Letter


Nature Biotechnology 25, 1177 - 1181 (2007)
Published online: 27 August 2007 | doi:10.1038/nbt1335

Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells

Alexander Meissner1,3, Marius Wernig1,3 & Rudolf Jaenisch1,2


In vitro reprogramming of somatic cells into a pluripotent embryonic stem cell–like state has been achieved through retroviral transduction of murine fibroblasts with Oct4, Sox2, c-myc and Klf4 (refs. 1, 2, 3, 4). In these experiments, the rare 'induced pluripotent stem' (iPS) cells were isolated by stringent selection for activation of a neomycin-resistance gene inserted into the endogenous Oct4 (also known as Pou5f1) or Nanog loci2, 3, 4. Direct isolation of pluripotent cells from cultured somatic cells is of potential therapeutic interest, but translation to human systems would be hindered by the requirement for transgenic donors in the present iPS isolation protocol. Here we demonstrate that reprogrammed pluripotent cells can be isolated from genetically unmodified somatic donor cells solely based upon morphological criteria.


Somatic-cell nuclear transfer and cell fusion with embryonic stem (ES) cells have been well-established approaches to achieve reprogramming of somatic nuclei into a pluripotent state5, 6, 7, 8, 9. Direct in vitro isolation of pluripotent ES cell–like cells from cultured somatic cells was achieved only recently by transduction of genetically modified fibroblasts with the four transcription factors Oct4, Sox2, Klf4 and c-myc (referred to hereafter as 'factors'). The selection for the rare reprogrammed iPS cells was based upon the reactivation of Fbx15 (ref. 1) or of Oct4 or Nanog2, 3, 4, all of which carried a drug-resistance marker inserted into the respective endogenous loci by homologous recombination1, 3, 4 or a transgene containing the Nanog promoter2. Although iPS-cell isolation based upon Fbx15 activation yielded cells that were pluripotent, they differed from ES cells at the molecular level and did not generate live chimeras. In these experiments selection was initiated 3 d after viral transduction. In contrast, selection for Oct4 or Nanog activation produced iPS cells that were epigenetically and biologically indistinguishable from normal ES cells. Reprogramming to pluripotency was, however, a slow and gradual process involving the sequential activation of the ES-cell markers alkaline phosphatase (AP), stage-specific embryonic antigen 1 (SSEA1) and Nanog over a period of 2–4 weeks after factor transduction3. The inverse relationship between the initiation of drug selection after factor transduction and the number of drug-resistant iPS cells is also consistent with the notion that the reprogramming process involves multiple stochastic events that convert the epigenetic state of a somatic cell to that of a pluripotent cell. Here we show that pluripotent iPS cells can be derived from normal, genetically unmodified donor cells.

In the first set of experiments, we used an enhanced green fluorescent protein (EGFP) marker inserted into the Oct4 locus to monitor the reprogramming process. Mouse embryonic fibroblasts (MEFs) carrying an internal ribosomal entry site (IRES)-EGFP cassette in the Oct4 locus10 were transduced with the four factors Oct4, Sox2, c-myc and Klf4 by retrovirus-mediated gene transfer, as described3. Three days after infection, the fibroblasts became morphologically more diverse than uninfected control cells, and foci of increased growth appeared. On day 6, small tightly packed and sharp-edged colonies resembling ES-cell colonies developed. During the following days these colonies continued to grow into large and more heterogeneous cell aggregates, with the changes in some sectors resembling ES cell–like growth (Fig. 1a and Supplementary Fig. 1 online).

Figure 1: Isolation and stability of iPS cells based on morphology.

Figure 1 : Isolation and stability of iPS cells based on morphology.

(a) A representative colony (EGFP negative) 16 d after infection (clone no. OG-9, Supplementary Table 1) is shown (10times magnification). (b) The colony in a was picked on day 16, 3 d later passaged and grew up to an ES cell–like morphology (shown on day 23 at passage 1). (ce) Nonselection-derived iPS lines stably express AP (c, inset), Nanog (d) and SSEA1 (e); (ce, 20times magnification; d and e insets shows DAPI). (f) Example of a heterogeneously growing line picked on day 11 (clone no. 8 shown on day 16; 10x magnification). (g,h) Example of two ES cell–like colonies from clone no. 8 on day 16 (20times magnification with digital zoom-in). (i,j) The colony in g was picked on day 16 and grew into a homogenous iPS line after three passages (shown on day 37, 10times magnification). The first EGFP-positive colonies in the population appeared around day 26 after infection. (k) The expression level of the Oct4-EGFP is comparable between the Oct4-EGFP ES cells and all iPS lines (iPS clone no. 7 shown at passage 2,5 and 9). (l) Stable expression of Oct-EGFP at passage 5 for two additional iPS clones (no. 9 and no. 10, respectively). (m) Summary of homogenous EGFP expression after infection (dashed line indicates time of picking, green indicates initiation of EGFP expression). The clone number is shown on the right. The asterisk indicates clones that were used for blastocyst injections and that generated chimeras or tetraploid embryos. (n) Representative postnatal chimera (agouti coat color indicates donor iPS cells (129SvJae/C57B6/L; clone no. 7). (o) Tetraploid blastocyst injection–derived live E14.5 chimeras (clone no. 14). Scale bars, 0.2 mm (a,i,j), 0.5 mm (b,f) and 0.1 mm (ce).

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Eight of these large colonies were picked on day 11 and ten additional colonies were picked on day 16 based solely upon their morphology (Fig. 1a and Supplementary Fig. 1). When examined under the fluorescence microscope, no EGFP expression was detectable at day 11, and only one of the ten colonies picked on day 16 showed weak EGFP expression (Supplementary Table 1 online). One of the eight colonies picked on day 11 and four of the ten colonies picked on day 16 gave rise to homogenous, ES cell–like cell lines (Fig. 1b). All five lines initiated Oct4-EGFP expression within 1–3 passages (derived from colonies no. 7, 9, 10, 11, 14; see Supplementary Table 1) and displayed homogenous AP activity as well as SSEA1 and Nanog expression (Fig. 1c–e), as would be expected for fully reprogrammed iPS cells.

Of the remaining colonies that had been picked initially, based on morphological criteria, ten gave rise to heterogeneous cultures containing mainly fibroblast-like cells interspersed with a few ES cell–like colonies (Fig. 1f, Supplementary Fig. 2a online and Supplementary Table 1). We investigated whether these heterogeneous cultures would yield additional iPS cell lines upon further passaging. For this we picked three ES cell–like colonies from each of five mixed cultures derived from the initial outgrowths and successfully established five additional iPS cell lines within 2–3 passages (these subcloned iPS lines were derived from the initial colonies no. 1, no. 5 and no. 8; see Fig. 1f–j,m and Supplementary Tables 1 and 2 online). To test whether the observed heterogeneity was a result of partly incomplete reprogramming or contamination by nonreprogrammed fibroblasts, we separated the EGFP positive and negative cells from clone no. 8 and the heterogeneous subclone no. 8.2 by fluorescence-activated cell sorting (FACS). Southern blot analysis demonstrated that the two cell populations had a similar pattern of c-myc proviruses and thus are derived from the same parental cell (Supplementary Fig. 2g). This suggests that the reprogramming process depends on stochastic epigenetic events and yields subclones with different biological characteristics. From the picked subclones that did not generate pluripotent iPS cells, three (6.1, 6.2 and 6.3; see Supplementary Tables 1 and 2) displayed an altered morphology (small cells, tightly grown colonies) but remained Oct4-EGFP negative over multiple passages and displayed no staining for AP, SSEA1 or Nanog, suggesting that these cells were not pluripotent (Supplementary Fig. 2h–j). The occurrence of ES marker–negative cells was rare, and these cells displayed subtle morphological differences from ES or true iPS cells, such as the shape of colony boundaries (compare Supplementary Fig. 2h–j,k). Because the cells were infected with all four retroviruses (Supplementary Fig. 2l), it is possible that the four factors may not have been expressed at the right levels, giving rise to transformed rather than pluripotent cells. For example, high c-myc/Klf4 and insufficient Oct4/Sox2 expression may lead to rapidly growing non-iPS cells, consistent with the notion that the role of Oct4 and Sox2 in the reprogramming process may be suppression of the c-myc- and Klf4-transformed phenotype11.

All iPS cell lines tested showed EGFP intensity comparable to the Oct4-EGFP ES cells (Fig. 1k,l), consistent with our previous observation that Oct4 protein levels were similar in different iPS cell lines3. To analyze whether the iPS cells isolated by morphological criteria remained phenotypically stable over time, we monitored EGFP fluorescence after multiple passages. The iPS cells exhibited nonvariable and robust Oct4-EGFP expression up to at least nine passages (Fig. 1k). These data clearly demonstrate that stable iPS lines can be efficiently derived without relying on drug selection. Figure 1m summarizes the appearance of EGFP after the initial infection, which is consistent with previous conclusions that the reprogramming is a slow and gradual process3.

We used the fraction of virus-infected input cells and the number of ES cell–like colonies to estimate the efficiency of reprogramming. In a typical experiment, about 100,000 cells were exposed to virus. We determined the fraction of Sox2-, Oct4- and c-myc-infected cells by immunohistochemistry and estimated that about 10% of the cells were infected with all four viral constructs, generating a total of 115 ES cell–like colonies. The efficiency for deriving iPS cells from the number of picked colonies was about 45% (eight iPS cell lines out of 18 picked colonies). Thus, the overall efficiency of reprogramming was estimated to be about 0.5%, which is roughly 5–10 times higher than what has been achieved with the drug-selection approaches2, 3. This supports the hypothesis that the reprogramming process is slow and gradual.

Finally, we evaluated the developmental potency of nonselected iPS cells by teratoma formation and by injections into diploid (2N) and tetraploid (4N) blastocysts (Fig. 1n,o and Table 1)12, 13. Three weeks after subcutaneous injection of the cells into severe combined immunodeficiency (SCID) mice, lines 8.1 and 14 developed tumors, which contained tissue types from all three germ layers as determined by histological analysis (data not shown). After injection of the cells into 2N blastocysts, we generated live postnatal animals with high coat-color chimerism (Fig. 1n and Table 1). Notably, iPS cells injected into 4N blastocysts, which is the most stringent test for developmental potency14, resulted in recovery of live E14.5 embryos (Fig. 1o and Table 1). These data demonstrate that screening for iPS cells based upon morphological criteria rather than selection for drug resistance can generate pluripotent iPS cells that display a biological potency similar to that of ES cells.


In the experiments described so far, the Oct4-EGFP marker was used to monitor the reprogramming process but not to screen for reprogrammed iPS cells. To assess whether iPS cells can be derived from genetically unmodified donor cells, we generated wild-type MEFs from BALB/c and 129SvJae/C57Bl6 (F1) mice and adult tail-tip fibroblasts from 129SvJae/C57Bl6 (F1) and C57Bl6/DBA (F1) 2- to 3-month-old mice. The cells were infected with retroviruses encoding the four factors, and colonies were picked at day 16 or later, as described above (Fig. 2a and Supplementary Fig. 1). As in the previous experiments, ES cell–like colonies became visible within one passage after picking of the primary colonies (Fig. 2b,e). Upon continued passaging or through subcloning, we readily established homogenous cell lines with ES-cell morphology and growth properties (Fig. 2c,f). Assuming that reprogrammed cells outgrow the donor fibroblasts, we attempted to generate iPS cells by passaging the entire plate instead of picking colonies following morphological criteria. Many small colonies perfectly resembling ES-cell colonies appeared within several days after the first passage of infected cell populations (Fig. 2h), and five out of six picked colonies grew into stable iPS lines (Fig. 2i and Supplementary Table 3 online). After 2–3 passages, using either direct picking or passaging the whole plate, followed by picking of individual colonies, we established one or more iPS lines from each background (Fig. 2c,f,i and Supplementary Table 3). All genetically unmodified iPS lines expressed AP, SSEA1 and Nanog (Fig. 2j-l). In addition we generated chimeric mice from 129/B6 MEF- and BALB/c-derived iPS lines, demonstrating that iPS cells from genetically unmodified fibroblasts are pluripotent (Fig. 2m–o and Table 1). It should be noted, however, that passaging of the factor-transduced cell populations, although representing a simplified isolation protocol, cannot exclude that individual iPS cell lines were derived from the same reprogrammed parental cell.

Figure 2: iPS cells from genetically unmodified embryonic and adult fibroblasts.

Figure 2 : iPS cells from genetically unmodified embryonic and adult fibroblasts.

(a) Primary colony 16 d after infection of wild-type F1 MEFs (129/B6 = 129SvJae/C57BL/6) and (d) wild-type (B6/DBA = C57BL/6/DBA) adult tail-tip fibroblasts. (b,e) Population 4 d after dissociation of the colonies in a and d. Some ES cell–like colonies are already visible. (c,f) Established iPS line from the F1 MEFs and F1 tailtip fibroblasts. (g) A few colonies on the primary infected F1 MEF (129/B6) plate on day 23. (h) ES cell–like colonies are readily visible 3 d after passaging the entire plate. (i) Established iPS line after picking colonies from the plate in h. (jl) All iPS lines derived from the unmodified fibroblast stained positive for AP (j), SSEA1 (k) and Nanog (l). (m) E12.5 chimera generated from the F1 MEF iPS line shown in c. (n) The F1 MEF iPS line was labeled with a EGFP expressing lentivirus. Shown are the same chimeras as in m. Left iPS and right control littermate. (o) Adult chimera derived from BALB/c iPS cells (white hair indicates donor contribution). Scale bars, 0.2 mm (a,c,d,f,g,i,j), 0.5 mm (b,e) and 0.1 mm (k,l).

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Our results demonstrate that in vitro reprogramming of fibroblasts occurs frequently enough to be detected in cultures of nontransgenic donor cells and is stable without selective pressure to express Oct4 or Nanog, as has been suggested recently4. Thus, four factor–induced reprogramming can be applied to wild-type cells. It appears that ectopic expression of Oct4, Sox2, c-myc and Klf4 initiates a gradual reprogramming process in multiple infected cells that ultimately leads to pluripotency over a time period of several weeks. Using Oct4 EGFP MEFs to monitor reactivation of the endogenous Oct4 locus, we found that all colonies but one were EGFP negative at the time of picking (Fig. 1m and Supplementary Table 2) and became EGFP positive only after several passages (Fig. 1m). This suggests that reprogramming is a slow process involving the sequential activation of ES-cell markers such as AP, SSEA1 and Nanog, with Oct4 activation representing one of the last epigenetic events in the process. Also, these observations are consistent with our previous finding that the numbers of reprogrammed colonies were lower when drug selection for Oct4 activation was applied early after viral transduction, but was substantially higher when drug selection was initiated later3. Finally, the slow reprogramming process induced by factor transduction may explain why drug selection for Fbx15 activation as early as 3 d after infection, as used in the initial iPS isolation protocol, yielded only cells that had undergone incomplete epigenetic reprogramming1. Our results predict that selection for Fbx15 activation at later times would generate iPS cells that are similar to iPS cells selected for Oct4 activation or isolated based on morphological criteria.

The generation of pluripotent cells by in vitro reprogramming of somatic cells into a pluripotent state is of potential significance for the generation of patient-specific cells that could be used for 'customized' transplantation medicine in various neurological, endocrinological and hematopoietic disorders15, 16, 17. Two experimental constraints of the original protocol for isolation of iPS cells seriously impeded the eventual translation of this approach to use in patients: the use of transgenic donor cells to isolate the reprogrammed cells and the need for retrovirus-mediated transduction of potentially harmful oncogenes that have been shown to induce tumors in iPS cell–derived mice2. Our results suggest that one of these impediments, the need for genetically altered donor cells, may not represent an insurmountable problem if the protocol can be adapted to human cells. A major unresolved issue that remains is to identify alternative strategies, such as small molecules, that could activate relevant pathways and thus would minimize or avoid the genetic alterations currently required for inducing reprogramming.

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Methods

Cell culture and viral infections.

ES and iPS cells were cultivated on irradiated MEFs in DME containing 15% FCS, leukemia inhibiting factor (LIF), penicillin/streptomycin, L-glutamine, beta-mercaptoethanol and nonessential amino acids. All cells were depleted of feeder cells for two passages on 0.2% gelatin before RNA, DNA or protein isolation. 2 times 105 MEFs at passage 3–4 were infected overnight with pooled viral supernatant generated by transfection of HEK293T cells (Fugene, Roche) with the Moloney-based retroviral vector pLIB (Clontech) containing the cDNAs of Oct4, Sox2, Klf4 and c-Myc together with the packaging plasmid pCL-Eco18.

Blastocyst injection.

Diploid or tetraploid blastocysts (94–98 h after HCG injection) were placed in a drop of DMEM with 15% FCS under mineral oil. A flat tip microinjection pipette with an internal diameter of 12–15 mm was used for ES-cell injection. A controlled number of ES cells were injected into the blastocyst cavity. After injection, blastocysts were returned to potassium simplex optimization medium and placed at 37 °C until transferred to recipient females.

Recipient females and caesarean sections.

Ten to fifteen injected blastocysts were transferred to each uterine horn of 2.5-d-postcoitum-pseudopregnant B6D2F1 females. Term pups were recovered at day 19.5 and fostered to lactating BALB/c mothers if necessary.

Viral integrations.

Genomic DNA was digested with SpeI overnight, followed by electrophoresis and transfer. The blots were hybridized to the radioactively labeled c-myc cDNA.

Immunofluorescence.

Immunofluorescence analysis was performed as described previously19. Briefly, cells were fixed in 4% paraformaldehyde for 10 min at 25 °C, washed 3 times with PBS and blocked for 15 min with 5% FBS in PBS containing 0.1% Triton-X. After incubation with primary antibodies against Sox2 (monoclonal mouse, R&D Systems), Oct4 (monoclonal mouse, Santa Cruz), c-myc (polyclonal rabbit, Upstate), Nanog (polyclonal rabbit, Bethyl) and SSEA1 (monoclonal mouse, Developmental Studies Hybridoma Bank) for 1 h in 1% FBS in PBS containing 0.1% Triton-X, cells were washed 3 times with PBS and incubated with fluorophore-labeled appropriate secondary antibodies purchased from Jackson Immunoresearch. Specimens were analyzed on an Olympus Fluorescence microscope and images were acquired with a Zeiss Axiocam camera.

Note: Supplementary information is available on the Nature Biotechnology website.

Author contributions

A.M. and M.W. performed the experiments. A.M., M.W. and R.J. conceived the experiments and wrote the manuscript.



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Acknowledgments

We thank Chris Lengner and Jacob Hanna for helpful comments on the manuscript. M.W. was supported in part by fellowships from the Human Frontiers Science Organization Program and the Ellison Foundation and R.J. by grants from the National Institutes of Health.

Received 29 May 2007; Accepted 9 August 2007; Published online 27 August 2007.

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References

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  1. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge Massachusetts 02142, USA.
  2. Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge Massachusetts 02142, USA.
  3. These authors contributed equally to this work.

Correspondence to: Rudolf Jaenisch1,2 e-mail: jaenisch@wi.mit.edu

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