extra view
Cell Cycle 9:10, 1893-1900; May 15, 2010; © 2010 Landes Bioscience
Cdks and cyclins link G1 length and differentiation
of embryonic, neural and hematopoietic stem cells
Christian Lange and Federico Calegari*
DFG-Research Center and Cluster of Excellence for Regenerative Therapies Dresden (CRTD); Medical Faculty; Technische Universität Dresden;
Dresden,Germany
I t is long known that stem cell differen-
Key words: cell cycle, G1 length, cdk/
cyclin complexes, stem cells proliferation/
differentiation
Abbreviations: cdk, cyclin dependent
kinase; ES cell, embryonic stem cell;
CNS, central nervous system; NSC, neu-
ral stem/progenitor cells; AP, apical pro-
genitors; BP, basal progenitors; VZ, ven-
tricular zone; SVZ, subventricular zone;
HSC, hematopoietic stem cells; LT-HSC,
long term HSC; ST-HSC, short term
HSC; BrdU, bromodeoxyuridine
Submitted: 02/19/10
Accepted: 02/23/10
Previously published online:
www.landesbioscience.com/journals/cc/
article/11598
*Correspondence to: Federico Calegari;
Email: federico.calegari@crt-dresden.de
www.landesbioscience.com Cell Cycle 1893
tiation correlates with a lengthening of
the cell cycle, in particular G1. Moreover,
models were proposed for mammalian
embryonic, neural and hematopoietic
stem cells whereby lengthening of G1 is a
cause, rather than a consequence, of dif-
ferentiation. These models are based on
the concept that time, i.e., G1 length, may
be a limiting factor for cell fate change
to occur because differentiation factors
require time in order to trigger a physi-
ological response. Despite the many cor-
relative studies, this hypothesis proved
difficult to demonstrate because most
trophic, signaling or transcription factors
involved in stem cell differentiation may
concurrently, but independently, also
have an effect on cell cycle progression,
which calls for a thorough review on the
differentiation role of genes whose best
characterized and long established func-
tion is exclusively to control G1. For this
reason, we here focus our attention on the
effects that the core molecular machin-
ery controlling G1 progression, i.e., the
G1-specific cyclin dependent kinase
(cdk)/cyclin complexes, have on stem
cell differentiation. In particular, we will
discuss the effects of G1-cdks/cyclins on
differentiation of embryonic, neural and
hematopoietic stem cells during develop-
ment and adulthood, for which a role of
G1 length has been proposed.
Introduction
All tissues are generated during develop-
ment by differentiation of stem cells. Stem
cells are defined by two key characteristics
extra view
distinguishing them from all other cells,
i.e., they can (1) unlimitedly divide to
generate at least one daughter that is iden-
tical to her mother and (2) divide to gener-
ate all differentiated cell types of a given
tissue, characteristics referred to as long
term self-renewal and multipotentiality,
respectively.1
In mammals, the first differentiation
event of embryonic stem (ES) cells that
contribute to all somatic tissues (pluripo-
tent ES cells) occurs at peri-implantation
stage, i.e., aproximately at embryonic
day (E) 5 in mouse, when differentiation
of ectoderm, mesoderm and endoderm
ensues.2,3 With the formation of these
three germ layers, stem cells lose pluri-
potency and become restricted to the
generation of cell types of specific organ
systems derived from a single germ layer.
For instance, the central nervous system
(CNS) and skin are both formed from
the ectoderm as blood, bone and muscle
arise from the mesoderm while, finally,
the gastrointestinal and respiratory tracts
are derived from the endoderm (Fig. 1).
Stem cells within each organ system are
defined as multipotent, including neural,
hematopoietic, intestinal, or any other
tissue-specific stem cell.
Starting with organogenesis, multipo-
tent stem cells start to generate cells that
are either (1) more restricted in their pro-
liferative potential and capacity to gener-
ate different cell types (progenitor cells)
or (2) do not divide anymore (postmitotic
cells). Importantly, multipotent stem cells
are not restricted to development, but also
persist during adulthood where they are
important for renewal of tissues with high
 Figure 1. Cell cycle length of stem cells during animal life (top; left). Color gradient to label cells according to the length of their cell cycle with black
and white indicating quiescent and postmitotic cells, respectively (middle; left). A very incomplete diagram representing some stem cell lineage (ar-
rows) from each germ layer. In many cases (dashed arrows) alternative progeny was not depicted for simplicity. Induced pluripotent stem (iPS) cells
were derived from many tissues, not necessarily the ones depicted here. Note the presence of the many gray cells whose cell cycle length is, to our
knowledge, still undefined (middle; bottom). Timeline, not in scale but aligned to the panel above, of the mouse life with (top to bottom) (1) stages in-
dicated in embryonic (E) or postnatal (P) days, months (M) or years (Y), (2) main phases and events in the animal life and (3) features of G1 in stem cells.
(rigth) Abbreviations for the cell types are indicated.

turnover such as skin, blood or the intes-
tinal epithelium, or may be involved in
repair upon injury.4-6 Also the adult mam-
malian brain, a tissue with remarkably low
turnover, contains stem cells in specialized
niches even though their function is not
entirely understood.7-9
In contrast to multipotent stem cells of
the developing embryo that are all cycling
to support tissue growth, most adult stem
cells leave the cell cycle and become qui-
escent to serve as a reservoir for tissue-
renewal and repair. Clearly, the concerted
timing of the switch of multipotent stem
cells to generate lineage restricted progeni-
tors or postmitotic cells together with the
control of their cell cycle length vs. qui-
escence determines the final cell number
within each organ as well as its cellu-
lar composition, thus, underlying tissue
formation, homeostasis and organ func-
tionality during animal life.
Many molecules can influence the cell
cycle and differentiation of stem cells and
many reviews have already highlighted the
role of trophic, signaling, and transcription
factors in different paradigms of stem cell
differentiation.4-6,10-12 Moreover, several
studies during embryonic development
have shown that a reduction in proliferative
and fate potential of stem cells tightly cor-
relates with a lengthening of the cell cycle,
thus, making of cell cycle regulation a fun-
damental aspect of stem cell biology.13-15
For the differentiation of stem cells, G1
is by far the most interesting phase of the
cell cycle. In fact, not only G1 is the phase
that changes the most during development
but it is also the phase during which cells
decide to progress through the cell cycle
or enter quiescence.16 More importantly, it
has been suggested that the length of G1
may by itself influence the differentiation
of certain stem cells, which is based on
the assumption that a long G1 may allow
the accumulation of factors needed for
differentiation to occur while a short G1
may not, thus, maintaining self-renewal
and pluripotency.13-15 However, despite
many correlative evidences this model has
proven intrinsically difficult to demon-
strate because manipulation of factors that
promote differentiation may, in addition,
also influence the cell cycle. Thus, corrob-
oration of this model requires manipula-
tion of genes whose main, if not exclusive,
role is to control G1 such as the G1-specific
cdk/cyclin complexes.
Cdk/cyclin complexes consist of a
catalytic subunit, a cdk, and a regulatory

1894 Cell Cycle Volume 9 Issue 10

subunit, a cyclin, whose binding is nec-
essary to activate the complex. Different
cdks/cyclins regulate progression through
G1 (cdk4/6 with D-cyclins), or G1-S
transition (cdk2 with E-cyclins) by
sequentially phosphorylating, and thus
inactivating, the inhibitors of S phase
entry of the pocket protein family (e.g.,
Rb and p107).17,18 D-cyclins trigger this
inactivation, which allows the expression
of genes essential for S phase entry, includ-
ing E-cyclins that enable further phospho-
rylation of the pocket proteins leading to
their complete inactivation.17
Notably, a role of G1 length in stem
cell differentiation has been indepen-
dently proposed by laboratories working
on embryonic,13 neural19 or hematopoi-
etic15 stem cells, thus, making it difficult
to merge the three models into an unifying
principle. Moreover, reports on stem cell
differentiation mainly discuss the role of
trophic, signaling and transcription factors
with multiple and complex effects,4-6,10-12
thus, making it difficult to ascribe differen-
tiation directly to G1 length. Therefore, we
here decided to focus our attention exclu-
sively on G1-cdks/cyclins in mouse embry-
onic, neural and hematopoietic stem cells
to investigate whether or not a common
picture emerges directly linking G1 length
and differentiation in the three systems.
G1-cdks/Cyclins
in Pluripotent ES Cells
In all phyla, the first division cycles after
fertilization are very rapid due to extremely
short, or even absent, G phases. The only
exception to this rule is observed in mam-
malian totipotent ES cells, which, in this
unique phylum, are mainly devoted to the
formation of extraembryonic tissues.3 Yet,
consistent with the rule, mammalian ES
cells contributing to the animal body, i.e.,
pluripotent ES cells, have remarkably short
G phases.3 For instance, mouse epiblast ES
cells divide every 4.5–9.0 hours2 with more
than half of this time being dedicated to
S phase.20 As a result of this unusual cell
cycle, pluripotent ES cells share a peculiar
cell cycle profile that distinguishes them
from more differentiated somatic cells.
Interestingly, differentiated cells that have
been reprogrammed to a pluripotent state,
such as embryonic carcinoma and induced
www.landesbioscience.com Cell Cycle 1895
pluripotent stem cells, display cell cycle
profiles that are similar to ES cells.13,21
The characteristic cell cycle of pluripo-
tent ES cells is probably due to a unique
pattern of cdks/cyclins activity. In par-
ticular, while it is unclear whether the
cdk4 and cdk6 are restricted to specific
phases of the cell cycle, cdk2 is constitu-
tively active due to continuous expression
of both cyclin E and A throughout the
cell cycle.22-24 Moreover, the cdk inhibitors
p16, p21 and p27 are silenced in ES cells.22-
24
Thus, as a result of constitutive cdks/
cyclins activity, the pocket proteins Rb
and p107 are almost exclusively found in
their hyperphosphorylated, inactive state,
which overruns G1 checkpoints and keeps
G1 as short as possible.22-24
Differentiation of pluripotent ES cells
into the three germ layers correlates with
a lengthening of their G1 and acquisition
of a canonical cell cycle profile.2,20,21 The
switch from a short G1 of pluripotent ES
cells to a longer G1 of lineage restricted
stem cells requires the establishment of
the G1-S checkpoint that restricts entry
into S phase and allows tuning the speed
of proliferation. Concomitant with the
introduction of this checkpoint, transcrip-
tion of cyclin E diminishes and becomes
restricted to G1-S transition, which may
be due to regulation by the pocket protein
p107. In addition, expression of cdk inhibi-
tors, such as p21 and p27, ensues decreas-
ing cdk4 and cdk2 activity.20,21
Importantly, decreasing cdk2 activity
by pharmacological inhibition or RNAi
was found to lengthen G1 establishing
a canonical cell cycle profile in ES cells.
Lengthening of G1 was followed by dif-
ferentiation as judged by cell morphology,
adhesiveness, as well as upregulation of
differentiation markers and loss of pluri-
potency under non-differentiating con-
ditions.25 Conversely, Kim et al.26 found
persistent pluripotency and defective dif-
ferentiation in ES cells with elevated cdk2
activity27 due to loss of its binding protein
cdk2ap1. This differentiation block proved
to be dependent on cdk2 since knockdown
of cdk2, or antagonizing its activity by
overexpression of constitutively active Rb,
alone was sufficient to induce differentia-
tion of both wildtype and cdk2ap1 defi-
cient ES cells.26,27 Similar results were also
shown for human ES cells.28,29
Altogether, not only lengthening of G1
and differentiation of pluripotent ES cells
temporally correlate but also manipulation
of genes known to primarily promote G1
length, in particular cdk2/cyclin E, alone
is sufficient to influence the cell cycle and
differentiation of ES cells. Thus, as recently
proposed by Singh and Dalton,13 lengthen-
ing of G1 appears to be a cause, rather than
a consequence of ES differentiation.
G1-cdks/Cyclins
during Development
G1-cdks/cyclins in neural stem cells of
the embryo. Neural stem and progeni-
tor cells (NSC) of the developing mouse
cortex are certainly the paradigm of stem
cell differentiation for which we have
the most detailed characterization of lin-
eage30,31 and cell cycle parameters14,32 dur-
ing development.
NSC can be classified in two cat-
egories: (1) polarized neuroepithelial and
radial glial cells [here referred together to
as apical progenitors (AP)] forming the
ventricular zone (VZ) and (2) progenitors
with more restricted potential [referred to
as intermediate or basal progenitors (BP)]
that are generated from AP, lose polar-
ity, and leave the VZ to form the subven-
tricular zone (SVZ).30,31 While AP have a
higher proliferative potential and gener-
ate both neurons and glia, BP divide up
to 3–4 times and only generate neurons.
Specifically, prior to the onset of neuro-
genesis (E11 in mouse) AP exponentially
expand in number by symmetric prolifer-
ative division. As development proceeds,
an increasing proportion of AP switches
to asymmetric division producing one AP
and either one BP or one postmitotic neu-
ron. In contrast, BP only undergo sym-
metric divisions to generate either two
neurons (ca. 90% of cases) or two addi-
tional BP. Finally, at the end of neurogen-
esis at E16 most AP become restricted to
a glial fate while a subpopulation of them
stops to proliferate and becomes quiescent
stem cells of the adult brain.30,31
Using cumulative labeling with nucle-
otide tracers, in particular cumulative
BrdU labeling, it has been shown that the
length of the cell cycle in the VZ increases
as development proceeds, specifically, from
8 to 18 hours at E11 and E16, respectively,
which is due to a lengthening of G1 from 3
to 12 hours.33 Moreover, this lengthening
was shown to correlate with neurogenesis
at the temporal33,34 and spatial34,35 level
and to specifically occur in progenitors
switching to neurogenic division.14,36
With regard to G1-cdks/cyclins activ-
ity, it has been observed that, similarly to
ES cells,25 pharmacological inhibition of
cdk2/cyclin E lengthened the G1 phase of
AP and induced their premature switch to
neurogenesis, suggesting that lengthening
of G1 may alone be sufficient to trigger
differentiation.19 Recent reports have cor-
roborated and extended this conclusion by
analyzing and genetically manipulating
G1-cdks/cyclins activity in vivo. In partic-
ular, deletion of cyclin D1, which is specif-
ically expressed by AP and downregulated
in BP,37-39 led to compensatory upregula-
tion of cyclin D2 in the VZ allowing a
normal cell cycle and differentiation of
cyclin D1 deficient AP. In contrast, dele-
tion of cyclin D2, which is enriched in BP,
was not compensated by upregulation of
cyclin D1, thus, causing a lengthening of
G1 in the VZ by 30%, premature neuro-
genesis and depletion of BP.37
Similar observations were also made
in another region of the CNS, the retina,
whose progenitors also lengthen the cell
cycle during development.40 Specifically,
deletion of cyclin D1, that in this tissue is
not compensated by upregulation of other
cyclins, lengthened the cell cycle of retinal
NSC and increased neurogenesis at the
expense of progenitor expansion,41 which
underlies hypocellularity of the cyclin
D1 knockout retina.42,43 Importantly, the
phenotype of cyclin D1 knockout mice
was partly rescued by either (1) knockin
of cyclin E into the cyclin D1 locus or
(2) codeletion of the cdk inhibitor p27,
thus, suggesting that the effects of cyclin
D1 deletion occur through a change in
G1, rather than to additional, cell cycle-
independent roles of cyclin D1.41
The advent of in utero electroporation
to acutely and tissue-specifically manipu-
late gene expression as an alternative to the
generation of transgenic lines has greatly
accelerated the study of mammalian CNS
development.44 Overall, cumulative BrdU
labeling after in utero electroporation
in control conditions has been shown to
faithfully reproduce cell cycle parameters
1896 Cell Cycle Volume 9 Issue 10
of unmanipulated cells (compare refs. 33,
38 and 45–47). However, it cannot be
excluded that under certain conditions
in utero electroporation may preferen-
tially target synchronized cells (noted by
Tsunekawa et al.48 Tabata et al.49 and Pilaz
et al.50 and Dr. Osumi and Dr. Nakajima,
personal communication), which should
be considered while assessing cell cycle
parameters.
Overexpression of cdk4/cyclin D1 by
in utero electroporation shortened G1
in the VZ by 30%, inhibited neurogen-
esis and promoted the generation and
expansion of BP.38 Conversely, RNAi
for cdk4/cyclin D1 induced the opposite
effects with a lengthening of G1 by 20%,
increase in neurogenesis and depletion
of BP.38 Importantly, since overexpres-
sion by in utero electroporation is tran-
sient and both cdk4 and cyclin D1 have
a particularly short half-life,51,52 neural
progenitors resumed physiological cell
cycle and differentiation few divisions
after manipulation. However, as a result
of a delayed neurogenesis and transitory
expansion of progenitors, an increase in
late-born neurons and cortical expansion
was observed.38
These findings are fully consistent with
several other reports. As mentioned above,
pharmacological inhibition of cdk2/cyclin
E1,19 or deletion of cyclin D2 lengthened
G1 and induced neurogenesis.37 In addi-
tion, Pilaz et al.50 have shown that in utero
electroporation with cyclin D1 or cyclin
E1 alone, without their cdks, induced sim-
ilar effects to those observed upon overex-
pression the cdk4/cyclin D1 complex.38 In
particular, a shortening of G1 by ca. 30%
(though in this report cumulative BrdU
labeling was not used due, apparently, to
inappropriate targeting of asynchronized
cells), delayed neurogenesis, and increased
the thickness of the SVZ.50
It should be noticed, however, that
this latter report50 differed from Lange et
al.38 in the sense that according to Lange
no effect was observed by overexpression
of cyclin D1 in the absence of cdk4, nor
by overexpression of the cdk2/cyclin E1
complex. We have no simple explanation
that would account for this discrepancy,
unless to assume that this may be due to
differences in developmental stage, over-
expression levels, and/or mouse strain.
Nevertheless, what we think is the most
important aspect of all these studies in the
cortex and retina19,37,38,41,50 is that whenever
a manipulation of either a cyclin alone37,41,50
or a cyclin together with its cdk19,38 leads
to a lengthening19,37,41 or shortening38,50 of
G1, this effect is always accompanied by an
increase19,37,41 or decrease38,50 in neurogen-
esis, respectively. Conversely, whenever a
manipulation of a cyclin, with or without
its cdk, has no effect on G1 length, neither
an effect on differentiation is observed37,38
(Table 1). Thus, we conclude that dif-
ferentiation of NSC upon manipulation
of cdks/cyclins is primarily caused by
the change in G1 length that, under cer-
tain conditions, cdks/cyclins may induce,
rather than being an additional effect
beyond cell cycle regulation as previously
suggested for both cyclin D and E.53-55
In fact, similarly to ES cells,13 Calegari
and Huttner have previously proposed
that G1 lengthening may be necessary
and sufficient to induce differentiation of
NSC.19 In this context, it is perhaps not a
coincidence that the first molecular mark-
ers of NSC switching to differentiation are
cell cycle inhibitors (such as Tis2156 and
BM8857) and that concomitantly with this
switch NSC physiologically downregulate
genes fostering cell cycle progression,58
including cyclin D1.37-39
G1-cdks/cyclins in hematopoietic
stem cells of the embryo. Another well
studied paradigm of stem cell differen-
tiation during embryonic development
is the hematopoietic lineage. During
hematopoiesis, long-term (LT) and short-
term (ST) hematopoietic stem cells (HSC)
coexists with the former being defined by
the ability to permanently reconstitute
the blood system in mice with ablated
hematopoiesis while the latter can only do
this for periods shorter than 4 months.6
LT-HSC give rise to all blood lineages by
generating ST-HSC that self-renew for a
limited number of divisions and then pro-
duce multipotent progenitors restricted
to either the lymphoid (B, T and natu-
ral killer cells) or myeloid (granulocytes,
macrophages, megakaryocytes and eryth-
rocytes) lineage.6
Interesting insights into the role of
G1-cdks/cyclins on differentiation of HSC
have been reported for double- and triple-
knockouts lacking either cdk4 and cdk6 or
 Table 1. Summary the effects of G1-cdks/cyclins in stem cells

Effects reported upon manipulation of G1-cdks/cyclins in stem cells of the developing and adult mouse. Red arrows indicate (left to right columns): (1)
inhibition of cdks/cyclins activity, (2) shortening of G1, (3) decreased differentiation and (4) decreased proliferation. Conversely, green arrows ­indicate
(left to right columns): (1) increased cdks/cyclins activity, (2) lengthening of G1, (3) increased differentiation and (4) increased ­proliferation. In some
instance, effects are not observed (=), detected (N.D.) or applicable (N.A.). Pharmacological (Ph), knockdown (RNAi), knockout (KO), ­overexpression (OE)
or knockin (KI) types of manipulations are indicated. Note the exact correlation between a lengthening of G1 and differentiation.

all three D-cyclins. Embryos deficient for
cdk4/cdk6 or cyclin D1/D2/D3 develop
normally until E13 but display severe
anemia at later stages causing embryonic
lethality.59,60 In these mutants, the liver
(the site of hematopoiesis at this stage) was
found to contain only about 20% of cells
as compared to wildtype embryos with an
even stronger reduction in most stem and
progenitor cell types but, in contrast, an
increased proportion of more differenti-
ated cells of the erythroid lineage, the
most abundant differentiated cell type at
this stage.59,60 Consistent with defective
expansion and premature differentiation,
mutant HSC displayed a reduced ability
to form colonies in vitro by more than
90% while D-cyclins deficient HSC failed
to repopulate the hematopoietic system of
irradiated hosts. Finally, while wildtype
embryonic HSC, in contrast to adult HSC,
are all cycling,61,62 deletion of D-cyclins in
embryonic HSC induced an increase in
diploid cells, which suggests lengthening
of G1 and/or entry into quiescence.60
Considering that the phenotype
induced by deletion of the two G1 cdks or
the three D-cyclins is essentially identical
(Table 1), we find it logic to assume that
this phenotype is attributable to a lack of
activity of the G1-cdk/cyclin complexes
rather than being caused by indepen-
dent, but coincidentally similar, effects
triggered by deletion of either cdk4/6 or
D-cyclins. Thus, we conclude that simi-
larly to ES cells13 and NSC14 of the devel-
oping embryo, G1 length may be involved
in controlling differentiation also in
HSC as recently proposed by Orford and
Scadden.15

www.landesbioscience.com Cell Cycle 1897

G1-cdks/Cyclins during Adulthood
While several studies have addressed the
effects of a manipulation of cdks/cyclins
in G1 length and differentiation of stem
cells during embryonic development, sim-
ilar reports in the adult are still very lim-
ited, if not completely lacking. This may
be due to several reasons, including early
lethality in certain mutants and that most
adult stem cells identified so far are quies-
cent or cycle at a extremely low frequency,
thus, making it difficult to discriminate
between G0 and G1. Nevertheless, a few
reports on G1-cdks/cyclins in vivo are
available.
G1-cdks/cyclins in adult NSC. Despite
their quiescence, adult NSC are reminis-
cent of their embryonic equivalent, the
AP, as they both have similar molecular
markers, are polarized, and generate more
committed progenitors that are later con-
sumed to generate neurons.31
In mouse, all three D-cyclins continue
to be expressed in NSC of the juvenile brain
but only cyclin D2 is expressed during
adulthood. Consistent with compensation
of certain D-cyclins, juvenile cyclin D2
knockout mice displayed normal prolifer-
ation and development in the neurogenic
niches while adult NSC, which are criti-
cally dependent on this only cyclin, were
found to have an almost complete block
in neurogenesis.63 As a result, the olfactory
bulb, a region critically dependent upon
the supply of new neurons to maintain its
size,9 was dramatically reduced in cyclin
D2 knockout mice.63
The increased neurogenesis observed
in cyclin D2 deficient embryos37 appears
to be in contradiction with the block in
neurogenesis in adult mice.63 However, it
should be noticed that in adult cyclin D2
knockout mice block in neurogenesis was
not associated with a lengthening of G1
but rather with a block in cell cycle pro-
gression, thus not allowing NSC to neither
expand nor undergo neurogenesis.63
We are not aware of any report show-
ing the effect of an acute manipulation of
G1-cdks/cyclins, in particular their over-
expression, in the adult brain. We believe
that such experiments are fundamental
to investigate whether manipulation of
G1 length, or quiescence, can be used to
control the expansion or differentiation of
1898 Cell Cycle Volume 9 Issue 10
adult NSC in vivo, which may be impor-
tant for regenerative therapy of the CNS.
G1-cdks/cyclins in adult HSC. In
contrast to the brain where cell turnover
is minimal, blood cells need to be con-
stantly replenished. Except for its differ-
ent location in the liver or bone marrow,
the hematopoietic lineage is similar in the
embryo and adult.6 Following the tenet
already seen in the CNS, adult LT-HSC
become quiescent and divide on average
every 1–5 months, with only 5% of cells
in S phase at steady state. In contrast, their
immediate progeny, the ST-HSC, divide
more frequently with about 20% of them
being in S phase at any given time point.64
Unfortunately, despite a thorough charac-
terization of the proportion of quiescent
cells in subpopulations of blood precur-
sors, their G1 length has, to our knowl-
edge, not been reported.
Quantitative analyses of D-cyclins
expression in HSC showed upregulation
of cyclin D1 in the physiological transi-
tion from LT- to ST-HSC and cyclin D3
upon induced, pharmacological expan-
sion of LT-HSC.64 Moreover, cyclin D2
has been show to be upregulated in cells
of the myeloid lineage, notably in granu-
locyte and macrophage progenitors, pop-
ulations known to contain the smaller
fraction of quiescent cells,64 and overex-
pression of cyclin D2 in HSC accelerates
myeloid reconstitution after transplanta-
tion.65 Finally, common lymphoid pro-
genitors upregulate cyclin D3,64 and loss
of cyclin D3 impairs expansion of B66
and T67 cells and generation of neutrophil
granulocytes.68 Finally, knockin of a non
degradable form of cyclin E was shown
to promote expansion of erythroid pro-
genitors at the expense of differentiated
erythrocytes.69
The studies discussed above indicate
that G1-cyclins have important roles in
HSC biology but more studies are neces-
sary to investigate whether these are lim-
ited to cell cycle control, differentiation or
both.
Conclusions
It is long known that factors that inhibit
differentiation tend to shorten G1 while,
conversely, molecules that trigger differen-
tiation tend to lengthen it.13-15 To explain
this correlation, at least three laboratories
have independently proposed a model
whereby the length of G1 may by itself
control the differentiation of embryonic,13
neural14 and hematopoietic15 stem cells.
Despite the fact that these models were
specific for different stem cell systems,
they turned out to be conceptually identi-
cal and, thus, we will here refer to them
by a common name, the cell cycle length
hypothesis, which has been originally for-
mulated for NSC of the developing cor-
tex.19 This model considers G1 a critical
period during which cell fate decisions
are made. Thus, increasing the length of
G1 may provide the additional time that
is necessary for certain differentiation fac-
tors to accumulate to the threshold needed
to trigger a cellular effect, whereas decreas-
ing the length of G1 would not.13-15 Clearly,
a sufficiently potent differentiation factor
may overrun a short G1 and trigger dif-
ferentiation even without a change in cell
cycle length. Yet, in general terms, a longer
G1 would promote differentiation while a
short G1 would inhibit it.
Despite its striking simplicity, the cell
cycle length hypothesis has proven intrinsi-
cally difficult to demonstrate because G1
lengthening can alternatively be consid-
ered a consequence, rather than a cause,
of differentiation as dogmatically viewed
for a long time. Clearly, testing this model
requires manipulation of genes whose
best characterized and long established
function is solely to control G1, such as
the G1-specific cdks/cyclins, which we
attempted to review in this article.
Even if reports on adult stem cells are
too limited to draw any conclusion, we
consistently found that during embry-
onic development a manipulation of
G1-cdks/cyclins triggers effects beyond
cell cycle progression by also influencing
the differentiation of embryonic, neural
or hematopoietic stem cells. In particu-
lar, manipulations of cdks/cyclins that
shorten G1 tend to prevent differentiation
while those that tend to lengthen G1 have
the opposite effect.
In relation to the cell cycle length hypoth-
esis, it could still be argued that certain
cyclins may also act without cdks to trig-
ger effects that are independent of cell
cycle regulation.53-55 However, the reports
discussed above show that whenever a

manipulation of a cyclin (with or without
cdks) influences stem cell differentiation,
this is always accompanied by a change in
G1 length. Conversely, whenever a manip-
ulation of a cyclin (with or without cdks)
has no effect on G1 length no effect on
differentiation is neither seen (Table 1).
Thus, effects on stem cell differentiation
and G1 length by cdks/cyclins are most
likely linked.
Clearly, strict logic cannot exclude
the possibility that this link is purely
coincidental, rather than causal. In fact,
G1-cdks/cyclins may simultaneously con-
trol cell cycle length and, independently,
differentiation through a yet to be char-
acterized mechanism. This would imply
that evolution has chosen G1-cdks/cyclins
as master regulators of cell cycle progres-
sion and, in addition, of stem cell differen-
tiation, which would be a very remarkable
coincidence indeed. An alternative, and to
our opinion more parsimonious, explana-
tion is that the effects of G1-cdks/cyclins
on stem cell differentiation are just sec-
ondary effects triggered by a change in
G1 length. That is to say, a change in G1
length may alone be a cause, rather than a
consequence, of stem cell differentiation,
as previously proposed.13-15
It would be tempting to speculate that
the cell cycle length hypothesis may be an
universal principle of stem cell differentia-
tion and that manipulations of G1-cdks/
cyclins may allow the control of prolif-
eration vs. differentiation of any stem cell
type, which will have an immense impact
in basic research and potential therapeutic
applications. However, in revising the lit-
erature we found, to our surprise, that pre-
cise assessment of G1-length in stem cells
and their progeny is, with the few excep-
tions here discussed, extremely scarce if
not completely lacking, thus making it
premature to extend the cell cycle length
hypothesis to other stem cell types.
With a better characterization of
molecular markers in different stem cell
niches4-6,31 and the realization that time,
i.e., G1 length, may alone play a criti-
cal role on stem cell differentiation,13-15
we expect, and hope, that analyses and
manipulations of cell cycle length similar
to those here described will soon be avail-
able for many other tissues.
www.landesbioscience.com Cell Cycle 1899
Acknowledgements
We thank Dr. Noriko Osumi and Dr.
Kazunori Nakajima for sharing informa-
tion about in utero electroporation and
Dr. Claudia Waskow and Dr. Marius
Ader for comments on HSC and retinal
progenitors, respectively. C.L. and F.C.
are supported by the DFG-funded Center
for Regenerative Therapies, the Medical
Faculty of the Technical University
Dresden and the Collaborative Research
Center SFB655 of the DFG (subproject
A20).
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