Stemgent Provides Proven iPS Cell Generation Technologies

 

Stemgent offers application-tested technologies to help you generate induced pluripotent stem (iPS) cells. iPS technology has been drawing increased interest since the method of cellular reprogramming was demonstrated in 2006. Reprogramming is a method of converting fully differentiated cells to an embryonic-like cell known as iPS cells. This breakthrough methodology was first described using mouse somatic cells by Dr. Shinya Yamanaka et al¹. Soon afterwards, both Dr. Shinya Yamanaka et al. and Dr. James Thomson et al. demonstrated reprogramming of human somatic cells². Later Dr. Rudolph Jaenisch et al. introduced additional innovations using inducible lentiviral expression vectors, providing more control over the reprogramming process³. In 2008, significant discoveries were made using small molecules to enhance reprogramming efficiency.

Stemgent's iPS cell technology program provides a resource where you can find validated, stem cell-tested products based on technologies and methods referenced in breakthrough publications and through the Stemgent Scientific Advisory Board. We strive to reduce the experimental timeline to bring high quality, thoroughly tested products to market so you can accelerate your stem cell research and generate publications faster.

Viral Reprogramming: Lentivirus, Retrovirus, or Adenovirus

The seminal cellular reprogramming publications demonstrated that four virally transduced transcription factors were sufficient to convert fibroblast cells to iPS cells. Stemgent offers pre-packaged high titer virus and plasmid sets that save you time and effort from not having to generate and validate viruses in your own lab (Figure 1):

• Stemgent® Human TF Lentivirus Set (Cat. No. 00-0005)
• Stemgent® DOX Inducible Mouse TF Lentivirus Set (Concentrated) (Cat. No. 00-0003)
• Stemgent® iPSC Generation Dox Inducible Mouse TF Lentivirus Set: OKSM (Cat. No. 00-0004)
• Stemgent® iPSC Generation Dox Inducible Mouse TF Lentivirus Set: OKSM + RG (Cat. No. 00-0021)
• Stemgent® Mouse TF Adenovirus Set (Cat. No. 00-0008)
• RiPStem™ Retroviral Mouse Plasmid Set (Cat. No. 00-0002)

Our lentivirus products have been fully tested to reprogram fibroblast cells to iPS cells (Figure 2 and Figure 3). Read a sample application note to learn more.

Small Molecule Reprogramming

Dr. Sheng Ding, Dr. Douglas Melton, and others have shown that adding small molecules can enhance reprogramming efficiency. Small molecules may act by affecting key epigenetic modifications and cell signaling pathways. Each small molecule in Stemgent's collection is of the highest purity (≥98%), structurally verified by NMR and mass spectrometry, and tested for cytotoxicity. Stemgent's small molecule collection for iPS cell generation includes:

• Valproic Acid (Cat. No. 04-0007) effectively inhibits HDAC activity, activating β-catenin expression, and can improve reprogramming efficiency from mouse somatic cells when used in combination with viral transduction of Oct-3/4, Klf4, Sox2, and c-Myc⁴.
• BIX01294 (Cat. No. 04-0002) effectively inhibits histone H3 K9 methyltransferase G9a, activating gene expression, and PD0325901 (Cat. No. 04-0006), an ERK suppressor, have been shown to help reprogram neural progenitor cells (NPCs) from mouse embryonic stem cells in combination with transduction of Oct-3/4 and Klf4⁵.
• R(+)BayK8644 (Cat. No. 04-0013) is an L-type Ca2+ channel activator that can enhance reprogramming efficiency when used together with Oct-4, Klf4, and BIX01294 (Cat No. 04-0002)⁵.
• Additional candidate small molecules for reprogramming studies include:
  • RG108 (Cat. No. 04-0001) effectively blocks DNA methyltransferase with no cytotoxicity, activating transcription.
  • A83-01 (Cat. No. 04-0014) and ALK5 Inhibitor (Cat. No. 04-0015) are selective inhibitors for ALK5.
  • Sodium Butyrate (Cat. No. 04-0005) is a known inhibitor of histone deacetylases⁶.
  • Stemolecule™ Tranylcypromine hydrochloride (Cat. No. 04-0033) is a monoamine oxidase inhibitor which has been shown to derepress Oct4 expression in P19 embryonal carcinoma cells

Primary Cells

Stemgent provides mouse embryonic fibroblasts (MEFs) with a selection marker or reporter gene for use as starting material for initiating reprogramming or to use as a control alongside your reprogramming experiments. All cell lines are early passage number and are tested with Stemgent® iPSC Generation Lentivirus products to validate reprogramming to iPS cell colonies. Additional testing includes mycoplasma, microbial contamination, a panel of 19 murine viruses, cell viability and genotype confirmation. Our current selection includes:

• Stemgent® Oct4-neo MEF (P2) (08-0014)

Cell Lines

Stemgent is building a collection of cell lines to study cellular reprogramming. Our growing collection contains cell lines for generating secondary iPS cells as well as control cell lines for monitoring primary iPS cell generation. All Stemgent® Cell Lines are thoroughly tested for mycoplasma and genetically characterized to validate cell type.

• Stemgent® Mouse Primary iPS Cells-NNeo (Cat. No. 08-0012)
• Stemgent® Mouse Primary iPS Cells-T1b (Cat. No. 08-0006)
• Stemgent® Mouse Primary iPS Cells-WP5 (Cat. No. 08-0007)

Shorten Your Time to Publication

Stemgent staffs highly trained stem cell scientists to support you every step of the way. We routinely reprogram cells and have proven these products in our own labs. For more information or to discuss your reprogramming project, simply contact Stemgent and ask for a scientist in our Reprogramming Group. You can reach us at www.stemgent.com or by calling 877-228-9783 (toll-free) or 617-245-0098 (international).

References

1. Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.
2. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., and Thomson, J.A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920.
3. Brambrink, T., Foreman, R., Welstead, G.G., Lengner, C.J., Wernig, M., Suh, H., and Jaenisch, R. (2008). Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151-159.
4. Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., and Melton, D.A. (2008). Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature Biotechnology 26, 1269-1275.
5. Shi, Y., Desponts, C., Do, J.T., Hahm, H.S., Schöler, H.R., and Ding, S. (2008). Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3, 568-574.
6. Sëemann, M.D., Böhmig, G.A., Österreicher, C.H., Burtscher, H., Parolini, O., Diakos, C., Stöckl, J., Hörl, W.H., and Zlabinger, G.J. (2000) Anti-inflammatory effects of sodium butyrate on human monocytes: potent inhibition of IL-12 and up-regulation of IL-10 production. FASEB J., 14(15), 2380-2382.

Figure 1.
A comparison of the amount of time and cost incurred when making lentiviruses for reprogramming or purchasing the ready-made and fully validated Stemgent® DOX Inducible Mouse TF Lentivirus Set for reprogramming shows a significant time and cost savings.

Figure 2.
Immunocytochemistry (ICC) analysis 48 hours post-doxycycline (DOX) induction to monitor transduction efficiency (200x). Nanog-GFP/rtTA mouse embryonic fibroblasts (MEFs) were transduced with each of four lentiviruses from the Stemgent® DOX Inducible Mouse TF Concentrated Lentivirus Set (Cat. No. 00-0003). The Nanog-GFP/rtTA MEFs contain the GFP gene knocked-in at the Nanog locus as well as a reverse tetracycline transcriptional activator (rtTA) expression cassette which is required for DOX inducible expression. The far left panel (-DOX) is a representative negative control for expression of the four transcription factors without DOX induction. Correctly expressed transcription factors were confirmed by corresponding antibodies (shown in red), stained with DAPI to visualize the nucleus.

Figure 3.
Analysis of iPS cell colonies generated using the Stemgent® DOX Inducible Mouse TF Concentrated Lentivirus Set (Cat. No. 00-0003). Nanog-GFP/rtTA MEFs were tranduced with all four viruses, carrying Oct4, Sox2, Klf4, and c-Myc cDNA. Expression of the four transcription factors was induced by adding doxycyline (DOX); initiating the reprogramming process. GFP expression reflects the endogenous Nanog expression level and is used for monitoring the pluripotent state. The emergent colonies were manually isolated and passaged for further characterization. (A) Phase contrast microscopy and alkaline phosphatase (AP) staining of an induced pluripotent stem (iPS) cell colony (200x). (B) Pluripotency marker analysis (100x): Left Panel - phase contrast overlay with GFP reprogramming reporter expression. Middle Panel - immunocytochemistry (ICC) staining for pluripotency marker (Nanog, Oct4, and SSEA1); Right Panel - DAPI staining to visualize the nucleus.