NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 10 OCTOBER 2007 1177
Direct reprogramming of genetically unmodified
fibroblasts into pluripotent stem cells
Alexander Meissner
1,3
, Marius Wernig
1,3
& Rudolf Jaenisch
1,2
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. 1–4). 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 loci
2–4
. 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 state
5–9
. 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 Nanog
2–4
,
all of which carried a drug-resistance marker inserted into the respec-
tive endogenous loci by homologous recombination
1,3,4
or a transgene
containing the Nanog promoter
2
. 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 transduc-
tion. 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 transduc-
tion
3
. 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) carry-
ing an internal ribosomal entry site (IRES)-EGFP cassette in the Oct4
locus
10
were transduced with the four factors Oct4, Sox2, c-myc and
Klf4 by retrovirus-mediated gene transfer, as described
3
. 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 con-
tinued 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).
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 fluo-
rescence 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 con-
taining 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 reprogram-
ming or contamination by nonreprogrammed fibroblasts, we separated
the EGFP positive and negative cells from clone no. 8 and the heteroge-
neous subclone no. 8.2 by fluorescence-activated cell sorting (FACS).
Southern blot analysis demonstrated that the two cell populations
1
Whitehead Institute for Biomedical Research and
2
Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge Massachusetts
02142, USA.
3
These authors contributed equally to this work. Correspondence should be addressed to R.J. (jaenisch@wi.mit.edu).
Received 29 May; accepted 9 August; published online 27 August 2007; doi:10.1038/nbt1335
LETTERS
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1178 VOLUME 25 NUMBER 10 OCTOBER 2007 NATURE BIOTECHNOLOGY
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 differ-
ences 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 phenotype
11
.
All iPS cell lines tested showed EGFP intensity comparable to the
Oct4-EGFP ES cells (Fig. 1k,l), consistent with our previous observa-
tion that Oct4 protein levels were similar in different iPS cell lines
3
.
To analyze whether the iPS cells isolated by morphological criteria
remained phenotypically stable over time, we monitored EGFP fluo-
rescence 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





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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 (10 σ 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). (c–e) Nonselection-derived iPS lines stably express AP (c, inset), Nanog (d) and SSEA1 (e); (c–e, 20 σ
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 (20 σ 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, 10 σ 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 (c–e).
L ETTERS
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