Proc. Natl. Acad. Sci. USAVol. 82, pp. 5390-5394, August 1985Cell Biology

Cell size as a determinant of the clone-forming ability ofhuman keratinocytesYANN BARRANDON AND HOWARD GREENDepartment of Physiology and Biophysics, Harvard Medical School, Boston, MA 02115

Contributed by Howard Green, April 29, 1985

ABSTRACT Keratinocytes isolated from human epi-dermis and subsequently cultured may form clones if they are11 jam or less in diameter but are irreversibly committed tofurther enlargement and terminal differentiation if they are 12pum or more in diameter. When a founding cell of 11 ,um or lessforms a small rapidly growing clone in culture, the cells of thatclone are able to found new colonies even when their diameteris as great as 20 pum. As the clone becomes larger and growsmore slowly, the maximal size of its clonogenic cells is reducedtoward that of the epidermis. A cultured cell of up to 20 pum indiameter can, when it divides, give rise to clonogenic progenysmaller than itself, thus reversing the process of enlargement.Cells larger than 20 pum cannot divide and therefore cannot berescued from terminal differentiation. It is concluded thatwhen keratinocytes multiply rapidly, they extend reversibly themaximal size at which they are capable of generating clones intothe range usually characteristic of terminally differentiatingcells. It is proposed that this mechanism enables thekeratinocyte to accommodate an increased rate of multiplica-tion to its need to attain a large size during terminal differen-tiation.

The basal layer of the epidermis contains multiplyingkeratinocytes. Terminally differentiating progeny leave thislayer and enlarge progressively as they move outwardthrough the spinous layer. The size of the cell is relatedtherefore to the degree of its terminal differentiation.

Studies of proliferation in the epidermis have been con-cerned with questions such as the following ones. (i) Whatcells of the basal and more superficial layer can multiply andwhat are their division rates (1-4)? (ii) What fraction of thecells possess sufficient growth potential as to be clonogenic(5-7)? Although there are protein markers characteristic ofbasal cells in general (8-14), there are no such markers bymeans of which one might discriminate between basal cellsthat are clonogenic and those that are not. Studies of growthdynamics in the epidermis are difficult to interpret, and therehave been differing estimates even for the average cell cycletime of human basal cells (2, 3).Under conditions elaborated over the past decade, human

epidermal keratinocytes form colonies in culture (15-19).Such colonies may be disaggregated and the cells subculturedmany times (19). In the course of its growth, each colonybecomes a mixture of proliferating and terminally differen-tiated cells (20). As in the natural epithelium, the two cellcompartments become separated by stratification, the pro-liferating cells occupying the basal position (15, 21). In youngcolonies the keratinocytes are in a hyperproliferative statecompared with the epidermis, and this affects the selection ofproteins they synthesize (22, 23).To identify factors affecting the growth potential of the

keratinocyte, we have studied the formation of clones byisolated single cells. The marker with the greatest signifi-

cance for estimating a cell's ability to found a clone is its size,but the relation between size and clonogenicity can shift; inhighly proliferating cultured cell populations, it is differentfrom that prevailing in the epidermis.

MATERIALS AND METHODSCell Culture. Human epidermal keratinocytes, derived

from foreskin of seven newborns, were cultivated as de-scribed earlier (24-26). One of the strains obtained (AY) wasstudied only in secondary and subsequent subcultures. Allexperiments were carried out with a single batch of serumtested for ability to support good colony formation. Themedium was changed every 4 days.

Inoculation and Sizing of Cells. Cells obtained aftertrypsinization of either epidermis or cultures were centri-fuged and resuspended in medium. Cells (500-1000) in 8-10ml of medium were placed in a 100-mm dish. Under a ZeissIM35 photo-invertoscope with x 10 phase objective, a singlerandomly selected cell was photographed, aspirated into aPasteur pipet, and inoculated into a culture dish alreadycontaining lethally irradiated 3T3 cells. A period of 1 hr wassufficient to isolate, photograph, and inoculate about 50 cells.

Cell diameters were measured on prints with a Bausch andLomb measuring magnifier. To obtain a frequency distribu-tion of the diameters of cells in a single colony, the colonywas trypsinized, and the cell suspension was introduced intoa phase hemocytometer chamber. The cells were photo-graphed through a x 10 phase objective with a Polaroidcamera. In each experiment a grid scale was photographedunder the same conditions, to correct for the microscopic andphotographic enlargement.

Determination of Cell Growth Rate by Successive Measure-ments of Colony Area. Five- to 10-day-old colonies werephotographed. The outline of each colony was traced on apaper, which was then weighed. The area was corrected formicroscopic and photographic magnification. The area ofsmall colonies was correlated with the number of cellscounted in the same photographs.Areas of 10- to 29-day-old colonies were measured without

enlargement. A Petri dish containing a living or fixed andstained colony was placed on paper ruled in millimeters, andthe area was determined by counting the number of squareswithin the colony. The cell number in a large colony wasobtained by trypsinizing the colony and counting. Over about4 orders of magnitude, extending from 102 to 106, the numberof cells was proportional to the area of the colony (1800 cellsper mm2).

RESULTSGrowth Rate of Epidermal Clones. In order to study the

behavior of clonogenic cells, most experiments were carriedout in 35- or 60-mm Petri dishes containing lethally irradiated3T3 cells and inoculated with a single keratinocyte. Colonieswere detectable macroscopically by about 6 days afterinoculation; thereafter they continued to increase in size for

5390

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more than 20 days (Fig. 1). By 14 days after inoculation,satellite colonies were commonly observed; these were muchsmaller than the principal colony and often situated close toit. Satellite colonies arise through cell displacement from theprincipal colony, a phenomenon that takes place in epidermalregeneration in animals (27). Satellite colonies were notincluded in any of the experiments to be described.

Cell growth could be measured from the increase in colonyarea. Since these measurements did not affect cell viability,growth curves could be obtained by consecutive measure-ments on the same colonies (Fig. 2). As shown for strain AY,colonies formed from cultured cells exceeded 1 cm2 in size by16-18 days, when they contained about 2 x 105 cells. Suchcolonies are larger than the macrocolonies thought to resultfrom clonogenic or stem cells in epidermal regenerationexperiments (5, 28). Founding cells capable of producingcolonies of at least 1 cm2 were defined as clonogenic; allclones were capable of further multiplication, as shown bythe continuing enlargement of the colonies or by the forma-tion of new colonies after subcultivation.

Keratinocytes isolated directly from epidermis (YF3) grewat virtually the same rate as strain AY (Fig. 2), but displace-ment of the curve by about 2 days on the time axis showedthat directly isolated epidermal cells were slower to startcolony formation than those that had already grown inculture.A plot of doubling time against age of the colonies sum-

marizes three experiments on strain AY (Fig. 3). The cellsmultiplied exponentially with a doubling time of about 16-18hr almost from the time of inoculation until about day 10.Thereafter growth was no longer exponential; the averagedoubling time increased progressively, reaching a value of200 hr by day 24. The slower growth was not due to changesin medium composition resulting from the presence of thelarge colony. When the doubling time of a principal colonywas as long as 150 hr, satellite colonies found in the sameculture dish were doubling in cell number in as little as 22 hr.The reduction in growth rate within a large colony wastherefore due to factors intrinsic to the colony.The ability of cells of 5- to 6-day-old clones to found new

colonies was extremely high (mean value, >60%). When theclones were 10-15 days old, an age at which cultures arecommonly transferred, the colony-forming efficiency wasslightly lower (mean, 30-40%). When the age of the coloniesexceeded 16 days, the colony-forming efficiency of the cellsdeclined markedly.

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FIG. 2. Growth curves of epidermal colonies deriv'ed froni singlefounding cells. Each curve was derived from the same coloniesmeasured consecutively. Scale of right hand ordinate was derivedfrom the value for the colonial cell density of 1800 cells per mm2; 0and *, two growth curves obtained from single AY colonies intertiary cultures made at different times. Each point gives the meansize of 7-10 colonies (o) or 6-9 colonies (e). A, Growth curveobtained from single YF3 colonies founded by cells obtained directlyfrom epidermis of foreskin obtained from a newborn and inoculatedinto primary culture. Each point is the mean size of 8 colonies, exceptfor the lowest point, which is the mean for 3 colonies.

Probability of Colony Formation by Founding Cells ofDifferent Size. Six- to 13-day-old secondary cultures of strainAY were trypsinized, and the cells were diluted for isolation.Single cells were photographed for measurement of diameterand then transferred, each to a separate Petri dish alreadycontaining lethally irradiated supporting 3T3 cells. The col-onies were scored 12 or more days later. Plotting size of the

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FIG. 1. Appearance of colonies after inoculation of single found-ing keratinocytes. Numbers indicate days between inoculation andfixation. Colonies are stained with 1% rhodamine. These colonies didnot give rise to satellites. The bar indicates a length of 1.0 cm in theoriginal. (Strain AY, tertiary cultures).

FIG. 3. Decreasing growth rate of keratinocytes in expandingclones. Pooled data from strain AY: o, data from Fig. 2; * and A, datafrom other experiments. In the descending portion of the curve, allcolonies are heterogeneous in that they contain a mixture ofproliferating and terminally differentiating cells.

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5392 Cell Biology: Barrandon andGreenP

inoculated cell against frequency of colony formation (Fig. 4)showed that cells of 11- to 13-,um diameter formed colonieswith an efficiency of 80%, but the probability of colonyformation by larger cells decreased, reaching zero at 21 ,um.Keratinocytes other than AY but also derived from newbornforeskin gave similar results,

Earlier experiments in which the frequency of DNAsynthesis as a function of cell size was determined by tritiatedthymidine incorporation had shown the same general features(29); however, a few cells with diameter greater than 21 gumwere able to undergo DNA synthesis. Colonies scored in Fig.4 possessed over 32 cells and therefore had undergone five ormore cell divisions. Thus, some cells able to initiate DNAsynthesis may be incapable of generating enough progeny toscore as a colony (see ref. 5).Because the size of a cell changes during its multiplication

cycle, the position ofthe declining portion of the curve in Fig.4 is subject to correction. Some cells capable of makingcolonies are likely to have been isolated in the G2 period,when their volume would approach twice that of the samecells measured early in the G1 period. If all of the cells couldhave been measured early in their multiplication cycle, thecutoff diameter for colony formation might have been as lowas 17 gm.

It was of interest to compare the relation of size tocolony-forming ability of cultured cells to that of cells in theepidermis. Foreskin of newborns was trypsinized, and singlecells were isolated and inoculated. For comparison, singlecells isolated from small and large clones of strain AY (thirdsubculture) were similarly inoculated. The size of each cellwas then correlated with the incidence of colony formationfor both classes of founding cell (Fig. 5).As in the previous experiment, cells of 12-day cultures of

AY formed colonies at a frequency that declined to zero at 21gm. Freshly isolated epidermal cells also showed an inverserelation between size and colony formation, but the curvewas strikingly shifted to the left: no colonies were founded bycells of >11-gmr diameter, and most colonies were formed bycells of 9 and 10 .m, a size range not even present in the

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FIG. 5. Colony-forming cells taken directly from epidermis orfrom large colonies are smaller than those of small colonies. Singlecells were isolated from trypsinized foreskins of newborns, photo-graphed, and inoculated. Among the cells inoculated, the presence ofsome cells other than keratinocytes could not be excluded. However,no colonies other than those of keratinocytes were obtained. Data onthe incidence of keratinocyte colony formation were pooled for sixindependent experiments (e). The size range of colony-forming cellsof 12-day colonies of strain AY (three experiments) (o) gave resultssimilar to those of the larger sample shown in Fig. 4. Cells from22-day colonies of the same plating (three experiments) (A) gave acurve displaced toward that of the cells isolated directly fromepidermis.

cultured cells. A large difference from the clone-formingability of cultured cells would remain even if the epidermalcells had all been measured early in their G1 period, since adoubling of the volume of an 11-,um cell would correspond toa diameter of only 14 ttm. The relation between size andclone-forming ability for cells taken front the much largercolonies of 22-day cultures of strain AY was intermediate inposition between the other two; virtually no clone formationwas obtained from cells larger than 16-,um diameter. Evi-dently, in older colonies the larger cells lose their clone-forming ability and approach the behavior of large cells in theepidermis.Growth ofProgeny ofLarge and Small Founding Cells. Cells

of 15-20 .tm, if they grow at all, might be expected to divideonly a few times before beginning terminal differentiation,thus forming abortive colonies. Such a founding cell wouldpossess the properties expected of a transit amplifyingpopulation (7). To test this possibility, we examined the sizeattained by colonies produced by large and small foundingcells isolated from the same culture.

Colonies obtained from founding cells of 11-20 gm weremeasured at 21 days. Most founding cells of all sizes werejudged to be clonogenic, in that their progeny had undergone>17 generations, producing a colony size larger than 100 mm2in area (Table 1). A minority of colonies failed to attain thissize, but there was, surprisingly, no significant difference intheir frequency between the founding cells of different size.It was concluded that the larger colony-forming cells do nothave the properties of a transit amplifying population. Al-though large founding cells form colonies with low frequency,those colonies appear to grow as rapidly and through as manycell generations as colonies founded by small cells. However,the comparison was not extended beyond 20 generations ofcolony growth, nor did it take account of any coloniescontaining <32 cells.Large Clone-Forming Cells Can Give Rise to Small Progeny.

The fact that a 20-aum cell, when it forms a colony, produces

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Table 1. Comparison of colony size for large and small foundingcells (f.c.)

Size of colonies at 21

Diameter Colonies, days, % of all colonies

of f.c., 'um no. >10 mm2 >100 mm2

11 7 86 8612 12 75 5813 24 75 7114 26 92 6515 5 80 6016 6 100 10017 5 100 8018 11 100 7319 3 100 6720 5 100 80

clonogenic progeny suggested that it may be possible for a

large cell to give rise to smaller cells. Since a cell reduces itsvolume by half at every cell division (and its diameter by20%), only reductions greater than that would be significant.Single cells of large and small diameter obtained from 8-daysecondary cultures were isolated and inoculated; 12 or 21-22days later, the resulting colonies were trypsinized and thesize of their cells was measured (Fig. 6). It was found thatcells of 18-20 gm, although able to found a colony onlyinfrequently, gave rise to progeny with about the same sizedistribution as did cells of 12 ,m. The modal diameter in allcases was 15 Mm. Some of the progeny were much smallerthan the founding cell. For example, the data of Fig. 6Ashowed that 40-50% of the progeny of 18- and 20-Mmfounding cells had volumes of <50% that of the foundingcells. The corresponding figures for the data of Fig. 6B are

16-44%. The smallest progeny cells had a volume of aboutone-sixth that ofthe founding cell (diameters of 11 and 20Mm,respectively).

It is evident that division of a cell can rescue it fromterminal differentiation; by 12 days after inoculation of a largefounding cell, the size distribution of the progeny seems

rather similar to that produced by a small founding cell. Untilan enlarging cell has passed the point of no return (after whichit cannot divide at all), it is able to reverse (perhaps com-

pletely) the process of enlargement it had previously under-gone.

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FIG. 6. Size distribution of progeny of large and small foundingcells (f.c.). (A) Size distribution of cells in 12-day colonies. Eighteen-and 20-Mm f.c. produced progeny whose size distribution was similarto that produced by 12-Mm f.c. (B) A similar experiment except thatthe size distribution was measured on cells of 21- and 22-daycolonies. The higher incidence of the largest cells (>20 Mum) indicatesmore advanced terminal differentiation in the larger colonies irre-spective of the size of the f.c. Each curve is based on one or an

average oftwo f.c. and a total of approximately 100 or 200 cells sized.

DISCUSSION

The keratinocyte is an unusual cell type in that its stage ofterminal differentiation is closely related to its size. This istrue not only for the keratinocytes of epidermis (30-33) butalso for those of internal squamous epithelia (34, 35), and forkeratinocytes in culture (20, 29, 36). The keratinocyte en-

larges progressively as it moves between the basal layer andthe granular layer; only at that point, the final destructiveprocess that creates the corneocyte results in some diminu-tion in volume (34).Enlargement of the keratinocyte may be important for its

differentiated functions and may even be a source of signalscontrolling the program of differentiation. While the nuclearsize and DNA content remain constant (20, 31), cytoplasmicenlargement is accompanied by the appearance of new

proteins that prepare for the final state of the cell, such as

keratins (37), involucrin (30, 35, 38), other proteins partici-pating in envelope synthesis (39), and filaggrin (40). Thesechanges require the accumulation of new mRNAs (30, 41-44)and probably depend on specific signals. Such signals mightoriginate from changing quantitative relations between thecytoplasm and the nucleus or between either of these and thecell membrane.The experiments described here deal with the proliferative

capacity of the cells rather than their differentiated proper-ties. The average colony-forming ability of cells we obtainedfrom human epidermis (<10%) was significantly lower thanthe proportion of basal cells in the epidermis (estimated at30%; ref. 33). This is consistent with earlier conclusions thatsome basal cells were not capable of proliferation and even

fewer were clonogenic (5, 45).The smallest cells were the most clonogenic. This was true

for freshly isolated epidermal cells as well as those alreadycultured. In both cases, the probability of clone formationbegan to decline when the cells reached a certain size, andultimately dropped to zero. In the epidermis, where multi-plication is infrequent or slow, clone-forming ability declinedwhile the cells were still quite small, whereas in the rapidlyproliferating cultures, colony-forming ability persisted over a

size range normally reserved for terminally differentiatingcells. Displacements of this sliding relation seem to bereversible because in large clones, in which the average rateof proliferation is much lower, the relation between cell sizeand colony formation shifted toward that of cells in theepidermis. The relation between size and colony-formingability in small colonies is likely to be found in the epidermisonly when the cells are highly stimulated to proliferate, forexample during epidermal regeneration (27, 45, 46).The question naturally presents itself, why is the system

designed in this way? Why not have multiplication alwaysconfined to the smallest cells? If, in the epidermis, multipli-cation were to become rapid and sustained and the transittime through the spinous layer were to be reduced (47), thecells in that layer would become smaller than normal, and thiswould likely interfere with their differentiated functions. Ifthe size of the multiplying cells were to increase, the cellsentering the differentiating compartment would be of a sizecloser to what is typical of that compartment. For example,in the epidermis, involucrin synthesis normally begins manycell layers above the basal layer, but in stratified cultures, itbegins in the relatively large cells of the first suprabasal layer(35).

If they do not begin terminal differentiation, large cells (upto 20 gm), like small ones, are a reservoir of clonogenicprogeny. A large cell has a low probability of forming a

colony, but if it succeeds in doing so, it gives rise to progenyof much smaller size and with clonogenic potential. Thismight occur as the result of unequal mitosis, in which thelarger daughter cell is destined for terminal differentiation, or

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as the result ofone or more cycles of mitosis occurring beforethe cell mass has doubled. The result of the reduction in cellsize is that young colonies, whether formed from small orlarge founding cells, come to contain cells of very similar sizedistribution, the modal diameter in both cases being 15 ,Jm.The experiments performed here have not demonstrated

the existence, in either the epidermis or in culture, of a classof nonclonogenic cells able to give rise to only a restrictednumber of progeny. Abortive colonies containing >32 cellswere encountered (Table 1) but were not numerous. Abortivecolonies containing <32 cells may have been more numerousbut were not identified because of difficulties in scoring. Inkeratinocyte cultures derived from older donors and subcul-tured repeatedly, both large and small abortive colonies arecommonly seen; the nature of the founding cells remains tobe explored.Whether the clonogenic cells described in these experi-

ments are identical to stem cells capable of self-renewal overthe natural life span of the organism (48) can only bedetermined by their behavior in regenerating epidermis aftertransplantation. It is already clear that transplanted clono-genic cells can continue to generate epidermal progeny overa period of years (49).

We are indebted to Dr. Mary Weiss for showing us a simple methodof cell isolation and transfer. These investigations were aided bygrants from the National Cancer Institute and the National Instituteof General Medical Sciences, and by a gift from Johnson andJohnson. Y.B. received a fellowship from the Institut National de laSantd et de la Recherche Mddicale.

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