Stem cells and the evolving notion of
cellular identity
rstb.royalsocietypublishing.org
Review
Cite this article: Daley GQ. 2015 Stem cells
and the evolving notion of cellular identity.
Phil. Trans. R. Soc. B 370: 20140376.
http://dx.doi.org/10.1098/rstb.2014.0376
Accepted: 1 June 2015
One contribution of 13 to a discussion meeting
issue Cells: from Robert Hooke to cell
therapya 350 year journey.
Subject Areas:
cellular biology, computational biology,
developmental biology, synthetic biology
Keywords:
embryonic stem cells, reprogramming, induced
pluripotent stem cells, cellNet
Author for correspondence:
George Q. Daley
e-mail: george.daley@childrens.harvard.edu
George Q. Daley1,2
1
Stem Cell Transplantation Program, Division of Hematology/Oncology, Howard Hughes Medical Institute,
Boston Childrens Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA
2
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston,
MA 02115, USA
Stem cells are but one class of the myriad types of cells within an organism.
With potential to self-renew and capacity to differentiate, stem cells play
essential roles at multiple stages of development. In the early embryo, plur-
ipotent stem cells represent progenitors for all tissues while later in
development, tissue-restricted stem cells give rise to cells with highly
specialized functions. As best understood in the blood, skin and gut, stem
cells are the seeds that sustain tissue homeostasis and regeneration, while
in other tissues like the muscle, liver, kidney and lung, various stem or pro-
genitor cells play facultative roles in tissue repair and response to injury.
Here, I will provide a brief perspective on the evolving notion of cellular
identity and how reprogramming and transcription factor-mediated con-
versions of one cell type into another have fundamentally altered our
assumptions about the stability of cell identity, with profound long-term
implications for biomedical research and regenerative medicine.
1. Background and history: from cells to stem cells
In his groundbreaking seventeenth century treatise Micrographia: or, Some
physiological descriptions of minute bodies made by magnifying glasses
(London: J. Martyn and J. Allestry, 1665) Robert Hooke described his visualiza-
tion of the tiny walled units of a thinly sliced film of cork, which he likened to a
honeycomb, and dubbed them cells. Some 350 years hence, cells are well
known as capable of living free as whole organisms or as building blocks of
living tissues within highly complex beings, and as remarkably malleable
and resilient fundamental units of life.
The twentieth century, arguably the century of genetics, began with the elu-
cidation of genes as units of heredity, mid-way through witnessed the discovery
of the double helix, and concluded with the completion of the rough draft of the
human genome sequence. Although the genome sequence provides the genetic
code, the principle research challenge of the twenty-first century is learning
how this code is read and interpreted by cellshow the information in the
single-celled human zygote is translated to produce the trillion-odd specialized
units within the human body. Developmental biology addresses many of the
questions inherent to formulating the body plan, but deciphering the expression
of the genetic code is increasingly the provenance of stem cell biology, which
seeks to understand epigenetic regulation and cell fate. Just as the gene is the
principle sub-unit of the genome, the stem cell is the chief unit of organization
for tissue formation and homeostasis.
The concept of developmental hierarchies of cells, with a stem cell at the top
of this hierarchy and multiple differentiated offspring derived from it, first
emerged in the late nineteenth century and has become an experimental main-
stay of the last 65 years, owing to the pioneering definitions of self-renewing
multipotential progenitors of all lineages of the blood by Till and McCulloch
in a series of pioneering papers [1,2]. In delineating the stem cell for the
blood-forming tissue the haematopoietic stem cell (HSC), Till and McCulloch
set the stage for understanding the developmental hierarchies of additional
tissues and organsthe skin, gut, lung, sperm and othersas well as the
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
emergence of whole organisms from the pluripotent stem
cells of the early embryo. To ascribe their unique proper-
ties, a stem cell must be defined clonallyat the single-cell
levelfor its ability to self-renew and differentiate, and
consequently stem cells represent but a tiny fraction of cells
within the tissues of adult organisms.
2. Developmental potency of stem cells
Stem cells are defined by their range of potential fatesa con-
cept termed developmental potency. The zygote is the most
developmentally expansive cell, yet is rarely considered a stem
cell in mammals because it cleaves into blastomeres of equal
developmental potency for at most three cell divisions, and
therefore manifests very limited self-renewal potential. The
zygote and early blastomeres form all the tissues of the
embryo proper as well as the supportive extraembryonic
tissuesthe fetal membranes and placentaa unique capacity
called totipotency. To date, no one has succeeded in propagating
the zygote or blastomeres continuously in cell culture. Embryo-
nic stem cells (ESCs), by contrast, can be isolated from the inner
cell mass of blastocysts and cultured as immortal cell lines.
When murine ESCs are aggregated with early blastomeres or
returned to the blastocoele cavity of the blastocyst by microinjec-
tion, they chimaerize all tissues within the embryo proper
but typically not the placenta, which reflects their developmen-
tal segregation from the trophectodermal lineage, which has its
own master cellthe trophoblastic stem cell. ESCs are con-
sidered pluripotential. The developmental potency of human
ESCs, which for ethical reasons cannot be determined by
embryo chimaerism, is instead assessed by observing the spec-
trum of tissue differentiation that results after subcutaneous
injection of cells into immune-compromised mice. Pristine
ESCs produce teratomas, encapsulated tumours consisting of
disorganized masses of differentiated tissues. Because elements
of the three primitive embryonic germ layersectoderm, meso-
derm and endodermcoexist in these tumours, human ESCs
share pluripotency with mouse ESCs.
When human ESCs were first described by Thomson et al.
[3], there was an oddity that went largely unnoticed for almost
a decade. The human-derived cells grew under distinct con-
ditions, stimulated by fibroblast growth factor instead of
leukaemia inhibitory factor (LIF), which nurtured the mouse
ESCs. Human ESCs appeared not to depend on LIF signalling,
nor to activate the downstream pathways driven by the LIF/
gp130 receptor complex, implying a distinct physiologic or
developmental state [4]. Moreover, the colonies of human
ESCs appeared flatter and were more prone to die when
passaged as single cells than the mouse ESCs, which grew as
domed colonies and could be readily dissociated and passed
as single cells. Only when stem cells from the epiblast stage
of mouse development were isolated and cultured did the dis-
tinctions become clear: human ESCs resembled more closely
the epiblast-derived stem cells (EpiSCs) of slightly later stage
embryos. Hence, murine EpiSCs appear to be a closer develop-
mental equivalent to human ESCs than murine ESCs [5].
Vigorous recent efforts to isolate human ESCs under con-
ditions that more closely mimic the mouse ESCs have
yielded a large number of papers that report slightly different
conditions and states of a putative naive human stem cell.
Such a cell, like the mouse ESC, is closer in its DNA methyl-
ation and gene expression patterns to the cells of the inner
cell mass of the blastocyst, and hence is termed naive [6,7], 2
in contrast to the cells that more closely resemble the epiblast
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stage of mammalian development, like human ESCs or
mouse EpiSCs, which are considered primed, as if poised to
differentiate. Indeed, several groups have claimed to have
identified combinations of transgenes or purely chemical
means to convert human primed pluripotent stem cells to
their naive state. If such naive human ESCs can indeed be
introduced as an alternative to classically derived hESCs oper-
ating since the first lines of Thomson, there may be advantages
in terms of growth rates, ease of passage from plate to plate,
and the receptivity to efforts at genome editing. Thus, the
Phil. Trans. R. Soc. B 370: 20140376
search for naive hESCs represents a holy grail of stem cell
biology and promises many interesting new insights into the
metastable or heterogeneous state of pluripotent stem cells [8].
3. Multipotential stem cells
While ESCs garner considerable interest due to their multi-
faceted developmental plasticity, and may one day prove
invaluable as a source for autologous, personalized transplants
of cells for tissue repair or regeneration, the best established
existing cell therapies typically involve another class of cells
deemed adult or somatic stem cells. Unlike embryo-derived
stem cells, tissue-restricted somatic stem cells are limited in
their potency to the cell types of the tissue in which they
reside. HSCs generate blood, gut stem cells the lining of the
intestines and skin stem cells the epithelial lining of the body.
Despite earlier claims of greater plasticity, somatic stem cells
are highly constrained and do not without considerable genetic
or chemical manipulation trans-differentiate into foreign cell
types or tissues, and do not represent repair kits for tissues
other than their own. The most widely exploited somatic stem
cellHSCscan be transplanted into patients with various
blood diseases, malignant and genetic, after the patients dis-
eased blood-forming system has been eradicated by high-dose
chemotherapy and irradiation. But blood cells will not readily
regenerate or repair other tissues like the heart, brain, liver or
kidney after they have been injured or afflicted by disease. Con-
sequently, multipotential stem cells represent highly valuable
sources for regenerative therapies within adult tissues, but on
account of their lineage-restricted nature, do not provide a
source for all potential tissue therapies in the adult. For tissues
that do not readily regenerate from stem cell pools in the
adultlike the heart, kidney, pancreatic beta cells and much
of the brainonly pluripotent stem cells directed to differentiate
along specific lineages that recapitulate embryonic development
provide hope for future cell replacement therapies.
4. Reverting differentiated somatic cells to
pluripotency
During much of history since the time of Hooke, cell biologists
have considered development a one way thoroughfare, with
the range of a cells fates becoming progressively restricted
as the zygote progresses from a totipotent state to the pluripo-
tent state of the inner cell mass of the pre-implantation
blastocyst, and ultimately devolves into the more limited
potential of tissue-restricted cells. Ultimately, most cells in
the adult organism attain a fully differentiated state, which
remains stable and immutable unless affected by pathologic
processes like metaplasia or frank malignancy. While today we
comprehend that virtually all cells, save lymphocytes, retain
the full complement of DNA as all other cells, and become
specialized by virtue of expressing certain subsets of genes
while silencing others, early thinkers like August Weissman
speculated that development proceeded by a series of quali-
tative divisions in which daughter cells differ as each
inherits a different set of factors that specify function. Later
Hans Spemann proved through a series of painstaking and
ingenious micromanipulations of early newt blastomere
nuclei that all had equal potential to generate viable organ-
isms, establishing the paradigm-shifting notion of nuclear
equivalence. Spemann envisioned but never succeeded in
performing his fantastical experimenttransplanting the
nucleus of a highly differentiated cell back to the egg to test
whether it would regain and manifest embryonic potential.
Reporting success in precisely that fantastical experiment,
Briggs and King showed in 1952 that when nuclei were trans-
ferred from the blastula stage cells of a frog embryo into a fresh
egg, a third resulted in swimming tadpoles, showing that later
embryonic stage cells showed nuclear equivalence. Ultimately,
John Gurdon proved the full developmental potential of
highly differentiated intestinal cells of the frog in generating
functional organisms, thereby unequivocally establishing the
principle of nuclear equivalence throughout development.
Somatic cell nuclear transfer (SCNT) ultimately succeeded in
proving the full developmental competency of the nuclear
genome of mammalian cells of the sheep [9], and now well
over a dozen distinct mammalian species. Indeed, SCNT
together with directed differentiation of murine ESCs rep-
resents a classical paradigm for customized gene and cell
therapy to treat genetic disease, as has been shown in
murine models [10]. While nuclear transfer has ultimately suc-
ceeded in humans [11], it has proven quite cumbersome,
requiring harvesting of fresh oocytes from women willing to
donate to research.
The impractical technical and controversial aspects of
human SCNT were bound to limit its impact. Indeed, in a
major paradigm-shifting demonstration that transcription fac-
tors (TFs) play central roles in specifying cell fates, Yamanaka
and colleagues [12] demonstrated that ectopic expression of
four TFs normally expressed in ESCs (Oct4, Sox2, KLF4 and
c-MYC) were sufficient to reset a somatic cells differentiated
state back to pluripotency. Within little more than a year, Yama-
nakas group as well as Thomsons and my own applied TF
reprogramming successfully to human cells, achieving the deri-
vation of induced pluripotent stem cells (iPSC; [1315]). We
used the reprogramming technology to assemble the first repo-
sitory of patient-derived iPSC representing a spectrum of genetic
disease, including immune-deficiency, Downs syndrome and
Huntingtons disease, among others [16]. Indeed, Yamanaka-
style reprogramming has revolutionized the field of stem cell
biology, rendering the notion that differentiated cellular state
can be efficiently reprogrammed to pluripotency, ushering in a
profound set of possibilities for modelling human disease and
developing new drug and cell-based therapies.
5. Diverting somatic lineages directly to
alternate cell fates
Inspired in part by the classical experiments of Weintraub,
Lassar and colleagues which exploited ectopic expression of
the MyoD TF to convert fibroblasts to muscle cells [17,18], 3
Yamanakas TF-based approach to reverting somatic cells to
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pluripotency also provoked a number of laboratories to test
whether TFs might drive conversions of one somatic cell
type directly to another, a process called transdifferentiation
or direct conversion. Among the first laboratories to demon-
strate this phenomenon was that of Doug Melton, who
succeeded in converting exocrine pancreatic cells to insulin-
producing beta cells by direct injection of mouse pancreas
with master regulators of beta-cell fate [19]. Wernigs group
later converted mouse and human fibroblasts to neurons
using a similar strategy, only that TFs were engineered into
Phil. Trans. R. Soc. B 370: 20140376
fibroblasts in vitro [20,21]. Subsequently, a flood of papers
have reported conversions of one somatic cell type to another
via this approach (reviewed in [22]). Indeed, these exper-
iments have further extended the concept that a cells
molecular composition and thus function are malleable and
can be altered for applications in research and potentially
for therapeutic purposes. But whether such cells function
equivalently to their native counterparts has remained a
lingering question among stem cell biologists.
6. CellNet: a metric for discerning cell identity
and enhancing cell engineering
Numerous laboratories within the field of stem cell biology
are consumed by attempts to convert various cell types
in vitro into distinct altered states: whether directing pluripo-
tent stem cells to differentiate along specific lineages towards
specialized terminal cells, directing the conversion of one
somatic cell type to another with enforced expression of
TFs, or reverting differentiated somatic cells to pluripotency
by expression of the Yamanaka factors. Such efforts promise
to validate cultures of human cells as improved models of
human pathology and physiology, and raise hopes that cell-
based target validation, drug-screening and mechanistic
research will be strengthened. Of some concern however,
are the standards by which cells engineered in vitro are com-
pared to native tissues for both molecular identity and
physiology. Most claims that cells engineered in a dish have
achieved a given cell fate equivalent to native tissues are typi-
cally founded on analysis of a small number of diagnostic
markers, on limited functional assays and on global assess-
ments of gene expression analysed by clustering algorithms
that provide at best a qualitative suggestion of similarity
without an objective metric of identity.
Given the momentous challenge of assessing the quality
and fidelity of cells engineered in vitro, and the lack of rigor-
ous metrics for assessing cell states, my laboratory has
worked closely with that of James Collins (MIT) to generate
a computational platform that defines how closely an engin-
eered cell approaches the identity of a target cell or tissue
type, and provides a set of candidate regulators of the cells
transcriptional state that require further modulation to push
the engineered cells ever closer to their target. CellNet is a
publicly available network-biology platform (http://cellnet.
hms.harvard.edu/) written by Patrick Cahan with input
from Hu Li and Edroaldo Lummertz de Rocha that has
been tested extensively against both published datasets [23]
and used within our own laboratory to enhance the in vitro
production of several cell types [24]. At its core, CellNet is
founded on the notion that cell- and tissue-specific gene
regulatory networks (GRNs) are major molecular determi-
nants of cell identity that govern both the steady-state
transcriptional programme as well as cellular responses to
the environment and to various contextual perturbations
like disease and ageing. We reasoned that defining the
status of GRNs within cells engineered in vitro would provide
a robust metric of cell identity as well as a means of determin-
ing which TFs were most dysregulated, thereby providing a
plausible set of candidates to be further manipulated in
future experiments. Using publicly available microarray
expression data and a modified version of the Context Like-
lihood of Relatedness algorithm [25], Cahan produced a
global GRN that defines regulatory relationships among all
annotated transcriptional regulators and target genes across
most cell types, and then refined these relationships into
cell and tissue type-specific GRNs that could be used to
build a cell-type classifier. Other metrics included quantitat-
ive assessments of GRN status and a network influence
score, which ranks TFs according to their effect on the GRNs.
With CellNet in hand, we evaluated the outcomes of 226
experimentally derived cell populations drawn from 56 pub-
lished studies, and gleaned several overarching lessons. First,
virtually all reprogrammed cellsi.e. those reverted to plur-
ipotency by virtue of Yamanaka-style reprogramming
achieved near identity to the gold standard ESC, whether
mouse or human. Second, efforts at directed differentiation
of pluripotent stem cells (either ESCs or iPSCs) using mor-
phogens and selective culture conditions in vitro achieved
on average higher classification scores relative to their
target tissues than experiments that endeavour to directly
convert one differentiated somatic cell type directly into
another via ectopic expression of master regulatory TFs
[23]. These analyses indicate that reprogramming is a remark-
ably robust transition in cell fates, perhaps in part due to the
highly selective culture conditions established for pluripotent
cell types. However, when making neurons or cardiomyo-
cytes or hepatocytes from pluripotent cells, we have much
to learn before we can claim great success in deriving
highly specialized cell and tissue types. Even more so, our
attempts to convert differentiated fibroblasts directly into
neurons or cardiomyocytes or hepatocytes without first
returning to the highly plastic state of pluripotency are lim-
ited by epigenetic modifications that stabilize the fibroblast
identity of the starting cells. Interestingly, a combination of
transient expression of Yamanaka factors with ectopic
expression of lineage specifying factorsa strategy some-
times referred to as primed conversion [26], appears in
some cases highly effective at converting one somatic cell
type directly into another, and bears further exploration.
In an effort anchored by Samantha Morris in my labora-
tory [24], we employed CellNets predictive features to refine
the conversion of B cells into macrophages by ectopic
expression of CEBP/a [27,28] and fibroblasts into hepatocytes
by enforced expression of Hnf4a and FoxA TFs [29,30]. In
converted macrophages, CellNet identified the persistence
of B-cell-associated TFs EBF1 and Pou2af1/OBF1, and their
knock-down enhanced macrophage functionality in vitro. In
the hepatocyte-like cells induced from fibroblasts (iHeps), Cell-
Net detected aberrant activation of cdx2, a hindgut-associated
transcriptional regulator, as well as low-level expression of the
intestinal regulators Klf4/5. When cdx2 was knocked down,
the putative iHeps became more robust in their liver-like prop-
erties of albumin synthesis and urea metabolism, whereas
enforced expression of Klf4/5 dampened liver marker 4
expression and fortified intestinal identity. Taken together,
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these data suggested that the iHeps might in fact be poised
as bi-potential progenitors of endoderm fates (semi-colon).
Remarkably, these semi-colon cells could be coaxed ever
closer to intestinal fates by engraftment in a murine colitis
model. When the colon-engrafted cells were recovered and
analysed by CellNet, they showed a high degree of colonic epi-
thelial identity. Our laboratory is further applying CellNet to
optimize our efforts to convert pluripotent stem cells along
various blood lineages.
CellNet (v. 1.0) has notable limitations. CellNet was
Phil. Trans. R. Soc. B 370: 20140376
trained on microarray data, which are plentiful enough to pro-
vide adequate power to discover GRNs for many but by no
means all tissues. The current version of CellNet is powered
to classify a query population of cells against a panel of
some 20 murine or 16 human target tissues. While many
medically relevant tissues currently being investigated by
researchers are included, e.g. neurons, heart, haematopoietic
stem/progenitor cells, the spectrum of human cell types is
vastly larger, especially when considering that the ideal train-
ing data would yield GRNs for every definable cell type at
every stage of development. Furthermore, the current training
data derive largely from parenchymal tissues and whole
organs, which encompass many cell types, while most engin-
eered cells are acclimated to culture in a dish. Despite these
limitations, when primary cells are cultured from whole
organs or tissues, purified, and then subjected to analysis via
CellNet, the de-differentiation effects of cell culture and the
heterogeneity of the target tissue does not compromise the uti-
lity of CellNet as a robust classifier of a cells identity [23].
CellNet will be augmented in future versions as transcriptional
profiles become available for additional target cells and
tissues at multiple stages of development. Moreover, as more
researchers turn to RNA sequencing as a mode of transcrip-
tional profiling, CellNet will need to be adapted to accept
these data (and such software refinement is underway). At
the theoretical limit, as RNA sequencing of single cells
becomes more robust, diagnostic GRNs might be discoverable
for every cell type at every stage of organismal development,
enabling algorithms like CellNet to define the transcriptional
roadmap for human development. Such a platform would
greatly facilitate efforts at directed differentiation of cell
types in vitro and accelerate the capacity for stem cells to be
translated for research and medical applications.
7. Prospects for the future
The definition of the cell has been refined at ever greater resol-
ution since its first description by Hooke 350 years ago. In
recent years, the capacity to reprogram and redirect cells such
that they morph from one identity to another has highlighted
the highly plastic and malleable nature of the genome and the
fluid state of cells. Indeed, cells have assumed a chameleon-
like character. Understanding the precise molecular mechan-
isms that define the formation, stabilization and transitions
among GRNs promises to inform future efforts to engineer
cells more faithfully in vitro and to diagnose and treat cellular
pathology and promote repair and regeneration in vivo. More-
over, understanding GRNs as modules of cellular function
offers the possibility that new types of hybrid cells with highly
engineered functions might be produced for biomedical
applications, the stuff of synthetic biology. Why be constrained biotechnology companies: True North, Solasia, Epizyme, Verastem, 5
to making a perfect hepatocyte or struggle to make a beta cell Ocata, Raze and MPM Capital. The author is an affiliate member of
the Broad Institute, and an investigator of the Howard Hughes Medical

when there might be advantages to engineering a hepatocyte
to perform glucose-responsive insulin secretion? It is provoca-
tive to consider what the cell will look like in another 350 years.
Competing interests. The author is a member of the scientific advisory
boards and receives consulting fees or holds equity in the following
rstb.royalsocietypublishing.org
Institute and the Manton Center for Orphan Disease Research.
Funding. The author is supported by grants from the US National Insti-
tutes of Health: R24-DK092760, RC4-DK090913, and UO1-HL100001
Progenitor Cell Biology Consortium; Alexs Lemonade Stand; Doris
Duke Medical Foundation and Ellison Medical Foundation.

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