare™ reagents enable live-cell assessment of RNA (rather than protein) expression. SmartFlares are gold nanoparticles conjugated to capture oligonucleotides bound to fluorescent probes. Hybridization of the capture oligos to a target RNA releases the probe, producing a fluorescent signal that can be used, for instance, to sort cells based on their mRNA profile. According to Asbrock, the company has SmartFlare reagents for many, but not all, reprogramming factors.
Finally, there’s Life Technologies’ TaqMan® hPSC Scorecard™ Panel. Developed in collaboration with Harvard University stem-cell researcher Alex Meissner, and based on a paper he published in 2011 [3], this panel tests expression of 93 genes for both pluripotency and differentiation, Lakshmipathy says. An associated cloud-based bioinformatics application enables researchers to compare their qPCR results to reference iPSC and ESC lines to score the user’s cells for functional pluripotency.
Long story short, if you’re interested in iPSCs, there’s no shortage of tools to make and characterize them.
References
[1] Takahashi, K, Yamanaka, S, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors,” Cell, 126:663-76, 2006. [PubMed ID: 16904174]
[2] Hou, P, et al., “Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds,” Science, 341:651-4, August 9, 2013. [PubMed ID: 23868920]
[3] Bock, C, et al., “Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines,” Cell, 144:439-52, 2011. [PubMed ID: 21295703]
Image: Confocal immunofluorescent analysis of neuroepithelial clusters differentiated from human iPS cells, showing multiple neurite extensions when labeled with Neurofilament-L (C28E10) Rabbit mAb #2837 (red) and ß3-Tubulin (TU-20) Mouse mAb #4466 (green). Blue pseudocolor = DRAQ5™ #4084 (fluorescent DNA dye). Courtesy of Cell Signaling Technology.