Nonstochastic Reprogramming from a Privileged … Reprogramming from a Privileged Somatic Cell State...

Please cite this article in press as: Guo et al., Nonstochastic Reprogramming from a Privileged Somatic Cell State, Cell (2014), http://dx.doi.org/10.1016/j.cell.2014.01.020

Nonstochastic Reprogrammingfrom a Privileged Somatic Cell StateShangqin Guo,1,2,* Xiaoyuan Zi,3,10 Vincent P. Schulz,4 Jijun Cheng,2,5 Mei Zhong,1,2 Sebastian H.J. Koochaki,1,2

Cynthia M. Megyola,1,2 Xinghua Pan,2,5 Kartoosh Heydari,6 Sherman M. Weissman,2,5,7 Patrick G. Gallagher,4,5,8

Diane S. Krause,1,2,9 Rong Fan,2,3,7 and Jun Lu2,5,71Department of Cell Biology2Yale Stem Cell Center3Department of Biomedical Engineering4Department of Pediatrics5Department of Genetics6Department of Immunobiology, Yale Flow Cytometry Core Facility7Yale Comprehensive Cancer Center8Department of Pathology9Department of Laboratory MedicineYale University, New Haven, CT 06520, USA10Department of Cell Biology, Second Military Medical University, Shanghai 200433, China

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.cell.2014.01.020

SUMMARY

Reprogramming somatic cells to induced pluripo-tency by Yamanaka factors is usually slow and ineffi-cient and is thought to be a stochastic process. Weidentified a privileged somatic cell state, from whichacquisition of pluripotency could occur in a nonsto-chastic manner. Subsets of murine hematopoieticprogenitors are privileged whose progeny cells pre-dominantly adopt the pluripotent fate with activationof endogenous Oct4 locus after four to five divisionsin reprogramming conditions. Privileged cells displayan ultrafast cell cycle of �8 hr. In fibroblasts, a sub-population cycling at a similar ultrafast speed isobserved after 6 days of factor expression and isincreased by p53 knockdown. This ultrafast cyclingpopulation accounts for >99% of the bulk reprog-ramming activity in wild-type or p53 knockdownfibroblasts. Our data demonstrate that the stochasticnature of reprogramming can be overcome in a priv-ileged somatic cell state and suggest that cell-cycleacceleration toward a critical threshold is an impor-tant bottleneck for reprogramming.

INTRODUCTION

Somatic cells can be reprogrammed into pluripotency by ex-

pression of defined transcription factors (Lowry et al., 2008;

Park et al., 2008; Takahashi et al., 2007; Takahashi and Yama-

naka, 2006; Wernig et al., 2007). Although most cell types can

be reprogrammed, this dramatic cell fate conversion occurs

only at low frequency following long latency, even when all cells

are engineered to express the reprogramming factors (Carey

et al., 2010; Stadtfeld and Hochedlinger, 2010; Wernig et al.,

2008). The prevailing theory for this low efficiency and long

latency is a stochastic model, which calls upon stochastic

changes to help subvert the various barriers limiting the fate tran-

sitions (reviewed in Hanna et al., 2010; Stadtfeld and Hochedlin-

ger, 2010; Yamanaka, 2009). Mathematic modeling suggests the

existence of a single major bottleneck event, although additional

non-rate-limiting events may also exist (Hanna et al., 2009, 2010;

Smith et al., 2010; Stadtfeld andHochedlinger, 2010; Yamanaka,

2009). However, the nature of such a bottleneck event has not

been clearly defined.

Although the reprogramming behavior of many cell types fol-

lows a stochastic model, it is possible that rare and/or transient

somatic cells may exist in a post-bottleneck state and can

progress toward reprogramming in a nonstochastic manner.

We term such putative post-bottleneck somatic cells the

privileged cells for reprogramming. Owing to the absence of

the rate-limiting stochastic events, these somatic cells should

display certain unique reprogramming behaviors (Figure 1): a

privileged somatic cell should produce progeny that mainly

progress toward pluripotency rather than adopt alternative

cell fates; their progeny should transition into pluripotency

rapidly in a largely synchronous fashion. Figure 1 depicts the

key differences between privileged and stochastic reprogram-

ming. Identification of a post-bottleneck cell state would help

to define the nature of the stochastic events restricting Yama-

naka reprogramming.

In this study, we provide evidence for the existence of privi-

leged somatic cells and describe a key feature of the privileged

cell state as an unusually fast cell cycle. The fast cycling cells

could exist naturally or could be induced from fibroblasts by

Yamanaka factors and are responsible for essentially all reprog-

ramming activities. Our data suggest a modified view for the role

of cell-cycle regulation in reprogramming and refine the conven-

tional stochastic versus elite models of reprogramming.

Cell 156, 1–14, February 13, 2014 ª2014 Elsevier Inc. 1

mailto:[email protected]


= Privileged somatic founder cell

= Pluripotent progeny= Progeny failed to reprogram

= Somatic founder cell

Latency(number of cell divisions)

% p

roge

ny

in p

lurip

oten

cy

0

50

100B Privileged

Stochastic

Stochasticreprogramming

Privilegedreprogramming

Few Most or all

Long Short

Asynchronous Largely synchronous

Reprogrammingbehavior

Progeny resulted in pluripotency

Latency

Kinetics

Hypothetic lineagescheme

A

Common RarePrevalence

Figure 1. Comparison between Stochastic and Privileged Reprogramming

(A) Hypothetic cell lineages with respect to the somatic founder cells and pluripotent progeny. The number of cell generations depicted is for illustration purpose

and does not represent the actual situations.

(B) Contrasting stochastic and privileged reprogramming with regard to their efficiency and latency.


RESULTS

Nonstochastic Reprogramming from a Subpopulation ofBone Marrow GMP CellsTo identify the existence of privileged somatic cells, we first took

a live-cell imaging approach, with which the behaviors of single

cells can be faithfully tracked with high resolution (Megyola

et al., 2013). We focused on the well-defined granulocyte mono-

cyte progenitors (GMP) because they support rapid and efficient

reprogramming (Eminli et al., 2009; Megyola et al., 2013) and are

more likely to contain privileged cells. Specifically, GMPs from

mice that carry both Rosa26:rtTA and Oct4:GFP alleles were

used as source cells for reprogramming (FACS-sorting scheme

in Figure S1B available online) so that activation of endogenous

Oct4 locus can be detected as green fluorescence in live cells.

The Yamanaka factors were introduced by a doxycycline

(Dox)-inducible polycistronic lentivirus (Carey et al., 2009) so

that factor expression could be initiated by adding Dox, with

image acquisition starting about 1 hr later (the time required to

calibrate the imaging system). The reprogramming cultures

were imaged at 5–15min intervals for�5 days, when Oct4:GFP+

cells display typical features of mouse pluripotent cells. These

Oct4:GFP+ cells, though still Dox dependent, progress with re-

programming highly efficiently (Table S1) without any obvious

bottleneck restrictions, reaching a pluripotent state that can

support chimeric mice formation and germline transmission

(Megyola et al., 2013).

Using this imaging approach, we mapped the entire fate tran-

sition process from single founder GMPs to Oct4:GFP+ progeny

2 Cell 156, 1–14, February 13, 2014 ª2014 Elsevier Inc.

(Movie S1 and Figures S1A and 2A) and constructed 14 success-

fully reprogrammed cell lineages from 5 independent experi-

ments. Strikingly, these reprogrammed GMP lineages displayed

a behavior that is consistent with features of the privileged state

(Figure 1). All live progeny from theseGMPs turned onOct4:GFP,

yielding multiple sister colonies with homogeneous Oct4:GFP

fluorescence (Movie S1). No progeny retained hematopoietic

appearance or became ‘‘stuck’’ in intermediate steps, which is

prevalent in the stochastic reprogramming systems (Chen

et al., 2013; Mikkelsen et al., 2008). Because we added Dox

within 1 hr of imaging and because it would take time before fac-

tor expression amounted to significant levels, we could ascertain

with good confidence that the initial somatic cell state was

captured in the imaging process. Taken together, live-cell imag-

ing at single-cell resolution supports that some GMPs exist in a

privileged state (Figures 2A and S1A and Movie S1).

To further validate that a subset of GMPs is privileged, factor-

transduced GMPs were FACS sorted as single cells into 96-well

plates to achieve clonal reprogramming (experimental scheme in

Figure S5A). When 420 total wells were examined after 5 days of

Dox induction, 71 wells (17%) gave rise to Oct4:GFP+ colonies.

The majority of wells (97%) positive for Oct4:GFP+ colonies did

not contain noticeable numbers of cells bearing hematopoietic

morphology (Figure 2B), confirming our observation that most

progeny became reprogrammed using the imaging approach

(Movie S1). Many of these wells contained multiple sister

colonies, with small numbers of round-shaped cells (also

Oct4:GFP+) in close proximity to the larger colonies (Figure 2B),

which we have described previously to result from the dynamic


migrative behavior associated with pluripotency (Megyola et al.,

2013). To ensure that all GMP-derived cells were scored, we

repeated single-cell reprogramming with GMPs from a trans-

genic mouse line that expresses an H2B-GFP fusion protein

(Hadjantonakis and Papaioannou, 2004) so that all progeny

could be identified based on their expression of H2B-GFP (Fig-

ure S1C). In wells that contained GMP-derived colonies, all

H2B-GFP+ cells were part of or close to mESC-like colonies

and were positive for alkaline phosphatase activity (Figure 2C,

top). In contrast, single lineage-negative-Kit+Sca+ HSPCs

(referred to as LKS cells, sorting strategy in Figure S1B) gave

rise to many H2B-GFP+ cells that scattered away from the

colonies and were negative for alkaline phosphatase activity

(Figure 2C, bottom). These data from clonal reprogramming con-

ditions indicate that a subset of GMPs produce essentially all-

reprogrammed progeny.

To further ensure that all GMP-derived progeny were

analyzed, including some that might have adopted alternative

cell fate, we trypsinized the entire reprogramming culture initi-

ated from GMPs and assessed the complete cellular constitu-

ents by FACS on day 6 of factor expression. We used a pan

hematopoietic marker, CD45, to mark the somatic cell state

and Oct4:GFP for the pluripotent state. Consistent with the

observation that the progeny of 17% of GMPs all turned on

Oct4:GFP+, the entire culture was largely binary, consisting of

two major populations: the CD45+ hematopoietic cells and the

Oct4:GFP+ reprogrammed cells (Figures 2D and S1D). These

two populations accounted for �97% of all cells in the culture,

with the remaining 3% likely being the feeder cells, as indicated

by their noncharacteristic scattering profile (Figure S1D). This

analysis demonstrates that alternative cell fates are not prevalent

in GMP-initiated reprogramming.

Another anticipated feature of nonstochastic reprogramming

is a synchronous short latency, which could be reflected

as either the number of cell divisions or the amount of time pre-

ceding endogenous pluripotency activation (Figure 1B). We

assessed these parameters using the imaging data and found

that most of these progeny (85%, total n = 38) activated endog-

enous Oct4 after four to five cell divisions (Figure 2E). The re-

maining 15% of events took six or seven cell divisions, but not

more. Expressed in absolute amount of time, all progeny

became Oct4:GFP+ (detectible by fluorescence microscopy)

within 46.0 ± 6.8 hr (n = 38), indicating a largely uniform fast

kinetics. Importantly, this latency is highly consistent among

the 14 GMP lineages obtained across five experiments. These

results support the privileged nature of the founder GMPs and

demonstrate that Yamanaka reprogramming could occur much

more rapidly and synchronously than previously appreciated

(Hanna et al., 2009).

Taken together, the data above demonstrate that a subset of

GMPs exist as privileged cells for Yamanaka reprogramming.

Privileged GMPs Display an Ultrafast Cell CycleWe next asked whether the privileged cells display any unusual

characteristics. We examined the live-cell imaging data, quanti-

fied the timing of events in the GMP-initiated reprogramming lin-

eages, and noticed that the first cell cycle (defined as the time

between the first and second mitotic divisions from the initial

cells; diagram in Figure S1A and Figure 2A) on average took

only�8 hr (Figure 3A). In contrast, a typical mammalian cell cycle

lasts 16–24 hr or longer (Lodish et al., 2000). mESCs and iPSCs

display one of the fastest cell cycles, which lasts�12 hr, owning

to a truncated G1 phase (Hanna et al., 2010; Wang and Blelloch,

2009). Thus, it was striking that the first cell cycle during privi-

leged reprogramming was even substantially faster than that of

mESCs. The three subsequent cell cycles were also fast but

became increasingly longer and stabilized at the speed of

�12 hr per cycle (Figure 3A). Using a cell-cycle phase reporter

(Sakaue-Sawano et al., 2008), we observed a short G1 duration

(�2 hr) for the first cell cycle (Figures 3B and S2 and Movie S2).

These data suggest that G1 shortening underlies the fast cell

cycle. Importantly, because this ultrafast cycle occurred within

the first 10 hr of Dox addition and the subsequent cycles actually

lengthened when factors were continued to be induced (Fig-

ure 3A), the fast cycling property should be intrinsic to the subset

of GMPs and independent from Yamanaka factors. Indeed,

�20% of all GMPs examined (n = 54) display such an ultrafast

cell cycle. Thus, privilegedGMPs are associated with an ultrafast

initial cell cycle.

If privileged GMPs are distinguished by an ultrafast cell cycle,

one would predict that prospectively isolated faster cycling

GMPs reprogram with higher efficiency than do their slower

cycling counterparts. To test this, we fractionated GMPs based

on their cell-cycle speed, using a dye dilution approach (Koche

et al., 2011; Takizawa et al., 2011). Specifically, GMPs were

labeled with a stable dye CFSE, and the number of divisions

that each cell experienced was indicated by the remaining

amount of dye after proliferation-based dye dilution (Figure S3).

Thus, the cells that retained the least amount of dyes repre-

sented the most rapidly cycling cells (Figure 3C). With this

strategy, we sorted factor-transduced GMPs into CFSE-low

and CFSE-high single cells (representing relatively fast and

slow cycling GMPs, respectively) (Figure 3C) and compared their

reprogramming efficiency (Figure 3D). The fast cycling GMPs re-

programmed with higher efficiency than the slower cycling ones

(Figure 3D and Tables S2A and S2B). To rule out the possibility

that this difference was due to the cycling status affecting viral

transduction (even though lentivirus infect both cycling and

quiescent cells), we confirmed this finding using GMPs from

a mouse strain harboring a knockin cassette of the same

polycistron (referred to as the iPS mouse) (Carey et al., 2010)

(Table S2C). Taken together, the data above support that

privileged GMPs are associated with an ultrafast cell cycle,

and the subpopulation of faster cycling GMPs reprogram more

efficiently.

Accelerating Hematopoietic Progenitor Cell CyclingIncreases Reprogramming Efficiency and Induces theEmergence of Privileged CellsThe unusual cell-cycle speed that associated with privileged

GMPs raises the possibility that the ultrafast cell cycle repre-

sents a path to overcome the bottleneck restricting reprogram-

ming. We reasoned that, for a cell that cycles slowly and

reprograms inefficiently, increasing its cycling speed may

enhance its reprogramming and privileged cells could be poten-

tially induced. LKS cells cycle slowly and contain much fewer


Oct4:GFP ON

Time

A

From a single GMP on Day6

B Phase Oct4:GFP

Div.1 Div.2 Div.3 Div.4

2:10

9:05

9:45

21:30

19:00

16:50

17:05

31:25

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28:50

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1st cycle 2nd cycle 3rd cycle 4th cycle

G1 G1 G1

200 μm 200 μm

0

20

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4 5 6 7

% o

f tot

al e

vent

s

# of divisions before Oct4:GFP ON

E

H2B-GFP AP

GMP

LKS

CC

D45D

Oct4:GFP

200 μm 200 μm

200 μm 200 μm

Figure 2. Nonstochastic Reprogramming from a Subset of GMPs

(A) A representative lineagemap from a single GMP toOct4:GFP+ progeny. The color of the circles corresponds to the color of arrows inMovie S1 and Figure S1A.

Lines denote lineage relationship. Filled green circles denote Oct4:GFP fluorescence as detectible by time-lapse imaging. The numbers (hr:min) at each

branching point indicate the time when mitosis occurred and were used to derive cell-cycle lengths. Red blocks on the horizontal block arrow indicate reporter

signals of a G1 phase reporter (see Figure S2 and Movie S2).

(B) GMPs were transduced with Dox-inducible Yamanaka factors and single cell sorted into 96-well plates in reprogramming conditions (scheme in Figure S5A).

Representative images of the reprogramming product of a single GMP are shown. Note the presence of multiple sister colonies and that many of the round-

shaped cells in the vicinity of Oct4:GFP+ colonies are also GFP+ (zoom in, red arrows).

(C) GMP and LKS cells from H2B-GFPmice were reprogrammed as single cells. Representative images of the reprogramming culture from a single GMP (top) or

LKS cell (bottom) after 6 days of Dox induction are shown. Note the presence of H2B-GFP+, alkaline phosphatase (AP)-negative cells in LKS-initiated culture, but

not in the GMP-initiated culture. A slight increase in colony sizes was noted after fixation/staining.

(legend continued on next page)



p = 4.4 x 10-8

RandomGMPsCell cycles of successfully

reprogrammed GMPs

Cel

lcyc

ledu

ratio

n(h

ours

)

0

5

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15

20

25

30

1st 2nd 3rd 4th

A

0

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* Unlabeled controlCFSE

Fast

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Post label

Post 24h dilution

0

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Fast Slow

D

Rep

rogr

amm

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effic

ienc

y(%

) GMP single cell reprogramming

Cell cycles of successfullyreprogrammed GMPs

%of

Max

%of

Max

23/360

7/360

Figure 3. Privileged GMPs Display an Ultrafast Cell Cycle

(A) Cell cycle lengths of successfully reprogrammedGMP lineages. Each dot represents amitotic event that gave rise to Oct4:GFP+ cells. More dots were scored

for cell cycles 2 and 3 due to cell number increase following previous divisions. Within the same imaging experiments, the first cell cycle of randomGMPs (n = 54)

was also measured. Pooled data of 14 lineages from five independent imaging experiments are shown. *p < 0.001.

(B) A G1 phase reporter was used to measure G1 duration by time-lapse imaging. Each dot represents a mitotic event that gave rise to Oct4:GFP+ cells.

(C) Representative FACS plots of GMPs immediately after CFSE label (top) and following 24 hr of dye dilution (bottom). The gating strategy for sorting fast and

slow cycling cells is shown.

(D) The fast and slow cycling GMPs were single cell sorted into 96-well plates in reprogramming conditions. The number of wells that contained Oct4:GFP+

colonies and the total number of plated wells are indicated, separated by a slash.

See also Figures S2, S3, Table S2, and Movie S2.


ultrafast cycling cells as compared to GMPs (Figures 4A, black

bars, and S4 and Table S3). Accordingly, LKS cells reprog-

rammed with much lower (7.6-fold) efficiency than did GMPs in

single-cell reprogramming assays (Figure 4B, black bars and

Table S4), consistent with a previous report (Eminli et al.,

2009). Importantly, LKS cells mostly reprogrammed in a sto-

chastic manner, i.e., single LKS cells gave rise to both reprog-

(D) GMP-initiated reprogramming culture were trypsinized after 6 days of Dox in

shows that CD45+ hematopoietic cells and Oct4:GFP+ cells make up �97% of

(E) The number of mitotic divisions before Oct4:GFP became detectible by imag

See also Figures S1, S2, S5A, Table S1 and Movies S1, S2.

rammed cells and substantial numbers of nonreprogrammed

cells (Figure 2C, bottom).

While the fast cycling of GMPs is required to sustain rapid tis-

sue turnover, stem cells remain largely quiescent to sustain life-

long tissue homeostasis and injury repair and only begin cycling

in response to proper cues (Greco and Guo, 2010). Thus, we

tested whether culturing LKS cells in growth factors/cytokines

duction and were stained with a CD45 antibody. A representative FACS plot

the culture.

ing (n = 38).


Rep

rogr

amm

ing

effic

ienc

y (%

)

Fresh cells CulturedB

LKS

GMP

4-8 8-11 9-15 >=12 Dead

A

% o

f pop

ulat

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% o

f pop

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ion

Phas

e O

ct4:

GFP

C

Progeny of a single LKS cellDay 5 on Dox0

5

10

15

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25

LKS GMP

0

20

40

60

0

20

40

60

HSPC cycle speed (hours/cycle)

77/420

2/42010/420

102/420

300 μm

300 μm

Figure 4. Accelerating HSPC Cycling Increases Reprogramming Efficiency and Induces the Emergence of Privileged Cells

(A) The cell-cycle speeds of LKS andGMP cells weremeasured, either fresh (black bars) or after 5 days of culture (red bars). The percentage of cells with indicated

cell-cycle speed is plotted (details in Extended Experimental Procedures and Figure S4). Note that fresh GMPs and cultured LKS cells contain ultrafast cycling

cells (red boxes), which were low/absent in fresh LKS cells and cultured GMPs.

(B) The reprogramming efficiency of fresh or cultured LKS andGMP cells were compared in single cell reprogramming assays. The number of wells that contained

Oct4:GFP+ colonies and the total number of plated wells are indicated, separated by a slash.

(C) Singly sorted LKS cells were cultured for 5 days and their somatic progeny transferred into reprogramming conditions for another 5 days (scheme shown in

Figure S5B). Shown is one representative well dominated by Oct4:GFP+ colonies and lacking hematopoietic-like cells.�15% of the wells containing Oct4:GFP+

colonies (or 3.6% of total wells) displayed this privileged reprogramming behavior.

Images were captured with 103 magnification. See also Figures S4 and S5 and Tables S3 and S4.


increases the ultrafast cycling cells (Figure S4). Indeed, after

5 days of culture, an ultrafast cycling population (>16%)

emerged from the LKS culture to a level even higher than freshly

isolated GMPs (Figure 4A, red bars). In contrast, GMPs cultured

under the same condition resulted in a drastic loss of the ultrafast


cells (Figure 4B, red bars). We then tested the reprogramming

efficiency of these cultured cells in the stringent clonal reprog-

ramming assay (scheme in Figure S5B). As a comparison, the

transduced LKS or GMP cells from the same experiment were

single cell sorted and reprogrammed directly without preculture


(scheme in Figure S5A). Consistent with the increase in the

ultrafast cycling population, cultured LKS cells became highly

efficient (24.3%) in reprogramming, more so than the freshly

isolated GMPs (18.3%) (Figure 4B, red bars, and Table S4). In

addition, the drastic decrease of the ultrafast cycling population

in cultured GMPs was accompanied by a steep drop of reprog-

ramming efficiency to 0.5% (Figure 4B). These data indicate that

changes in cycling speed parallel the changes in reprogramming

efficiency.

The cultivation turned a single LKS cell into tens to hundreds of

cells (scheme in Figure S5B). Sampling a pool of the mixed prog-

eny from many LKS cells revealed ultrafast cycling behavior in a

fraction of them (Figure 4A). It is conceivable that, if the ultrafast

cycling behavior is shared by the daughters of a common LKS

cell, privileged reprogramming might occur simultaneously

from the many daughter cells. To test this, we transferred the

somatic progeny from single LKS cells into reprogramming con-

ditions (Figure S5B). After 5 days of Dox induction, we observed

the astounding appearance of privileged reprogramming (Fig-

ure 4C and Table S4). In �15% of the wells that contained

Oct4:GFP+ colonies (or 3.6% of total wells), hematopoietic-like

cells were absent. Instead, numerousOct4:GFP+ colonies domi-

nated these wells. Again, we confirmed that all progeny were

accounted for by using clonal LKS cells from the H2B-GFP-

transgenic mice (Figures S5C and S5D). As such, reprogram-

ming from these somatic cells was no longer a slow or rare event

but, instead, fast and prevalent. These wells provide further vali-

dation for the existence and unique behavior of privileged cells.

Thus, mitogenic activation of the slowly cycling LKS cells

increased the ultrafast cycling population as well as their reprog-

ramming efficiency. This activation also induced the emergence

of privileged cells for reprogramming.

Ultrafast Cycling Cells Emerge from Fibroblasts afterProlonged Factor Induction and Harbor the Majority ofReprogramming ActivityTo extend our finding of the ultrafast cycling cells in the hemato-

poietic system, we examined reprogramming from mouse em-

bryonic fibroblasts (MEFs). Because it is well established that

MEFs reprogram with low efficiency (�0.1%) and long latency

(>8 days), we did not anticipate that a substantial fraction of

MEFs naturally cycle at the ultrafast speed. Instead, we asked

whether an ultrafast cycling population can be induced by

Yamanaka factors. Because MEFs do not proliferate well at

clonal density, we estimated their cycling speed following a

similar dye dilution approach (Figure S3). Specifically, we trans-

duced MEFs with the Dox-inducible factors and added Dox to

induce their expression. On day 4 of Dox treatment, the cultures

were trypsinized, labeled with CFSE, and plated back in the

presence of Dox to allow the dye to be diluted for 2 additional

days (Figure S6A). This time frame was chosen based on the

observation that >7 days of Dox induction led to the appearance

of a small percentage of Oct4:GFP+ cells (although Oct4:GFP+

colonies were not visible at this time), whereas 6 days of Dox

treatment yielded no such cells (Figure S6B). Consistent with

MEFs being a more heterogeneous population, the CFSE peak

immediately following dye labeling was wider than that of

hematopoietic progenitors (compare Figures 5A and 3C). Strik-

ingly, a small population of cells (1%–8%) that retained much

less CFSE emerged after a total of 6 days of Dox treatment (Fig-

ure 5A, red box). In contrast, such a population was not observed

in the absence of Dox from the same transduced MEFs (Fig-

ure S6C), indicating that the CFSE-low population was induced

by factor expression. The mean CFSE fluorescence intensity

for this minor population was at least 20-fold less than that of

the bulk cells, suggesting that the fast cells had undergone, on

average, four more divisions than the bulk MEFs. Given that

the bulk MEFs divide every 25–30 hr (Sage et al., 2000; Wang

et al., 2001; White and Dalton, 2005) and would divide, on

average, once to twice during 48 hr, the CFSE-low cells must

have undergone approximately five to six divisions during the

48 hr of dye dilution period. This estimation suggests that

some of the CFSE-low cells had divided at a speed close to

8 hr per cycle. Thus, an ultrafast cycling population emerges

from MEFs in the presence of prolonged factor expression.

We then asked whether the ultrafast cycling cells are more

efficient in reprogramming. Cells of three different CFSE levels

(reflecting different cycling speed)were FACSsorted and their re-

programming efficiencies compared. Due to their poor ability to

endure clonal culture and overall low reprogramming efficiency,

we were unable to perform clonal reprogramming assays with

MEFs. Instead, we used the generally adopted definition for re-

programming efficiency, which is the number of Oct4:GFP+ col-

onies divided by the number of cells plated. The fast cycling cells

displayed �1,150 -fold increase in reprogramming efficiency as

compared to the cells cycling at a medium speed characteristic

of the bulk culture (Figures 5B and 5C). The slow populations

completely lacked reprogramming activity (Figures 5B and 5C).

Overall, we estimated that the minor but fast population con-

tained�99.7%of the reprogrammingactivity of theentire culture.

Taken together, an ultrafast cycling population was induced in

MEF-initiated reprogramming after 6 days of factor expression.

The reprogramming activity was exclusively confined to the

rapidly cycling cells.

p53 Knockdown Expands the Ultrafast CyclingPopulation, which Harbors the Majority ofReprogramming Activity among p53 Knockdown MEFsAlthough genetic perturbations that result in hastened prolifera-

tion have been well documented to increase reprogramming

efficiency (Banito et al., 2009; Hong et al., 2009; Ruiz et al.,

2011; Utikal et al., 2009), it remains unclear whether the role of

cell-cycle acceleration is to increase the number of daughter

cells, each of which possesses an independent probability to

reprogram, or to confer a higher probability selectively to a sub-

set of cells to accomplish the fate transition.

To distinguish these two possibilities, we examined reprog-

ramming in the context of p53 knockdown, which enhances re-

programming via a cell-division-dependent mechanism (Hanna

et al., 2009). Indeed, higher reprogramming efficiency occurred

with p53 knockdown (Figures 6A and 6B). We then performed

CFSE dilution assay to measure the ultrafast cycling fractions

(Figure 6C). Consistent with a role of p53 in regulating cell cycle,

p53 knockdown led to overall decreased CFSE level (i.e.,

increased cycling) among the bulk population, as evidenced by

a shift of the main CFSE peak toward left (Figure 6C). As in


Nega ve Post label Post dilu on

>4 more div.

Fast Med SlowA

CFSE

B

Rep

rogr

amm

ing

effic

ienc

y (%

)

0

0.2

0.4

0.6

0.8

Fast Med Slow

1150x

% o

f Max

C

Phase

Oct4:GFP

AP

Fast Med Slow

300 μm

300 μm

300 μm

300 μm

300 μm

300 μm

(legend on next page)



A

β-actin

p53

shCtrl shp53B

CFSE

Post

labe

lPo

st48

hdi

lutio

n

shControl

shp53

C

0123456

shControl shp53

Fast

cells

(%)

D

0

0.5

1

1.5

2

ControlFast

ControlSlow

shp53Fast

shp53SlowR

epro

gram

min

gef

ficie

ncy

(%)

E

0

0.01

0.02

0.03

0.04

shControl shp53

Rep

rogr

amm

ing

effic

ienc

y(%

)

shControl shp53

Unlabeled MEFs

Fast

Fast

Slow

Slow

Figure 6. The Ultrafast Cycling Population from MEFs Is Increased by p53 Knockdown and Accounts for Most Reprogramming Activity

MEFs were transduced with either control (shCtrl) or shRNAs targeting p53 (shp53), along with the Dox-inducible reprogramming factors.

(A) p53-knockdown increased reprogramming in unfractionated MEFs.

(B) Western blot analysis confirmed p53 protein downregulation by p53-targeting shRNAs.

(C and D) Factor-transduced MEFs on 4 days of Dox treatment were labeled with CFSE (top) and were allowed to dilute the dye for another 48 hr. The cultures

were then trypsinized andCFSE intensity analyzed by FACS. (C) Representative FACSplots are shown for CSFE levels right after labeling or following 48 hr of dye-

dilution. Gating for fast and slow cells is shown. (D) Quantification of fast cycling cells (n = 3 per condition).

(E) Reprogramming efficiency of the sorted fast and slow cycling MEFs as shown in (C) (n = 3 per condition).

Error bars indicate SD.


control knockdown, a distinct fast cycling population retaining

much less CFSE was clearly identifiable in p53 knockdown cells.

Importantly, this fast cycling population increased with p53

knockdown as compared to the control (Figures 6C and 6D).

These data indicate that p53 knockdown increases the number

of cells cycling past a certain threshold speed.

We then asked whether reprogramming activity is confined

within the fast cycling population or nondiscriminatively in p53

knockdown cultures. With p53 knockdown, the great majority

of the reprogramming activity was again confined to the fast

cells, similar to the situation in controls (Figure 6E). These data

Figure 5. Ultrafast Cycling Cells Emerge from Fibroblasts after Yaman

Activity

Factor-transduced MEFs were treated with Dox for 4 days, labeled with CFSE, a

(A) The levels of CSFE in nonlabeled MEFs (negative) right after labeling (postla

medium (med), and slow cycling cells are shown. Note that the fluorescence inten

division (div.) differences.

(B) Reprogramming efficiency of cells sorted on CFSE. Oct4:GFP+ colonies we

indicate SD).

(C) Representative images of the reprogramming cultures from cells of different cy

and alkaline phosphatase (AP)-stained dishes were from whole 60 mm plates.

See also Figure S6.

demonstrate that p53 impacts reprogramming by controlling

the emergence of the ultrafast cycling cells. Outside of the fast

cycling population, however, other cells do not become more

likely to reprogram. Taken together, our data suggest that cell-

cycle acceleration toward a critical threshold could be an impor-

tant bottleneck for reprogramming.

Molecular Characterization Confirms Enhanced CellCycle as the Predominant Feature of a Unique Cell StateTo gain insights into the molecular nature of the unique cell state

associated with the extraordinary efficiency to transition into

aka Factor Expression and Harbor the Majority of Reprogramming

nd allowed for dye-dilution for 48 hr in the presence of Dox.

bel) or following 48 hr of dilution (postdilution) are shown. The gates for fast,

sity difference between fast and medium populations indicates more than four

re scored on day 20 from initial Dox induction (n = 3 per condition; error bars

cling speed. Phase and Oct4:GFP images were captured at 103magnification,


0

0.0001

0.0002

0.0003

0.0004

SlowMEF1

FastMEF1

SlowMEF2

FastMEF2

iPS1 iPS2

Endogenous Sox2

Nor

mal

ized

exp

ress

ion

(2Δ

CT )

BA First 2 PCA components

GO Category Name Related Cellular ProcessDNA_REPLICATION Cell CycleCHROMOSOME Cell CycleDNA_DEPENDENT_DNA_REPLICATION Cell CycleM_PHASE Cell CycleCHROMOSOMAL_PART Cell CycleMITOSIS Cell CycleM_PHASE_OF_MITOTIC_CELL_CYCLE Cell CycleREPLICATION_FORK Cell CycleDNA_PACKAGING Cell CycleCELL_CYCLE_PROCESS Cell CycleCELL_CYCLE_PHASE Cell CycleDNA_METABOLIC_PROCESS Cell CycleCONDENSED_CHROMOSOME Cell CycleSPINDLE Cell CycleSPLICEOSOME RNA ProcessingDNA_REPAIR Cell CycleCHROMOSOMEPERICENTRIC_REGION Cell CycleRNA_PROCESSING RNA ProcessingRIBONUCLEOPROTEIN_COMPLEX RNA ProcessingSPINDLE_POLE Cell CycleSMALL_NUCLEAR_RIBONUCLEOPROTEIN_COMPLEX RNA ProcessingMITOTIC_CELL_CYCLE Cell CycleSTRUCTURAL_CONSTITUENT_OF_RIBOSOME Protein TranslationCHROMOSOME_SEGREGATION Cell CycleNUCLEAR_PART Cell CycleCHROMATIN_BINDING RNA Transcription

C

D F

β-actin

p57

shCtrl shp57

00.20.40.60.8

11.2

E

Rep

rogr

amm

ing

effic

ienc

y (%

)

0

0.1

0.2

0.3

0.4

shCtrl shp570

0.5

1

1.5

shCtrl shp57

*LKS GMP

p57 RNA

Nor

mal

ized

exp

ress

ion

(legend on next page)




pluripotency, we performed transcriptome analysis by RNA

sequencing (RNA-seq) (Pan et al., 2013). The transcriptomes of

fast GMPs and the fast cells arising from MEFs after 6 days of

Dox induction (referred to as ‘‘fast MEFs’’ from now on) were

compared to slow GMPs, slow MEFs, bulk MEFs, and estab-

lished iPSCs. Principle component analysis (PCA) showed that

biological replicates of the same cell states cluster in close prox-

imity (Figure 7A). Furthermore and as expected, fast MEFs are

enriched for genes associated with MEFs permissive to reprog-

ramming, whereas the slow MEFs display signatures associated

with MEFs refractory to reprogramming (Polo et al., 2012) (Fig-

ure S7A). These data indicate that our RNA-seq data are of

good quality.

Activation of endogenous Sox2was known to mark the begin-

ning of the nonstochastic phase of reprogramming (Buganim

et al., 2012). However, neither GMPs nor fast MEFs expressed

endogenous Sox2, contrasting iPSCs (Figures 7B and S7B).

Thus, the fast cycling cells exist in a cell state earlier than that

marked by endogenous Sox2 activation, indicating that the non-

stochastic trajectory of reprogramming can be attained earlier

than that demonstrated previously (Buganim et al., 2012).

The high reprogramming efficiency of both fast GMPs and fast

MEFs implies that they may share certain molecular similarities

that contribute to their unique reprogramming behavior even

though they are well separated on the PCA plot (Figure 7A). To

identify such similarities, we performed gene set enrichment

analysis (GSEA) comparing the fast populations with the bulk

MEFs and the slowMEFs, which were similar in gene expression

(Figure 7A). Strikingly, when all gene ontology (GO) categories

were examined, those associated with enhanced cell prolifera-

tion appear to be the predominant feature (Figures 7C and

S7C). Other enriched GO categories reflect enhanced RNA tran-

scription/processing and protein translation, which are consis-

tent with molecular needs in association with fast cell cycle. Of

note, enhanced cell cycle and protein translation were also en-

riched in fast GMPs as compared to slow GMPs, albeit to a

lesser extent (data not shown), due to their overall similarity in

gene expression (Figure 7A). In addition to unbiased GSEA anal-

ysis, we also examined enrichment in mesenchymal-epithelial

transition (MET), apoptosis, and metabolism. Results show that

fast GMPs and fast MEFs are depleted for mesenchymal signa-

tures (Table S5). There is no consistent difference in apoptosis

gene sets, whereas metabolism gene sets enriched in fast cells

are predominantly related to nucleoside metabolism, again sup-

porting a difference primarily in cell cycle (Table S5). These

molecular analyses suggest that fast cell cycle is indeed the

major attribute shared between the two cell states capable of

Figure 7. Molecular Characterization of the Cellular States

(A) Principle component analysis (PCA) was performed on RNA sequencing da

independent reprogrammed cells (iPS) and bulk MEFs (MEF) were included as c

(B) The endogenous Sox2 level was measured by qRT-PCR using primers speci

(C) Gene set enrichment analysis (GSEA) was performed comparing the fast cells

gene ontology (GO) categories enriched in both fast cell populations are shown.

(D) qRT-PCR analysis of p57 mRNA level in HSPCs.

(E) Western blot analysis confirms p57 protein downregulation by p57 targeting

(F) Reprogramming efficiency of LKS and GMP cells following treatment with co

Error bars indicate SD. See also Figure S7.

high-efficiency reprogramming. Loss of mesenchymal gene

expression appears as an additional contributor, which is impor-

tant in early reprogramming (Li et al., 2010; Samavarchi-Tehrani

et al., 2010).

To further understand the molecular regulator(s) of the privi-

leged cell state, we thus focused on cell-cycle regulators and

asked which cell-cycle genes are differentially expressed be-

tween LKS and GMP cells, which display drastically different

cell-cycle and reprogramming behaviors (Figure 4). We exam-

ined all expressed cyclins, cyclin-dependent kinases (CDKs),

and CDK inhibitors using published data (Krivtsov et al., 2006).

The only gene that showed a major difference was Cdkn1c, or

p57 (Figures S7D–S7F). p57 is a G1/S-blocking CDK inhibitor

that was expressed >10-fold higher in the slow cycling LKS

cells as compared to GMPs, a result confirmed by qRT-PCR

(Figure 7D). We next tested whether reduced p57 promotes

LKS cell reprogramming. We inhibited p57 expression by

sequence-specific shRNAs and confirmed downregulation of

the protein (Figure 7E). p57 shRNAs in LKS cells led to signifi-

cantly higher reprogramming (Figure 7F, LKS), consistent with

previous reports that loss of p57 leads to enhanced HSC cycling

(Matsumoto et al., 2011; Zou et al., 2011). In contrast, similar

treatment in GMPs did not yield further increase in reprogram-

ming (Figure 7F, GMP), suggesting that the endogenous p57

level in GMPs is already sufficiently low. These data support

p57 as one of the molecular roadblocks limiting HSPCs to enter

a privileged cell state.

DISCUSSION

We demonstrated that Yamanaka reprogramming could occur

nonstochastically from a privileged somatic cell state. Our data

support a model in which privileged and stochastic reprogram-

ming coexist as part of a continuum (Figure S7G). Because the

privileged cell state is highly efficient in reprogramming, it might

suggest an ‘‘elite’’ status. However, the privileged state differs

from the conventional ‘‘elite’’ cells in several key aspects. First,

the conventional ‘‘elite’’ model assigns different probability of re-

programming to different cells and assumes it as a fixed property

(Hanna et al., 2009, 2010; Yamanaka, 2009). Ourmodel suggests

that the privileged state is rather dynamic, which could exist

naturally but could also be gained via alternative means. Yama-

naka factors may facilitate the emergence of a similar state that

could be viewed as an ‘‘acquired privilege.’’ Our data also sug-

gest that the exact molecular means to reach a privileged state

might be different in different cell types. Specifically, p57 pre-

vents LKS cells to become privileged, but it is conceivable that

ta. Isolation of fast and slow cells was performed as described above. Dox-

ontrols. Three to five replicates were used for each cell type.

fic for its 30UTR region. Two biological replicates for each cell type are shown.

(FastGMP and FastMEF) versus the slow cells (MEF and SlowMEF). The top 25

shRNAs.

ntrol (shCtrl) or p57-targeting shRNAs (shp57).



other cell types may utilize alternative means to reach the

extreme speed of cycling. Although inhibition of p57 in HSCs

enhances their cycling (Matsumoto et al., 2011; Zou et al.,

2011), MEFs deficient in p57 do not become overtly proliferative

(Takahashi et al., 2000). Consistently, shRNA inhibition of p57 in

MEFs resulted in no detectible increase in ultrafast cycling cells

or iPS colony numbers (data not shown). Second, the conven-

tional ‘‘elite’’ cells are considered for their ability to produce

any pluripotent progeny without a specific latency. In contrast,

our privileged cells produce essentially all pluripotent progeny

with a synchronous short latency. Depending onwhen andwhich

cells reach a privileged state (somatic or acquired) within a cell

lineage, reprogramming could occur with varying efficiency

and latency (Figure S7G). Privileged somatic cells represent an

extreme case when the bottleneck is overcome prior to their

exposure to Yamanaka factors.

One of the key features of the privileged cell state is an unusu-

ally fast cell cycle, a mechanism that is distinct from previously

suggested ones, as GMPs are nonepithelial (Aasen et al.,

2008; Li et al., 2010; Samavarchi-Tehrani et al., 2010), do not

endogenously express any of the Yamanaka factors (Kim et al.,

2008; Krivtsov et al., 2006; Wakao et al., 2011), are more differ-

entiated than stem cells (Eminli et al., 2009; Stadtfeld and

Hochedlinger, 2010), and do not have reduced MBD3 levels

(Luo et al., 2013; Rais et al., 2013) (Figure S7H–S7J). The cellular

states of naturally fast cycling somatic cells, such as GMPs, and

those that arise fromMEFs in response to Yamanaka factors are

drastically different (Figure 7A). However, the fact that both types

of fast cycling cells display extraordinarily high reprogramming

efficiency suggests that a fast cell-cycle kinetic has a dominant

role in determining how the epigenome responds to remodeling.

A connection between cell-cycle and fate outcome has been

noted in other cellular systems (Kueh et al., 2013; Pauklin and

Vallier, 2013; Pop et al., 2010; Tsunekawa et al., 2012). Detailed

studies are required to reveal the connections linking cell-cycle

dynamics to epigenetic remodeling.

Although ultrafast cell cycle is a key feature of the privileged

state and ultrafast cycling cells reprogrammuchmore efficiently,

we also notice that not all fast cycling cells become reprog-

rammed. A number of reasons, both biological and technical,

could limit the observation of reprogramming even though the

major bottleneck may have been overcome. Biologically, this

may relate to proper establishment of E-cadherin-mediated

cell-cell adhesion (Chen et al., 2010; Li et al., 2010; Megyola

et al., 2013; Redmer et al., 2011; Samavarchi-Tehrani et al.,

2010) and optimal level or stoichiometry of continued factor

expression (Carey et al., 2011; Papapetrou et al., 2009). Such

events could impact a large number of cells that collectively

reduce the overall reprogramming efficiency. Technically,

although the CSFE dilution method greatly enriches for ultrafast

cycling cells based on divisional history, it is not an absolute or

instant measure of cell-cycle speed. The resolving power of

this method is heavily influenced by the inherent populational

heterogeneity and the duration of dye dilution. This explains

why much larger difference was seen between populations

isolated from MEFs as compared to that between different

GMPs (GMPs are more homogeneous and were allowed a

shorter period for dye dilution). Defining the additional require-


ments for the ultrafast cycling cells to secure the transition into

pluripotency will help to further elucidating the mechanisms of

reprogramming.

EXPERIMENTAL PROCEDURES

Mice and Cells for Reprogramming

All mouse work was approved by the Institutional Animal Care and Use

Committee of Yale University. The Oct4:GFP 3 Rosa26:rtTA mice were

derived by crossing Oct4:GFP mice with Rosa26:rtTA mice (Eminli et al.,

2009; Megyola et al., 2013) and were used to isolate/derive hematopoietic

cells, MEFs, and ESCs. The iPS mouse (R26rtTA;Col1a14F2A) (Carey et al.,

2010) (stock# 011004) and H2B-GFP transgenic line (stock# 006069) were

purchased from the Jackson Laboratory.

Cell Sorting, Culture, and Reprogramming

Hematopoietic populations were sorted as previously (Guo et al., 2010).

Hematopoietic growth factors included 100 ng/ml mSCF, 50 ng/ml mIL3,

50 ng/ml Flt3L, and 50 ng/ml mTPO (PeproTech) and were added to a

serum-free base medium X VIVO15 (BioWhittaker). Reprogramming condi-

tions included inactivated MEF feeders, complete mESC medium, and

2 mg/ml of Dox (Sigma). CFSE was purchased from Life Technologies. A work-

ing concentration of 7.5–8 mM was used following manufacturer’s instruction.

CFSE-labeled GMPs after 24 hr of dye dilution were sorted for the brightest

and dullest 15%–20% of cells as slow and fast GMPs, respectively. CFSE-

labeled MEFs were cultured in mESC medium with Dox for 48 hr before sort-

ing. MEFs cultured in the absence of Dox were used as controls.

Transcriptome Analysis by RNA Sequencing

1,000 of each cell types were directly sorted into lysis buffer in 96-well

plates for RNA isolation. The mRNA selection and reverse transcription were

performed as described previously with some modifications (Pan et al.,

2013). Additional details are described in the Extended Experimental

Procedures.

ACCESSION NUMBERS

The RNA-seq data described in Figures 7 and S7 have been deposited at the

GEO database with accession number GSE53074.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, seven

figures, five tables, and two movies and can be found with this article online at

http://dx.doi.org/10.1016/j.cell.2014.01.020.

ACKNOWLEDGMENTS

This work was supported by NIH grants K01 DK082982 (to S.G.),

R01CA149109 (to J.L.), P30 DK0724429 and R01 DK086267 (to D.S.K.),

R01HL106184 (to P.G.G.), U54 CA143798 (to R.F.), P01GM099130 (to

S.M.W.), the State of Connecticut (to J.L., R.F., and S.M.W.), and the Packard

Fellowship for Science and Engineering (to R.F.).

Received: June 7, 2013

Revised: November 4, 2013

Accepted: January 10, 2014

Published: January 30, 2014

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Nonstochastic Reprogramming from a Privileged … Reprogramming from a Privileged Somatic Cell State...

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