STEMCCA™ Lentivirus Reprogramming Kits



Figure: Successful generation of iPS cells from human foreskin fibroblasts after infection with single-vector Human STEMCCA Cre-Excisable Lentivirus (Cat. No. SCR545), as indicated by expression of characteristic pluripotency markers. Resulting passage 3 human iPS cells exhibit high alkaline phosphatase activity (A), Oct-4 expression (B), SSEA-3 expression (C), and TRA-1-60 expression (D). Nuclei are stained with DAPI (blue).
A ready source of induced pluripotent stem cells (iPS cells) is critical to the effective study of differentiation pathways or the investigation of the therapeutic potential of iPS cells.

More convenient and reliable than embryonic stem cells, which are often more heterogeneous, hard to culture and subject to regulation, iPS cells offer a significant opportunity to not only propel, but dramatically enhance the efficiency and effectiveness of stem cell research overall.

Merck (Millipore) STEMCCA lentivirus reprogramming kits make it easier than ever to obtain and work with induced iPS cells, addressing the key challenges facing iPS cell generation.

STEMCCA Vector Advantages:

  • Efficient: uses a single vector with four transcription factors rather than co-transducing four separate expression vectors
  • Minimizes viral integrations: single vector reduces the risks of insertional mutagenesis and viral reactivation
  • Excisable: Cre/LoxP-regulated version enables removal of reprogramming transgenes

The Challenge: Efficiently Generating Reproducible iPS Cells

Traditionally, adult cells are reprogrammed through the co-infection of the four Yamanaka transcription factors (Oct-4, Klf4, SOX-2, and c-Myc (OKSM)) in four separate expression vectors3-7. For successful reprogramming, a sufficient number of each virus must deliver the four factors simultaneously to the same cell, raising concerns over the high number of integration sites and the difficulty in removing these viral integrations from genomic DNA. Moreover, the inability to predict whether cells receive one, two, three, or all four factors has created heterogeneous cell populations, further complicating detailed study into the mechanism and timing of reprogramming.

The Solution: Single-Vector Reprogramming with STEMCCA Technology

Unlike traditional iPS generation, which requires simultaneous co-infection by four separate expression vectors, the STEMCCA kits use a single polycistronic lentiviral vector to improve efficiency and reduce the number of viral integrations. It also enables the creation of more homogeneous, reproducible iPS cell populations—in some cases, iPS clones which possessed only a single viral integrant were isolated1. This polycistronic cassette technology has also been applied toward generating singlegene transgenic mouse strains8.

Furthermore, removing the reprogramming vector (an option with the STEMCCA Cre-excisable kits) improves the developmental potential of iPS cells and significantly increases their capacity to undergo directed differentiation in vitro. This is a step towards safer iPS cell technology for studying disease models and clinical therapies2.

STEMCCA vector
Figure: The STEMCCA vector is comprised of the transcription factors Oct-4, Klf4, SOX-2, and c-Myc (OKSM), separated by the self-cleaving 2A peptide and IRES sequences driven by the EF-1α constitutive promoter1,2. It is also available with flanking LoxP sites incorporated for Cre-mediated excision of the exogenous reprogramming transgenes.

STEMCCA Technology to Advance Your Research

STEMCCA kits are available in two formats, for reprogramming either rodent or human cells, and include lentivirus that expresses either mouse or human OKSM factors from a single polycistronic transcript. Both human and mouse STEMCCA lentivirus kits are available in constitutive and Cre/LoxP-regulated formats.

Whether mouse or human, the STEMCCA advantage means safer, more efficient and more consistently reproducable iPS cells than traditional systems.

Cat. No.DescriptionAmount
SCR544Human STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit1 kit
SCR548Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKS/L-Myc) Lentivirus Reprogramming Kit1 kit
SCR545Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit1 kit
SCR545-CREHuman STEMCCA/TAT-Cre Bundle1 kit
SCR518Mouse STEMCCA Cre-Excisable Constitutive Polycistronic (OKS) Lentivirus Reprogramming Kit1 kit
SCR513Mouse STEMCCA Cre-Excisable Dox-Inducible Polycistronic (OKSM) Lentivirus Reprogramming Kit1 kit
SCR512Mouse STEMCCA Dox-Inducible Polycistronic (OKSM) Lentivirus Reprogramming Kit1 kit
SCR510STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit1 kit
SCR530STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit1 kit
SCR511STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit1 kit
SCR531STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit1 kit
MAB4360Anti-TRA-1-60 Antibody, clone TRA-1-60100 ug

References

  1. Sommer CA, et al. iPS cell generation using a single lentiviral stem cell cassette. Stem Cells. 2009 Mar;27(3): 543-9.
  2. Sommer CA, et al. Excision of Reprogramming Transgenes Improves the Differentiation Potential of iPS Cells Generated with a Single Excisable Vector. Stem Cells. 2010 Jan;28(1): 64-74.
  3. Takahashi K and Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4): 633-676.
  4. Okita, et al. Generation of germline-competent induced pluripotent stem cells. Nature. 2007 Jul 19;448(7151): 313-7.
  5. Wernig M, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007 Jul 19;448(7151): 318-24.
  6. Takahashi K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov 30;131(5): 861-72.
  7. Yu J, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec 21;318(5858): 1917-20.
  8. Stadtfeld M, et al. A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nature Methods. 2010 Jan;7(1): 53-5.
  9. Somers A, et al. Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells. 2010 Oct;28(10):1728-40.


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Brands: Merck (Millipore)