RESEARCH ARTICLE SUMMARY◥

CELL REPROGRAMMING

Cell competition duringreprogramming gives riseto dominant clonesNika Shakiba*, Ahmed Fahmy*, Gowtham Jayakumaran, Sophie McGibbon,Laurent David, Daniel Trcka, Judith Elbaz, Mira C. Puri, Andras Nagy,Derek van der Kooy, Sidhartha Goyal, Jeffrey L. Wrana, Peter W. Zandstra†

INTRODUCTION:Recent advances in lineagetracking using DNA-based barcoding technol-ogies have provided new insights into thedevelopmental dynamics of cell populations.Nevertheless, the mechanisms that governthese dynamics and their relation to clonaldominance remain unclear. Understandingthe factors that determine the appearanceand disappearance of individual clones andtheir derivatives in dynamic, heterogeneoussystems has broad implications for normaland aberrant tissue development, providinga new lens with which to understand emer-gent behaviors in multicellular systems.The process of reprogramming, whereby

somatic cells are converted to induced pluri-potent stem cells (iPSCs) through the over-

expression of key transcription factors, servesas a tractable model to probe the connectionbetween intrinsic and extrinsic factors in-fluencing cell population dynamics. The fieldof reprogramming has largely accepted the“clonal equipotency” paradigm, in which so-matic cells have an equal potential to attain aniPSC state; however, it remains unclear whetherinequalities between the fitness of reprogram-ming cells drive non-neutral clonal drift.

RATIONALE: This study aimed to quantita-tively explore the impact of competitive inter-actions in driving clonal dynamics within areprogramming population. A cellular barcod-ing strategy, whereby heritable DNA tags areinserted into the genome of mouse embryonic

fibroblasts (MEFs), was used to track the rel-ative contribution of clonal derivatives to thereprogrammingpopulation.Mathematicalmod-elingwasused todecouple the effect of stochasticreprogramming latencies on clonal dynamics,

enabling the identificationofnon-neutral clonaldom-inance. Finally, a mousemodel was developed totrace the developmentalorigins of the heteroge-neous MEF compartment

and investigate the mechanism of cell fitnessinequalities in reprogramming.

RESULTS: Population reprogramming pro-moted highly selective clonal dynamics, leadingto the elimination of up to 80% of clones after aweek of reprogramming. Single-cell reprogram-ming studies, on the other hand, suggested thatall MEF-derived clones had a propensity to re-program when cultured in isolation. Further-more, an elite subset of dominating clones wasfound to drive the dynamics and compositionof the bulk reprogramming pool. Early dominat-ing clones that emerged after a week of re-programming exhibited a robust and predictablepropensity to overtake the cultureandcontributeto the final successfully reprogrammed iPSCpool. Our modeling results reveal that neutraleffects didnot capture the evolution of clone sizedistributions. Indeed, parallel reprogrammingcultures grown froma commonpool of barcodedMEFs demonstrated a correlation in clonal out-comes, driven by a subset of reprogrammingMEFs. This subset ofMEFs exhibited an a prioripropensity for reprogramming and dominance.Lineage tracing revealed that these MEFs ariseduring embryonic development from aWnt1-expressing population associable with the neu-ral crest compartment.

CONCLUSION: This study reveals cell com-petition as an important parameter that arises inthe population context as a result of geneticallyencoded inequalities in cell fitness. Competi-tion serves as amechanism bywhich otherwisehidden cellswith context-specific eliteness emergeto occupy and dominate the reprogrammingniche. Given the propensity for a small subsetof clones to overtake the population, it is likelythat bulkmeasurements of the reprogrammingprocess represent a biased view of the repro-gramming trajectory. The findings of this studyreveal that cell competition may be a general-izable and controllable parameter for under-standing and controlling the dynamics ofmulticellular developmental and disease sys-tems in a quantified manner.▪

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The list of author affiliations is available in the full article online.*These authors contributed equally to this work.†Corresponding author. Email: peter.zandstra@ubc.caCite this article as N. Shakiba et al., Science 364,eaan0925 (2019). DOI: 10.1126/science.aan0925

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Competition during reprogramming results in dominant clones derived from elite cells.(Top) During reprogramming, when MEFs transition to pluripotency, competition betweencells leads to clonal selection and dominance. Numbered circles represent individualreprogramming clones; circle size indicates the relative abundance of cells derived from aclone in the overall population. (Bottom) Mathematical modeling reveals that clonal dynamicsare not driven by stochastic reprogramming latencies, but rather by a subset of elite MEFsderived from the neural crest (NC) that have an enhanced a priori reprogramming propensity.

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RESEARCH ARTICLE◥

CELL REPROGRAMMING

Cell competition duringreprogramming gives riseto dominant clonesNika Shakiba1*, Ahmed Fahmy2*, Gowtham Jayakumaran2,3†, Sophie McGibbon4,Laurent David5,6,7, Daniel Trcka3, Judith Elbaz3, Mira C. Puri3,8,Andras Nagy3,9,10,11, Derek van der Kooy2,12, Sidhartha Goyal1,4,Jeffrey L. Wrana2,3, Peter W. Zandstra1,12,13,14,15‡

The ability to generate induced pluripotent stem cells from differentiated cell types hasenabled researchers to engineer cell states. Although studies have identified molecularnetworks that reprogram cells to pluripotency, the cellular dynamics of these processesremain poorly understood. Here, by combining cellular barcoding, mathematical modeling,and lineage tracing approaches, we demonstrate that reprogramming dynamics inheterogeneous populations are driven by dominant “elite” clones. Clones arise a priorifrom a population of poised mouse embryonic fibroblasts derived from Wnt1-expressingcells that may represent a neural crest–derived population. This work highlights theimportance of cellular dynamics in fate programming outcomes and uncovers cellcompetition as a mechanism by which cells with eliteness emerge to occupy and dominatethe reprogramming niche.

The ability to engineer gene regulatory net-works to control cell fate holds great po-tential for enabling regenerative medicinetechnologies. Perhaps the most prominentexample to date is the discovery that somatic

cells can be reprogrammed to generate inducedpluripotent stem cells (iPSCs) by the overexpres-sion of four key transcription factors—Oct4, Klf4,c-Myc, and Sox2 (OKMS) (1). Patient-specific iPSCs

serve as an unlimited source of cells that give riseto all cell types in the body, with applications intissue engineering and drug screening. Addi-tionally, reprogramming has enhanced our in-sight into the nature of cellular plasticity.Our understanding of reprogramming comes

largely from population-level analyses of the celltranscriptome, proteome, and epigenome (2–5).These analyses have revealed three distinct andwell-characterized phases of bulk reprogramming:Cells exit a somatic state, transition through atransgene-dependent state (pre-iPSCs), and finallyacquire a stable transgene-independent state(iPSCs) (2–4). These studies do not, however,provide insight into individual cell trajectoriesduring reprogramming (5–7). Indeed, the rela-tionship between cell population outcomes andsingle-cell reprogramming events is poorly under-stood. Analyses of single isolated cells undergoingreprogramming (also known as clones) (8, 9)support the concept of “clonal equipotency,”wherein all cells are able to reprogram, albeitwith stochastic reprogramming latencies (8)(fig. S1A). As a result, individual clones exhibitingasynchronous state changes are expected to giverise to heterogeneity in population reprogram-ming. The apparent contradiction between theprogression of reprogramming through deter-ministic, stepwise phases at the population leveland the stochastic nature of clonal reprogram-ming has yet to be resolved.Clonal interactions give rise to both active

competition, during which cells may directlyinduce apoptosis in their neighbors (10, 11), aswell as passive competition for limited resources

and space (12). Understanding the role that cellcompetition plays during reprogramming iscritical to interpreting the abundance of popula-tion omics data being generated, yet to date, ithas not been investigated.Here we present data on clonal competition in

a population context, revealing how reprogram-ming can occur in a stepwise manner in spite ofthe stochastic nature of clonal reprogramming.We quantified heterogeneity in clonal fitness—therelative survival potential of a reprogrammingclone—using barcode-based cell tracking in pop-ulation reprogramming. In the context of re-programming, fitness is a function of a cell’sreprogramming potential as well as its abilityto outcompete other clones. By using cell com-petition as a new lens, we dissect the link be-tween clonal “fitness” and reprogramming“eliteness.” Although eliteness has been used todescribe clones with enhanced reprogrammingpotential (8), we expand this definition to de-scribe clones that are dominant (where dominanceis quantified by the number of cells in that clone)in the context of large heterogeneous populations.In particular, our data allowed us to examinewhether clonal dynamics during reprogrammingare governed by stochastic reprogramming laten-cies of equipotent clones or by selective clonal lossor dominance of heterogeneous clones (Fig. 1A).Given the role of cell competition in PSC culture(13, 14), we hypothesized that clonal contribu-tions to the reprogramming pool would not bea result of neutral dynamics. Additionally, weexplored whether cellular heterogeneity is anexisting property of somatic cells or emergesduring reprogramming.

Population homogeneity emergesin spite of clonal heterogeneity

In a preliminary study, we assessed the degree towhich single-cell and population reprogrammingdynamics may differ. We used a secondary re-programming system with somatic cells thatcontain a doxycycline (DOX)–inducible cassetteto drive exogenous OKMS factor expression.Briefly, primary iPSCs (harboring DOX-inducibleOKMS transgenes) were used to generate second-ary mice via tetraploid complementation, hereinreferred to as the 1B tetr system (15). The result-ant mice contain secondary mouse embryonicfibroblasts (MEFs) that are readily induced toreprogram via DOX induction. We used ourpreviously reported marker strategy (6) to trackreprogramming in these MEFs, in terms ofthe contribution of isolated clones to pre-iPSC(transgene-dependent, CD24high/SSEA1+) andiPSC (transgene-independent, CD24low/SSEA1+)states on day 18 (fig. S1B). Although clones inbulk cultures gave rise to a homogeneous pop-ulation of pre-iPSCs, as previously reported (5, 6),we observed a high degree of clone-to-clone het-erogeneity in the pre-iPSC fraction produced byindividual isolated clones (Fig. 1B).We next determinedwhether the high fraction

of pre-iPSCs previously seen in population re-programming (6) could be recapitulated in clonalmixing studies, combining distinct clones with

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1Institute of Biomaterials and Biomedical Engineering(IBBME), University of Toronto, Toronto, Ontario M5S 3E1,Canada. 2Department of Molecular Genetics, University ofToronto, Toronto, Ontario M5S 1A8, Canada. 3Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital,Toronto, Ontario M5G 1X5, Canada. 4Department ofPhysics, University of Toronto, Toronto, Ontario M5S 1A7,Canada. 5SFR-SANTE, iPSC Core Facility, INSERM, CNRS,UNIV Nantes, CHU Nantes, Nantes, France. 6CRTI,INSERM, Université de Nantes, Nantes, France. 7ITUN,CHU Nantes, Nantes, France. 8Department of MedicalBiophysics, University of Toronto, Toronto, Ontario M5T3H7, Canada. 9Department of Obstetrics and Gynecology,University of Toronto, Toronto, Ontario M5G 1E2, Canada.10Institute of Medical Science, University of Toronto,Toronto, Ontario M5T 3H7, Canada. 11AustralianRegenerative Medicine Institute, Monash University,Melbourne, Victoria, Australia. 12The Donnelly Centre forCellular and Biomolecular Research (CCBR), University ofToronto, Toronto, Ontario M5S 3E1, Canada. 13School ofBiomedical Engineering, University of British Columbia,Vancouver, British Columbia V6T 1Z3, Canada. 14TheBiomedical Research Centre, The University of BritishColumbia, Vancouver, British Columbia V6T 1Z3, Canada.15Michael Smith Laboratories, The University of BritishColumbia, Vancouver, British Columbia V6T 1Z4, Canada.*These authors contributed equally to this work.†Present address: Department of Pathology, Memorial SloanKettering Cancer Center, New York, NY, USA.‡Corresponding author. Email: peter.zandstra@ubc.ca

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varying fractions of pre-iPSCs (fig. S1C). Homo-geneous pre-iPSC populations emerge from co-cultures of two or more heterogeneous clones(fig. S1, D and E). This finding revealed thepossibility that a subset of clones could drivethe population’s dynamics andmotivated furtherstudies to understand the underlying clonal com-petition during reprogramming.

Cellular barcoding strategy revealsclonal selection during reprogramming

To probe long-term clonal dynamics over the30-day reprogramming process, we adapted apreviously reported cellular barcoding strategy(16). In brief, before inducing reprogramming, avariable DNA sequence—or “barcode”—was in-tegrated into the MEFs’ genome by lentiviraltransduction (Fig. 2A). To ensure that our ob-servations were not an artifact of the system andrepresented the reprogramming process, we con-ducted experiments with four reprogrammingsystems containing DOX-inducible OKMS factorgene cassettes: the secondary 1B tetr system (15),the secondary iRep1 system [derived by previousmethods (15)], a primary reprogramming system(figs. S2 and S3), and the secondary 9DT system[similar to that previously reported (17), fig. S4].Barcoded MEFs from the four systems were

sampled for initial barcode profiling before in-ducing reprogramming. Throughout the repro-gramming process, cells were sampled regularlyto assess barcode abundance at time points basedon pluripotency characterization studies eitherpublished in the 1B tetr system (5) or conductedhere (figs. S2 to S4). At day 14, half of the cells in

the 1B tetr and iRep1 systems were transitionedto DOX-free (DOX−) culture to assess clonal con-tributions to transgene-independent iPSCs (5)(Fig. 2A). A detailed description of the experi-mental and bioinformatics pipeline can be foundin the methods section and figs. S5 and S6.To assess clonal selection, the number of

clones present in the population was tracked onthe basis of their unique barcodes. We observeddramatic clonal loss in the 1B tetr and iRep1systems, with the majority lost in the first weekfollowing DOX induction (Fig. 2B). The numberof clones continued to decrease in DOX+ con-ditions, with less than 10% surviving 30 days ofreprogramming. This was accompanied by a no-table increase in cell death (fig. S7A). The removalof DOX at day 14, which forces silencing of ex-ogenous OKMS factor expression, was expectedto drive another wave of clonal selection (18) andalso led to clonal loss (Fig. 2B). Analogous bar-coding experiments conductedwith the 9DT andprimary reprogramming systems produced con-sistent trends of clonal selection and dominance(see supplementary text and figs. S8 and S9).Notable clonal selection in noninducedMEF cul-tures was not observed, and there was no cor-responding reduction in cell number duringreprogramming, indicating that rapid clonalloss was a result of competition in reprogram-ming (fig. S7, B to D).Combining cellular barcoding with our pub-

lished surface-marker profiling strategy (6), wemeasured the fraction of clones that contributeto the MEF, pre-iPSC, and iPSC fractions (seesupplementary text and fig. S10). Although the

number of clones in an iPSC state increasedover time during population reprogramming,not all clones contributed to the iPSC fractionand even fewer survived DOX removal (figs. S7Eand S10D). By contrast, our earlier isolated clonalreprogramming study suggested that all cloneshave iPSC-producing capabilities (Fig. 1B). Thisdiscrepancy in clonal iPSC potential in isolatedversus mixed conditions signaled that clonalinteractions are involved in population dynamicsduring reprogramming.We next examined whether clonal loss is ac-

companied by the survival of a dominant clonalsubset. After 30 days of DOX induction, two clonesexhibited a clear advantage over their competitors,occupying more than 50% of the culture (Fig.2C). The primary reprogramming and secondary9DT systems also gave rise to a few dominantclones, emerging as early as day 14 (figs. S8Cand S9C). The surprising observation that afew clones overtook the majority of the cell poolprovides a possible basis for the homogeneityseen inmixed clonal populations (Figs. 1B and 2Dand fig. S7F). These dominant clones can attainan iPSC state (fig. S10E) and in most, but not all,cases maintain their dominance after DOX re-moval (Fig. 2C and figs. S7G, S8C, and S9C).

Mathematical model of clonalreprogramming challenges the clonalequipotency hypothesis

It remained unclear whether clonal loss anddominance were due to stochastic reprogram-ming latencies of MEFs or selective advantagesof a subset of clones. To address this question, we

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Fig. 1. Isolated reprogramming clones exhibit heterogeneity.(A) Clonal competition in population culture affects selection, resultingin clonal loss due to either stochastic selection against smaller clones(clone size is a function of stochastic reprogramming latency) ornonstochastic selection for clones exhibiting eliteness (enhanced repro-gramming rate or ability to outcompete other clones). Numberedcircles represent individual reprogramming clones, starting from asomatic cell state and finally acquiring an iPSC state following theoverexpression of the OKMS factors. Circle size indicates relative clonesize, that is, the relative abundance of cells derived from a clone inthe overall population. (B) Comparison of heterogeneity in isolated clonalversus population reprogramming. Ability of individual secondary reprogramming MEFs (2oMEFs) versus population MEF reprogramming (1B tetrsecondary reprogramming system) to give rise to pre-iPSCs (CD24high/SSEA1+) and iPSCs (CD24low/SSEA1+) is shown, assayed by flow cytometryat day 18 (D18) of reprogramming. Reprogramming clones were derived from DOX-induced secondary MEFs that were single-cell sorted on D8.Population reprogramming data represent means ± standard deviation of n = 3 biological replicates.

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analyzed clone size distributions (19). Theclonal equipotency model of reprogrammingpredicts neutral clonal dynamics, whereby dom-inant clones stochastically initiate reprogram-ming earlier and therefore have more time tocontribute to the reprogrammed population. Weobserved a bimodal distribution of dominantversus nondominant clones (Fig. 2D and figs.S7H, S8D, and S9D), which is incongruous withthe assumption of clonal equipotency becauseneutral dynamics are expected to produce a uni-modal clone size distribution (fig. S1A) (8), lead-ing us to hypothesize that clonal dominance is notexclusively driven by stochastic neutral dynam-ics. To test this, we developed a stochastic modelof cellular reprogrammingwith clonal resolution(fig. S11) and compared the predictions of our

model with experimental observations (seemethods and figs. S11 to S13).In silico simulations of clone size distribu-

tions in stochastic reprogramming diverged sig-nificantly from experimental observations byday 8 (fig. S11, C and D). Notably, the in silicomodel gave rise to a unimodal distribution ofclone sizes, as expected. Given that the mostdramatic changes in clone size distributionsin vitro occurred within the first week of repro-gramming (fig. S11, C and D), accompanied bythe greatest clonal loss (Fig. 2B), we next testedwhether the neutral model could capture clonaldynamics following the first week of reprogram-ming. We ran the model with initial conditionsfrom day 8 clone distributions and found thatthe neutral model did not reproduce the exper-

imental observations following the first weekof reprogramming (fig. S11E). These findingssupport the selective nature of clonal dynamicsand led us to examinewhether the experimentallyobserved dynamics could be driven by underlyingheterogeneity in clonal fitness.

Clonal eliteness arises in the first weekof reprogramming

To determine when larger clones emerge andacquire dominance, we further probed the ex-perimental results of our initial barcoding study(Fig. 2) and compared outcomes based on clonesize.We sorted clones into four bins of increasingsize while keeping the clone size range (in log-arithmic scale) consistent between bins (Fig. 3A).This sorting was performed at different time

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Fig. 2. Cellular barcoding reveals clonal selection in reprogramming.(A) The barcoding pipeline, in which lentivirus containing unique DNAbarcodes are generated and used to infect secondary MEFs from the 1B tetrand iRep1 reprogramming systems [multiplicity of infection (MOI) ~ 0.05].Cells are subsequently reprogrammed and regularly sampled (20%) forbarcode sequencing to track the temporal behavior of clones throughoutreprogramming. DsRed is a red fluorescent protein. (B) Number of uniqueclones (barcodes) over time following DOX induction in the 1B tetr andiRep1 systems. Representative data are from n = 4 biological replicates.

(C) Frequency of individual clones over time following DOX induction in the1B tetr and iRep1 systems.Clones showing extreme dominance are highlightedin red and blue and indicate the same clone in DOX+ and DOX− cultures.Representative data are from n = 4 biological replicates. (D) Fraction of clonesexhibiting relative dominance (versus nonzero, nondominant clones) over timefollowing DOX induction in the 1B tetr and iRep1 systems. Representativehistogram of clone sizes is from the 1B tetr reprogramming system at D24of DOX+ culture. Bar graph data represent means ± standard deviation ofn = 4 biological replicates (from 1B tetr and iRep1 systems).

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points, and clone sizes were tracked in each binover time and then normalized to initial clonesize at the time of binning. We tested the pro-pensity for bins with larger clones to becomedominant later in culture. The largest clones(bin 4) at days 8 and 14 went on to becomerelatively dominant compared with the clonesin the other three bins (Fig. 3B and fig. S14A).By contrast, clones that were larger at day 0(owing to stochastic differences in lentiviralbarcoding) did not become dominant.We found that early dominating clones (in bin

4 at day 8) were able to contribute to all four binsthroughout reprogramming (fig. S14B). Neverthe-less, the majority of late-stage dominant clones(in bin 4 at day 30) were also dominant at day 8.Simulated clone size fluctuations based on theneutral stochastic model also predicted that earlydominating clones maintain their dominance(fig. S14B), though the neutral model was unableto capture their dynamics (fig. S11E). Further-more, early dominating clonesweremore likely tosurvive competition over time in DOX+ culture(Fig. 3C). Similar observations were made inexperiments grouping clones into four bins ofincreasing clone size while keeping the num-ber of clones constant between bins (fig. S15, A toE). Applying our binning strategy to the datafrom the primary reprogramming and secondary9DT systems, we found similar trends (fig. S16, Aand B). Larger clones (in bins 3 or 4) also ex-hibited an enhanced propensity to contributeto transgene-independent iPSCs in both DOX+(figs. S14C and S15F) and DOX− culture (Fig. 3Dand figs. S15G and S16C), further highlighting

their enhanced fitness. Thus, although not allearly dominating clones (bin 4 at day 8) do-minate in late-stage reprogramming, they havean enhanced probability of doing so.

A priori propensity for clonal eliteness ispresent in the initial MEF pool

In light of the heterogeneous nature of MEFs(20), we examined the possibility that a subset ofcells exhibits an a priori eliteness that drivesthe emergence of dominant clones by day 8 ofreprogramming. Following an average of threecell divisions, barcoded MEFs were split intothree equally distributed pools: One pool wasreserved for day 0 barcode sequencing, whereasthe remaining pools were seeded into two flasksand subjected to identicalDOX treatment (Fig. 4Aand figs. S8A and S9A). Following induction ofreprogramming, we tracked clonal loss and con-firmed similar selection dynamics in the parallelflasks (fig. S17A).To determine whether clonal dominance is

inherited from the MEF state, we first assessedthe correlation in clone sizes between parallelflasks containing MEFs from a common paren-tal origin. In scatter plots (Fig. 4B), the corre-lation of clones (identified by unique barcodes)between parallel flasks was high (Fig. 4C and figs.S8, E to G; S9, E to G; and S17B)—consistentlyhigher than that expected by chance (fig. S17, Dand E).We applied our binning strategy to the splitt-

ing experiment to determine whether a sub-population of clones was driving the correlationbetween flasks. We reasoned that if a priori

poised clones existed, they would be prominentin the early dominating clones. Our binningstrategy revealed that although smaller clonesdisplayed barcode heterogeneity and thus poorcorrelation, the largest clones were relativelywell correlated (fig. S18A). Indeed, removal of bin4 clones substantially reduced correlationbetweenparallel flasks (fig. S18B). Furthermore, bin 4clones gave rise to more dominant clones laterin culture with comparable outcomes in parallelflasks (figs. S18C and S19A). Similar correlationswere observed in primary and secondary 9DTsystems (figs. S16, D andE, and S19B). Thus, earlydominating clones arise from a subpopulation ofpoised MEFs, inheriting their potential for dom-inance and driving the correlations observed inour parallel reprogramming flasks, while also ex-hibiting reproducible dominance (see supplemen-tary text and fig. S20).Taken together, our results suggest that a

priori eliteness associated with parental MEFstatus determines a clone’s ability to initiate re-programming quickly and dominate, confirmingthat the heterogeneous MEF population displayscell-specific reprogramming rates (21). Further-more, the rigorous analysis of clonal selection anddominance dynamics presented here may alsoinform iPSC production strategies (see supple-mentary text and fig. S21).

MEFs derived from Wnt1-expressing cellsare poised to dominate thereprogramming pool

Our barcoding analysis revealed the existence ofa subpopulation of elite MEFs. To uncover the

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Fig. 3. Early dominating clones exhibit eliteness. (A) Binning strategywith clones binned according to clone size (clone size range constantbetween bins) and tracked to assess their dominance and survival inDOX+ and DOX− culture. (B) Mean clone size in bins, with binningperformed at D0 and D8 for comparison. Means ± standard deviation ofn = 4 biological replicates are shown (from 1B tetr and iRep1 systems).(C) Percent of clones per bin surviving until D30 in DOX+ culture.Bar graph data represent means ± standard deviation of n = 4 biologicalreplicates (from 1B tetr and iRep1 systems). Bins containing less than50 clones were excluded (bin 4 for D0 and D14). (D) Percent ofclones in bins surviving DOX removal at D14. Bar graph data representmeans ± standard deviation of n = 4 biological replicates (from 1B tetr andiRep1 systems). Bins containing less than 50 clones were excluded(bin 4 for D0 and D14). For (B) to (D), statistical significance was assessedby paired, two-tailed Student’s t test (*P < 0.10; **P < 0.05; ***P < 0.01).

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identity of these MEFs, we tested whether theneural crest (NC) population in skin has an apriori ability to dominate reprogramming (seesupplementary text).We developed a lineage tracing strategy to

retrospectively identify MEFs based on Wnt1expression, which has been suggested to markNC-derived cells (22, 23) (see methods and fig. S22).Upon isolation and serial passaging, the frac-tion of NC-derived MEFs became increasinglyprominent in nonreprogramming culture, sug-gesting their competitive advantage (fig. S22B).We next infected the population containing bothNC and non-NC–derived MEFs with lentiviruscarrying the OKMS factors and scored the originof emerging SSEA1+ clones. After 3 weeks of re-programming, 100% of reprogramming coloniesoriginated from NC-derived MEFs (fig. S22C).Furthermore, upon cell sorting, we found a higherefficiency of reprogramming induction in NC-derived clones (fig. S22D).To validate these preliminary findings from low-

efficiency primary reprogramming experiments,we next explored the dynamics of NC-derivedMEFs in an efficient secondary reprogrammingsystem (Fig. 5A and methods). The prominenceof NC-derived [yellow fluorescent protein posi-tive (YFP+)] MEFs increased in reprogramming

culture, where the YFP+ fraction overtook thepopulation (Fig. 5B and fig. S22E). This suggestedthat NC-derived cells exhibited a competitiveadvantage in both MEF and reprogrammingculture and prompted us to further assess thedynamics of their dominance. We conductedmixing studies, controlling the initial fraction ofYFP+ to YFP− cells while keeping the total cellnumber constant (Fig. 5C). The fraction of YFP+cells increased over the course of reprogrammingand overtook the population in almost all cases,regardless of seeding frequency. Given the pro-pensity of YFP+ cells to overtake reprogrammingpopulations, we next assessed their reprogram-ming potential in isolated clonal studies andfound that NC-derived MEFs exhibited a signif-icantly higher ability to initiate reprogramming(Fig. 5D).Given the results of our isolated clonal exper-

iments, we next asked whether reprogrammingsuccess was dependent on NC-derived MEF fre-quency at day 0 of reprogramming. As expected,higher initial levels of YFP+ MEFs gave rise tohigher levels of SSEA1+ cells at earlier time points(Fig. 5E). Whereas the fractions of SSEA1+ cellsarising from the YFP+ and YFP− subpopulationswere comparable in these studies (fig. S22F), theoverall SSEA1+ pool was predominantly composed

of YFP+ cells (fig. S22G). This suggests thatalthough YFP−MEFs can give rise to reprogram-ming cells, the YFP+ cells overtake the repro-gramming compartment. The eliteness of YFP+reprogramming clonesmay be linked to a higherreprogramming propensity as well as a higherbaseline competitive advantage, as seen from theDOX− controls (fig. S22B). The observation thatYFP− cells can reprogram in isolation but havelimited contribution to population-based repro-gramming underscores the effects of competi-tion on reprogramming dynamics.As a final validation,we expanded our stochastic

model of reprogramming in which all cloneshave the same potential to reprogram (fig. S11),which did not accurately capture experimentaldynamics, to a two-population stochastic model.In our new model, two MEF subpopulations aredefined (A and B), where B has an a priori pro-pensity to dominate the reprogramming pool, asin NC-derivedMEFs (Fig. 5F). This two-populationreprogramming model accurately captured clonaldynamics (Fig. 5G), representing adrastic improve-ment over our original model. The model quanti-fied the relative eliteness of NC-derived clones anddemonstrated that these cells are consistentlymore competitive than their nonelite counterparts(Fig. 5H). We also found no notable improvement

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Fig. 4. A subpopulation of a priori elite MEFs gives rise to earlydominating clones. (A) Population splitting experiment in which barcodedsecondary MEFs (expanded for 3 days) from the 1B tetr and iRep1reprogramming systems are split at D0 of DOX induction into parallelflasks and sampled regularly for barcode sequencing. Parallel flasks wereassessed for similarity in outcomes of common clones. (B) Scatter plots of

individual clone sizes (percentage of total barcoded cells) in parallelreprogramming flasks over time following DOX induction in the1B tetr and iRep1 systems. Color shows the distance of each pointto the diagonal (blue is shorter; red is longer). (C) Correlationbetween clone sizes in parallel flasks over time following DOX inductionin the 1B tetr and iRep1 systems.

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Fig. 5. NC-derivedMEFs dominate thereprogramming popu-lation. (A) Secondaryreprogramming systemlabeling NC-derivedMEFs. (B) Fraction ofNC cells after 30 daysof reprogramming.Means ± standard devi-ation of n = 3 biologicalreplicates are shown.(C) Controlled mixingstudies of NC and non-NC MEFs. The heatmapshows the fractionof NC (YFP+) cells overtime. Representativeresults are from n = 2biological replicates.(D) Reprogrammingefficiency of NC andnon-NC MEFs, assayedby isolated single-cellreprogrammingassay. Statistical signif-icance was assessedby Z test (**P < 0.01;***P < 0.001).(E) Reprogrammingfractions in controlledmixing studies, assayedby SSEA1 expression.Representative resultsare from n = 2 biologicalreplicates. (F) Compart-mental probabilisticmodel of reprogram-ming, including an eliteMEF fraction (sub-population B,corresponding to NC-derived MEFs).Expected clone sizedistribution plots forthe new (elite sub-population) and old (noelite subpopulation)models are shown.(G) Experimental clonesize distributions fromthe 1B tetr system, withmodel-generatedcurves overlaid.Bayesian informationcriterion (BIC) quanti-fies improved fit ofthe two-populationmodel. Means ±standard deviationof n = 4 biological rep-licates are shown(from 1B tetr and iRep1 systems). (H) Relative eliteness of subpopulation B. Data points represent n = 4 biological replicates; the gray bar shows themean (from 1B tetr and iRep1 systems). rA and rB, effective growth rates of subpopulations A and B.

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in the model’s ability to accurately recapitu-late experimental data by adding more thantwo MEF subpopulations (fig. S23A), supportingthe observation that the single identified NC-derived MEF fraction is the primary source forelite clones. Furthermore, when applied to ourday 0 population splitting experiment, the two-population model recapitulated the range of ob-served positive correlations between parallel flasks(fig. S23B) as well as the observation that largerclones make the main contribution to the corre-lation (fig. S23C), whereas the neutral model onlyproduced correlations of zero. Fitting the exper-imental correlations to the two-population modelalso allowed us to estimate the frequency of eliteMEFs (fig. S23D), which was bound by the mea-sured fraction of NC-derivedMEFs.We also notedthat the model was able to accurately capture thedynamics of clonal loss we observed experi-mentally (fig. S23E). We next used the two-population model to understand if there are twophases to clonal dynamics—an early phase gov-erned by clonal selection and a later phase re-sulting from neutral or stochastic, clonal loss.Indeed, our earlier observation that clone sizedistributions shiftmost dramatically betweendays0 and 8 (fig. S11D) suggested this possibility. Asexpected, themodel predicted the selective advan-

tage of the elite reprogramming subpopulationto be larger than the nonelite fraction (fig. S23F).Furthermore, the time-varying nature of theeffective growth rates of the two subpopulations(see methods) suggested that the entire repro-gramming process is governed by selective dynam-ics, consistent with our earlier finding that thelater phase of reprogramming could not be cap-tured by neutral clonal dynamics (fig. S11E).These observations confirmed that individual

MEFs exhibit unequal propensities for repro-gramming induction. This led us to uncover amechanism for the observation of a priori re-programming eliteness, which is driven by a sub-population ofMEFs derived fromWnt1-expressingcells, likely corresponding to an NC population.Additionally, an exploration of growth and deathdynamics in isolated versus coculture of NC andnon-NC cells provided some evidence for directinteractions between these competing popula-tions (see supplementary text and fig. S24).

Discussion

We aimed to uncover the connection betweenintrinsic cell identity, the genetically encoded po-tential of a cell, and cell-extrinsic communityeffects (Fig. 6). During reprogramming, whetherinequalities in cell fitness drive non-neutral clo-

nal drift in interacting cells remains unclear.Wefound a poised subpopulation of somatic cellsthat gives rise to dominant clones within thefirst week of population reprogramming. Theseearly dominating clones are fitter, exhibiting ahigher probability of dominance in later cultureand an increased propensity to reprogram. In-deed, our lineage tracing experiments uncoveredthe identity of a previously unknown subpop-ulation of MEFs from the Wnt1+ (neural crest)lineage that preferentially gives rise to elite re-programming clones. These observations alignwith previous reports of the heterogeneous re-programming potential of MEFs (21, 24, 25). Ourdata suggest that the eliteness of NC-derivedMEFs may be linked to their ability to initiatereprogramming more efficiently, allowing themto transition to an iPSC state more rapidly andescape the MEF and pre-iPSC states, which ex-perience increased cell death.Our findings make clear that competitive in-

teractions between reprogramming clones drivepopulation dynamics in a manner that is dif-ferent from isolated clonal studies, suggest-ing that the clonal equipotency paradigm—inwhich somatic cells have an equal potential toattain an iPSC state in isolation—may not governclonal dynamics in the population setting. These

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Fig. 6. Competition in reprogrammingemerges as a relevant parameter thatbridges our understanding of populationdynamics and single-cell potential.By exploring the dynamics of clones withinmulticellular populations, cell competitionemerges as a mechanism by whichcells with hidden, context-specific elitenessemerge to occupy and dominate thereprogramming niche. Cell competitionserves to connect our understanding of thegenetically encoded potential of a cell,quantified by single-cell studies, and thebehavior of these cells within the communitycontext. This paradigm can help to inform ourunderstanding of developmental and diseasesystems as well as strategies for controlling thecompetitive potential of cell therapies.

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interactions may be influenced by indirect com-petition for limited culture nutrients and spaceor by direct competition between cells. Indeed,direct cell competition has been reported todrive population homogeneity in PSCs in vivo(11) and in vitro (13, 14), where Mychigh cellsdirectly induce apoptosis in their Myclow neigh-bors. Given that Myc overexpression is involvedin the induction of reprogramming,Myc-mediatedcompetition between reprogramming cells isnot unexpected. Indeed, population-level Mycexpression reportedly increases over the re-programming process in the presence of exog-enous OKMS factor expression, suggesting thatthese cells may overtake the culture (5). To gainclarity on the role of Myc in this process, weneed a deeper understanding of the molecularmechanisms of fitness in reprogramming, whichrequires as yet undeveloped tools for the isolationand single-cell sequencing of rare populations ofspecific barcoded cells. This technical challengecurrently poses a barrier to the omics character-ization of elite reprogramming clones.We found that a few clones overtake the re-

programming population. Although it remainsunclear whether these clones inherit their elite-ness potential from the MEF state, it is possiblethat they acquire their dominance in a culture-induced manner. Indeed, the predictable behav-ior of the clones in parallel reprogramming flaskscould be due to the acquisition of heritablechanges that confer enhanced cellular fitness.Previous reports have pointed to genomic aber-rations in PSCs acquired in culture (26), and ithas been reported that passaging results in theloss of most of these mutations (27). Indeed,active competition in PSC culture has been shownto remove genomic aberrations (13). These ob-servations underline that emergence of culture-induced eliteness is a potential secondary sourceof competition to consider in long-term culture.Our data also support the possibility that NC

stem cells present in the MEF population arethe source of these top dominating clones. NCstem cells are developmentally related to neuralstem cells, which have been shown to reprogramto an iPSC state through the overexpression ofOct4 alone (28). Notably, NC stem cells can beexpanded long-term and express core pluripo-tency factors, which may poise them to over-take the population. Indeed, our datamay supportat least three time-dependent selection events,all of which may involve NC cells: (i) NC MEFsdominate somatic culture before reprogramming,(ii) NC clones are further selected through com-petition with non-NC cells during early repro-gramming, and (iii) reprogrammedNC stem cellsand their progeny become dominant and over-take later cultures. Overall, it is likely that iPSCsare not equivalent in terms of fitness, thus merit-ing the careful selection of PSC clones for regen-erative medicine applications (29).This study uncovers cell competition as a

mechanism for cellswith context-specific elitenessto occupy the reprogramming niche. These find-ings support a paradigm shift, turning com-petition into a generalizable parameter for

understanding multicellular developmentaland disease systems (Fig. 6). To this end, re-programmingmay serve as amodel to probe thegenetic and epigenetic basis of cell fitness, pro-viding key insights toward controlling competi-tion for regenerative medicine (29). This will becritical for synthetic biology strategies that aimto engineer cells with predictable and desirablebehaviors in multicellular contexts (30).

Methods summary

For reprogramming, DOX-inducible secondaryMEF 1B cells [isolated as previously described(15) from tetraploid complementation], MEFiRep1 cells [derived by previous methods (15)],MEF 9DT cells [similar to that previously re-ported (17)], or primary MEFs were used. Follow-ing cellular barcoding by viral infection using anadapted version of a previously reported cellularbarcoding strategy (16), MEFs were subsequent-ly passaged once to allow for an average of~threefold expansion and subsequently split,whereby cells were sampled for initial barcodeprofiling and the remaining cells were seededfor reprogramming induction. Following DOXinduction, cultures were sampled regularly forsequencing. Cells were transitioned to DOX-freeculture to assess clone contributions to the gen-eration of transgene-independent iPSCs.To assess barcode survival and abundance,

we conducted targeted DNA paired-end sequenc-ing followed by analysis with an in-house bio-informatics analysis pipeline using custom Perland MATLAB scripts.For NC lineage studies, we created a mouse

strain containing the transgene Wnt1-Cre andfloxed ROSA-tdTomato. Upon Cre expression,Wnt1-expressing cells and their progeny becomeirreversibly labeled. To develop a more efficientreprogramming system with NC tracing, a homo-zygous ROSA-YFP mouse was crossed to a heter-ozygousWnt1-Cre. TheWnt1-Cre/ROSA-YFPmicewere then crossed to the highly efficient sec-ondary reprogramming system iRep2 [derivedby previous methods (15)] mouse line.Mathematical modeling of clonal dynamics

was conductedwith customPython andMATLABscripts. Calculations for statistical significancewereperformed using Excel and MATLAB software.

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ACKNOWLEDGMENTS

The authors thank the Bystrykh laboratory for sharing theirbarcoding plasmids. We also thank C. L. Bauwens for her editing,K. Chan for assisting with sample sequencing, M. Parsons forassisting with flow cytometry, T. Yin for performing cell culture andsample preparation for flow cytometry, and J. Ma for artisticcontributions to the figures. We acknowledge the assistance andsupport of laboratory colleagues and collaborators. Funding: Thiswork has been supported by Medicine by Design (University ofToronto), CFREF program, CIHR (MOP 57885), and CIHRFoundation grants to P.W.Z., D.v.d.K., A.N., and J.L.W. N.S. is the

recipient of the NSERC Vanier Canada Graduate Scholarship.A.N. is the Tier 1 Canada Research Chair in Stem Cells andRegeneration. P.W.Z. is the Canada Research Chair in Stem CellBioengineering. Author contributions: N.S., A.F., G.J., J.L.W.,D.v.d.K., and P.W.Z. conceived and designed the experiments. N.S.and P.W.Z. wrote the manuscript. A.N. contributed to study design.N.S. and G.J. performed all barcoding experiments, cell culture,sequencing, and bioinformatics work. A.F. developed theneural crest lineage tracing system. S.M. and S.G. conducted insilico simulations and mathematical modeling studies. L.D.developed the lentiviral barcoding system. J.E. and M.C.P. createdthe 1B tetr, iRep1, and iRep2 secondary reprogramming systems.G.J. and D.T. designed and characterized the primary andsecondary 9DT reprogramming systems. Competing interests:The authors declare that they have no competing interests.

Data and materials availability: All data are available in themanuscript or the supplementary materials.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/364/6438/eaan0925/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S24Table S1References (31–39)

3 March 2017; resubmitted 2 August 2018Accepted 25 February 2019Published online 21 March 201910.1126/science.aan0925

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Cell competition during reprogramming gives rise to dominant clones

Puri, Andras Nagy, Derek van der Kooy, Sidhartha Goyal, Jeffrey L. Wrana and Peter W. ZandstraNika Shakiba, Ahmed Fahmy, Gowtham Jayakumaran, Sophie McGibbon, Laurent David, Daniel Trcka, Judith Elbaz, Mira C.

originally published online March 21, 2019DOI: 10.1126/science.aan0925 (6438), eaan0925.364Science 

, this issue p. eaan0925; see also p. 330Sciencenervous system.normally emerge during embryonic development and migrate into various tissues, including the skin, muscle, and Wolff and Purvis). The ''winners'' are a special class of skin cells originating from the neural crest. Cells of this typereprogramming, eliminating one another as the population progresses toward the stem cell state (see the Perspective by

used experimental and mathematical approaches to show that skin cells compete duringet al.understood. Shakiba thus gaining the ability to become any cell type in the body. But what happens during reprogramming is not completely

winning discovery showed that specialized cells can be genetically reprogrammed into stem cells,−A Nobel PrizeDomination in the stem cell world

ARTICLE TOOLS http://science.sciencemag.org/content/364/6438/eaan0925

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CONTENTRELATED http://science.sciencemag.org/content/sci/364/6438/330.full

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