There may be a way to avoid the moral controversies surrounding the use of human embryos in stem cell research — induced pluripotent stem (iPS) cells. With the help of the laboratory mouse, the technologies for efficiently producing iPS cells that can safely be used to treat human diseases are evolving quickly. Several JAX® Mice strains may be particularly useful.
First produced in 2006 (Takahashi and Yamanaka 2006), iPS cells are a type of pluripotent stem cell generated by virally transducing certain transcription factors into non-pluripotent cells, forcing them to express certain genes. They appear to be identical to natural pluripotent cells – such as embryonic stem (ES) cells – in many ways: they express stem cell genes and proteins, exhibit the same DNA methylation patterns and doubling time as stem cells, and form embryoid bodies, teratomas and viable chimeras.
They are an important breakthrough in therapeutic stem cell research because of their potential to substitute for ES cells, eliminating the ethical dilemmas associated with using pluripotent stem cells derived from human embryos. Additionally, because they are derived entirely from a patient's somatic cells, they are less likely to be rejected than are ES cells.
Technologies are improving for producing safe iPS cells
Current technologies for producing iPS cells impart risks that must be overcome. Because viral transduction is generally used to introduce transcription factor genes into the host cell genome, the genome of the resulting iPS cells can be altered by integrating the exogenous viral DNA. Moreover, c-Myc, one of the transcription factor genes commonly used to produce iPS cells, is a proto-oncogene. Methods to reduce these risks are being developed (see Lysiottis et al. 2009).
Initially, reprogramming somatic cells to become pluripotent required combinations of four transcription factor genes – either Oct4 (new name is POU domain, class 5, transcription factor 1 – Pou5f1), Klf4, Sox2, and c-Myc or Oct4, Sox2, Nanog, and Lin28. Later, a three-gene combination of Oct4, Sox2, and Klf4 was developed, eliminating the need for c-Myc. More recent improvements include the use of endogenously expressed factors, excisable vectors, non-integrating vectors, and transient transfection approaches.
Particularly promising is the possibility of replacing transcription factors with chemicals that can force the expression of desired genes. For example, one new induction method uses valproic acid and requires only two factors – Oct4 and Sox2 – eliminating the need for both c-Myc and Klf4. Recently a high-throughput chemical screen revealed a suitable chemical substitute for Klf4 (Lysiottis et al. 2009). The identification of additional chemical substitutes will likely eliminate the risks of viral transduction, increase the efficiency with which iPS cells are produced, better our understanding of the reprogramming process and lead to new disease therapies.
JAX® Mice for iPS cell research
The Jackson Laboratory is distributing several mouse strains that can be used for iPS cell research. Mouse embryonic fibroblasts (MEFs) derived from several of these strains can be used as high-throughput screens for discovering pluripotency-inducing chemicals.
- Heterozygous for a construct that contains the laboratory mouse Nanog homeobox (Nanog) gene, in which most of exon 2 is disrupted by a firefly luciferase gene, an SV40 polyadenylation signal sequence, and a floxed PGK-neo cassette (homozygotes are not viable).
- MEFs may be used in high-throughput in vitro screens to identify compounds that induce iPS reprogramming.
- MEFs reprogrammed into iPS cells can be easily detected because they express luciferase (Lysiottis et al. 2009).
- Heterozygous for an Oct4 gene disrupted by an IRES-GFP-neo fusion cassette downstream of exon 5 (homozygotes are not viable).
- MEFs treated with transcription factors OCT4, SOX2, C-MYC, and KLF4 and reprogrammed into iPS cells express green fluorescent protein.
- May be used alone or in conjunction with strain 008214 to fluorescently label embryonic stem cells or generate iPS cells (Wernig et al. 2007).
- Homozygous for an Oct4 gene disrupted by an IRES-EGFP fusion cassette downstream of the indigenous Oct4 gene stop codon.
- MEFs treated with transcription factors OCT4, SOX2, C-MYC, and KLF4 and reprogrammed into iPS cells express enhanced green fluorescent protein.
- May be used alone or in conjunction with strain 008204 to fluorescently label embryonic stem cells or generate iPS cells (Lengner et al. 2007).
STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm3(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J (011004)
- Expresses the optimized form of the reverse tetracycline-controlled transactivator (rtTA-M2) protein in multiple tissues, under the direction of the Gt(ROSA)26Sor promoter.
- Additionally, harbors a doxycycline (dox)-inducible polycistronic 4F2A cassette containing four mouse reprogramming genes (Oct4, Sox2, Klf4, and c-Myc) controlled by the Col1a1 locus.
- When cultured with dox, the following cell types may be reprogrammed to iPS cells: MEFs, adult liver cells, keratinocytes, adult tail-tip fibroblasts, CD11b+ splenic macrophages, CD19+ bone marrow pro-B cells, intestinal epithelial cells and mesenchymal stem cells (Carey et al. 2010).
STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm4(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J (011011)
- Similar to Stock No. 011004, but tetO-4F2A cassette is flanked by loxP sites.
- Exposure to Cre recombinase excises the reprogramming transgene, allowing the generation of vector-free iPS cells (Carey et al. 2010).
STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm5(tetO-Pou5f1,-Klf4,-Myc)Jae/J (011013)
- Similar to Stock No. 011004, but the cassette harbors only three reprogramming genes (Oct4, Klf4, and c-Myc).
- Expression of these three transcription factor genes alone does not reprogram somatic cells into iPS cells, but other factors (such as SOX2) may be added to reprogram multiple dox-cultured somatic cell types into iPS cells.
- Somatic cells may be used to screen for chemical replacements for Sox2 (Carey et al. 2010).
Because the reprogramming factor genes in the previous three strains are carried on a single construct, the strains can be easily maintained and the transgenes can be easily transferred to other genetic backgrounds.
Carey BW, Markoulaki S, Beard C, Hanna J, Jaenisch R. 2010. Single-gene transgenic mouse strains for reprogramming adult somatic cells. Nat Methods 7:56-9.
Lengner CJ, Camargo FD, Hochedlinger K, Welstead GG, Zaidi S, Gokhale S, Scholer HR, Tomilin A, Jaenisch R. 2007. Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 1:403-415.
Lyssiotis CA, Foreman RK, Staerk J, Garcia M, Mathur D, Markoulaki S, Hanna J, Lairson LL, Charette BD, Bouchez LC, Bollong M, Kunick C, Brinker A, Cho CY, Schultz PG, Jaenisch R. 2009. Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4. Proc Natl Acad Sci U S A.106:8912-7.
Wernig M, Meissner A, Foreman R, Brambrink T, Ku M; Hochedlinger K, Bernstein BE, Jaenisch R. 2007. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318-24.
Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–76.