Abstract
It has been very difficult, if not impossible, to establish mouse induced pluripotent stem cells (iPSCs) from differentiated cells, such as fibroblasts, without leukemia inhibitory factor (LIF). We have established and maintained LIF-independent iPSCs for longer than 120 days with modified Oct4 along with Sox2, Klf4, and c-Myc. The iPSCs will provide a novel tool to investigate the roles of the LIF-Stat3 signaling pathway in mouse pluripotent stem cells.
Resource Table.
Biological reagent: induced pluripotent stem (iPS) cell
| Name of Stem Cell construct | M3O-lenti-iPSC-LIF(−) |
| Institution | University of Minnesota |
| Person who created resource | Hiroyuki Hirai |
| Contact person and email | Nobuaki Kikyo, kikyo001@umn.edu |
| Date archived/stock date | 2014–2015 |
| Origin | Mouse embryonic fibroblasts |
| Type of resource | Biological reagent: induced pluripotent stem (iPS) cell |
| Sub-type | Cell line |
| Key transcription factors | M3O, Sox2, Klf4, c-Myc |
| Authentication | Identity and purity of cell line confirmed (Figure 1) |
| Link to related literature (direct URL links and full references) | Hirai H., et al. In press, Stem Cell Research |
| Information in public databases |
Resource Details
Reprogramming factors: M3O (the first 186 nucleotides of MyoD were fused to the amino terminus of full-length Oct4), Sox2, Klf4, and c-Myc were used.
Vector system: M3O was transduced with the TetO-FUW inducible lentivirus vector; Sox2, Klf4, and c-Myc (SKM) were transduced with the pMXs-IP retrovirus vector.
Evidence for the silencing of the reprogramming factors in iPS cells: Transduced SKM were silenced as shown by RT-PCR (Fig. 1A). Expression of M3O was maintained by doxycycline during establishment and maintenance of M3O-lenti-iPSCs-LIF(−).
Figure 1. Characterization of mouse M3O-lenti-iPSCs-LIF(−).
(A) RT-PCR of the Klf4 transgene (Exo-Klf4) comparing MEFs transduced with SKM (positive control), O-retro-iPSCs -LIF(+) (negative control), M3O-lenti-iPSCs-LIF(−), and MEFs (negative control). GAPDH was used as a loading control.
(B) Relative expression levels of endogenous Oct4 and Sox2. The values obtained with O-retro-iPSCs-LIF(+) was defined as 1.0 for each gene. Average + SD of three independent experiments are shown.
(C) Immunofluorescence staining and alkaline phosphatase staining of M3O-lenti-iPSCs-LIF(−) cultured without LIF for 120 days. GFP was immunostained due to quenching of the signal during fixation. Bar, 100 μm.
(D) Hematoxylin and eosin staining of teratoma sections obtained with M3O-lenti-iPSCs-LIF(−). Bar, 100 μm.
(E) Karyotype analysis of M3O-lenti-iPSCs-LIF(−).
Expression of pluripotency markers: Expression of endogenous Oct4 and Sox2 were verified with quantitative RT-PCR (qRT-CPR) (Fig. 1B). Expression of Nanog, SSEA-1, and alkaline phosphatase was confirmed with cell staining (Fig. 1C).
Capacity to differentiate into three germ layers: Teratomas containing tissues derived from three germ layers were formed after injection into immunocompromised mice (Fig. 1D).
Materials and Methods
M3O is a fusion gene between full-length Oct4 and the transactivation domain of mouse MyoD (1). M3O with the FLAG sequence DYKDDDDK at their carboxyl terminus was cloned into the TetO-FUW inducible lentivirus vector (Addgene, modified #20726). Lentivirus encoding M3O was prepared by transfecting TetO-FUW-M3O, psPAX2 (Addgene #12260), and pCMV-VSV-G (Addgene #8454) into 293T cells with Lipofectamine 2000 (Life Technologies). Lentivirus encoding FUW-M2rtTA (Addgene, #20342) (2) was prepared in the same way. Polycistronic Sox2, Klf4, and c-Myc (SKM) were inserted into the pMXs-IP retroviral vector (3) and transfected into Plat-E cells (4) to prepare retrovirus. M3O-lentivirus and SKM-retrovirus were transduced with 10 μg/ml polybrene into mouse embryonic fibroblasts (MEFs) harboring the Oct4-GFP transgene (B6;129S4-Pou5f1tm2Jae/J; Jackson Laboratory, stock #008214) (5). Transduced MEFs were subcultured onto irradiated MEF feeder cells in iPSC medium (DMEM, 20% FBS, 100 μM MEM non-essential amino acids, 100 μM 2-mercaptoethanol, 2 mM L-glutamine, and 2 μg/ml doxycycline) on the next day. The resulting iPSCs called M3O-lenti-iPSCs-LIF(−) were maintained on feeder cells without LIF for longer than 120 days. O-retro-iPSCs-LIF(+) were prepared from MEFs with retroviral OSKM in the presence of LIF and used as a control in Fig. 1A and B.
Immunofluorescence and alkaline phosphatase staining
iPSCs were fixed with 4% formaldehyde in PBS and stained with antibodies against GFP (Santa Cruz Biotechnology, sc-8334), Nanog (R&D Systems, AF2729), and SSEA-1 (Alexa Fluor 555-labeled, BD Biosciences, 560119). Secondary antibodies included Alexa Fluor 488-labeled anti-rabbit IgG (Life Technologies, A11304) and Cy3-labeled anti-goat IgG (Jackson ImmunoResearch, 805-165-180). DNA was counter stained with Hoechst 33342. Fluorescence images were captured with an AxioCam camera attached to an Axiovert 200M microscope (Zeiss). Alkaline phosphatase was detected with a Millipore kit (SCR004).
RT-PCR
Total RNA was prepared with RNeasy Micro Kit (QIAGEN) and cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies). Following primers were used for PCR. Exogenous Klf4: 5′-GGGAAGTCTTCTAACATGCG-3′ and 5′-GAAGGACGGGAGCAGAG-3′. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5′-TGCACCACCAACTGCTTAG-3′ and 5′-GATGCAGGGATGATGTTC-3′.
Quantitative RT-PCR (qRT-PCR)
cDNA prepared from total RNA of M3O-lenti-iPSCs-LIF(−) was applied for qPCR with a GoTaq qPCR Master Mix (Promega) using the following primers. Endogenous Oct4: 5′-TCTTTCCACCAGGCCCCCGGCTC-3′ and 5′-TGCGGGCGGACATGGGGAGATCC-3′. Endogenous Sox2: 5′-AAAGGAGAGAAGTTTGGAGCCCGA-3′ and 5′-GGGCGAAGTGCAATTGGGATGAAA-3′. GAPDH was used for normalization of the levels of mRNA.
Teratoma formation
Two-month-old NOD/SCID mice (Charles River) were intramuscularly injected with 1 × 106 M3O-lenti-iPSCs-LIF(−). Four weeks after injection, teratomas were fixed with 10% formalin and embedded in paraffin. Sections of 5-μm thickness were stained with haematoxylin and eosin solution.
Karyotyping
Cells were arrested with colcemid, harvested, treated with 75mM KCl hypotonic solution, and fixed with methanol and acetic acid at 3:1. The cells were spread onto glass slides and stained with Wright-Giemsa stain. G-banded metaphases were imaged and karyotyped using Applied Spectral Imaging (ASI) software.
Verification and Authentication
Normal karyotype of 40, XY (Fig. 1E).
Acknowledgments
We thank Toshio Kitamura for Plat-E cells and the pMXs-IP vector. Histology and karyotyping were performed at the Comparative Pathology Shared Resource and the Cytogenomics Shared Resource, respectively at the University of Minnesota Masonic Cancer Center with the support by the NIH grant P30 CA077598. M.F. and N.K were supported by Richard M. Schulze Family Foundation. N.K. was supported by the NIH (R01 GM098294 and R21 CA187232); Engdahl Family Foundation; Grain-in-Aid of Research, Artistry and Scholarship, University of Minnesota (22802); and the University of Minnesota Foundation (4160-9227-13).
Footnotes
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References
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