ISSN 0012-4966, Doklady Biological Sciences, 2007, Vol. 416, pp. 406–408. © Pleiades Publishing, Ltd., 2007.Original Russian Text © E.S. Chermnykh, E.A. Vorotelyak, S.B. Tkachenko, A.V. Vasil’ev, V.V. Terskikh, 2007, published in Doklady Akademii Nauk, 2007, Vol. 416, No. 4,pp. 555–558.

406

The niche of epithelial stem cells of epidermis is sit-uated in the upper part of the hair follicle referred to asthe bulge. Numerous studies have shown that the cells inthis region are precursors of all epithelial cells of the hairfollicle during the hair growth cycle, cells of interfollicularepidermis, and cells of the sebaceous gland [1].

Epidermal stem cells express specific keratins, suchas keratin 15 [2] and keratin 19 (K19) [3]. K19 isexpressed in the human fetal skin in the cells of thebasal layer [4], whereas in the adult skin, it is expressedonly in the outer sheath of the hair follicle [5]. Michelet al. [3] found that

ä19

+

cells retain labeled thymidinefor a long time. In this study, we showed that K19 isexpressed in the hair follicle and is absent in the inter-follicular epidermis. The skin parts devoid of hair folli-cles have

ä19

+

-expressing cells at the top of the epider-mal crest.

ä19

+

cells have a high level of expression of

β

1-intergrin, which is a marker of epidermal stem cells[6]. The number of

ä19

+

cells in the epidermis of theforeskin is considerably higher in newborns than inadults [7]. The hypothesis that K19 may serve as amarker of stem cells was confirmed by studies on thehuman eye cornea. K19 was not expressed in the basallayer of corneal epithelium, which contains differenti-ated cells. This marker was expressed in the limberbasal layer containing stem cells [8]. In addition, it wasestablished that the apical surface of all

ä19

+

cells ofthe limbal basal layer, which is subjected to ultravioletirradiation [8], contains melanin granules, which serve,presumably, for protection of the stem cells. These datasuggest that K19 is a marker of epidermal stem cells.

Homeostasis of the cell population in the culture ofepidermal keratinocytes, as in the epidermis in vivo, ismaintained by the stem cells. The possibility of themaintenance of stem cells in culture is confirmed by thepractice of autografting grown layers of keratinocytes

to the wound surface [9]. Hair follicles may serve as asource of stem cells during culturing.

ä19

+

cells inhaired skin regions of adults are located only in the hairfollicles; hence, K19 expression may indicate that thesecells have originated from the compartment of stemcells of the hair follicle.

We studied the population dynamics of

ä19

+

cellsin culture and some characteristics of these cells.

Earlier, we showed [10] that

ä19

+

cells are main-tained in culture during at least two passages, and thecell clusters formed after passaging contain

ä19

+

cells.They have the same sizes as K19

–

cells: they includevery small cells, which corresponded to stem cells, andlarge cells, which may correspond to differentiated ele-ments.

The formation of hair at the stage of anagen is deter-mined by proliferation of cells of the matrix of hair fol-licles. The duration of this stage determines the finallength of the hair, and the human hair follicle mayremain at the anagen stage for eight years. This dura-tion of the proliferation period shows that the matrixcells in vivo have a high proliferative potential. Thisfact suggests that stem cells in the bulge of the hair fol-licle migrate to the matrix during the anagen and returnback during the catagen [11]. However, there are manyother hypotheses on the cycle of hair growth.

Identification of the exact location of stem cells inthe hair follicle is important for understanding the roleof the bulge cells in the homeostasis of the epidermis.

We studied hair follicles extracted from the humanskin at the stage of anagen and found the expression ofkeratin 19 in two areas of the external sheath of the hairfollicle: the upper one-third of the bulge area and thelower part of the hair follicle, the matrix (Fig. 1). Thisdistribution of

ä19

+

cells in the hair follicles duringanagen is in agreement with other studies [12].

We believe that part of shaft cells of the hair follicle(

ä19

+

) migrate during the hair growth cycle into thematrix of the hair follicle, where these cells proliferateand form precursors, transitional cells with a high pro-

CELLBIOLOGY

Proliferation of K19

+

HumanEpidermal Keratinocytes in vitro

E. S. Chermnykh, E. A. Vorotelyak, S. B. Tkachenko, A. V. Vasil’ev, and V. V. Terskikh

Presented by Academician N.G. Khrushchov April 12, 2007

Received May 3, 2007

DOI:

10.1134/S0012496607050250

Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 117808 Russia

DOKLADY BIOLOGICAL SCIENCES

Vol. 416

2007

PROLIFERATION OF K19

+

HUMAN EPIDERMAL KERATINOCYTES 407

liferation potential involved in the formation of the hairstem.

We studied the distribution of

ä19

+

cells in a pri-mary culture. Keratinocytes were cultured as describedpreviously [13]. The cells were extracted from skinfragments cut during surgical operations. The epider-mis was separated from the derma after incubation ofthe skin fragments in a dispase solution. The suspen-sion of keratinocytes obtained using enzymatic disag-gregation of epidermis was cultured in DMEM : F12(1 : 1) supplemented with 10% fetal calf serum,L-glutamine, insulin, isoproterenol, and EGF and incu-bated at 37

°

C in a CO

2

incubator.Study of the primary culture at early stages of cul-

turing in the medium with a physiological Ca

2+

contentshowed that single

ä19

+

-expressing cells were presentin clusters or located singly. The single

ä19

+

cells hada small diameter and a high nucleus-to-cytoplasm ratio.The number of these cells was less than 1%. The diam-eter of

ä19

+

cells in cell aggregates was larger. We alsofound aggregates formed only by

ä19

+

cells. Culturingof epidermal cells for six days increased the size andnumber (to 20%) of

ä19

+

cells.It is known that the degree of differentiation of kera-

tinocytes is very sensitive to culturing conditions,including the extracellular calcium concentration. Cul-turing of keratinocytes in a low-calcium medium helpsto maintain cells in the undifferentiated state. There-fore, we hypothesized that the calcium concentrationmay affect the number of

ä19

+

cells during culturing.However, culturing in a low-calcium medium had asimilar effect: 24 h after the start of culturing, we foundsingle

ä19

+

cells (1%), and six days later, their numberincreased to 20%. The increase in the proportion of

ä19

+

cells may be determined by several factors: first,their proliferation, second, their predominant survival,and third, initiation of K19 expression by K19

–

cells.

Hence, we studied the proliferation of these cells. Dou-ble immunohistochemical staining for K19 and Ki-67showed that

ä19

+

cells cultured for one day were notstained with antibodies against Ki-67 (Fig. 2); i.e., theydid not proliferate, whereas K19

–

cells proliferated.However, on the sixth day in culture, 15% of

ä19

+

keratinocytes were Ki-67-positive (Fig. 3), whichapproximately corresponded to the proportion of K19

–

proliferating cells. This indicates that the proliferativeactivity of

ä19

+

cells increases in culture, which pre-sumably results in an increase in the proportion of thesekeratinocytes.

Study on

ä19

+

cells was performed also in a popu-lation of basal keratinocytes. In contrast to the primaryculture, the subpopulation of basal keratinocytes ismore uniform and less differentiated and contains stemand progenitor cells with a high proliferative and mor-

500 µm 50 µm

Fig. 1.

Expression of keratin 19 in the hair follicle at thestage of anagen. Keratin 19-expressing cells (marked byarrows) are situated in the upper one-third of the bulgeregion and in the lower part of the hair follicle, the matrix.

Fig. 2.

Expression of keratin 19 and Ki67 in one-day-oldprimary culture of keratinocytes.

K19

+

cells marked byshort arrows do not proliferate, whereas K19

–

cells prolifer-ate (marked by long arrows).

50 µm

Fig. 3.

Expression of keratin 19 and Ki67 in a primary cul-ture of keratinocytes after six days of culturing. Some kera-tin 19-expressing cells are stained with antibodies againstKi67 (marked by arrows); i.e., they proliferate.

408

DOKLADY BIOLOGICAL SCIENCES

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2007

CHERMNYKH et al.

phogenetic potential. To obtain cells of the basal layer,the multilayer sheet of keratinocytes was incubated in amedium with a low Ca

2+

content.

ä19

+

cells in the cul-ture of basal keratinocytes were grouped and had alarge size. The proportion of these cells increased to20%. The number of

ä19

+

cells increased to 30% 21 hafter passaging of the basal cells. Study on the cellslabeled with bromodeoxyuridine (BrdU) showed thatthe number of BrdU-positive

ä19

+

cells was notincreased. This suggests that culturing conditions mayinduce K19 expression in K19

–

cells. It is known thatkeratins of monolayer epithelium are induced in cul-tured epidermal keratinocytes, and the expression ofkeratins specific for differentiation of keratinocytes issuppressed. On the other hand, K19

–

cells may morefrequently undergo apoptosis or stratification.

On the basis of these data, we hypothesized thatthere are several subpopulations of epidermal kerati-nocytes, which can maintain themselves in culture.Both populations are heterogeneous. After several divi-sions,

ä19

+

cells form groups of cells in the culture.The proportion of

ä19

+

cells in the culture increases. Itmay be hypothesized that

ä19

+

keratinocytes aredescendants of the cells of the hair follicle in the sys-tems cultured for a long time, or culturing promotesadditional synthesis of keratin 19. The data obtainedsuggest that both mechanisms are possible.

ACKNOWLEDGMENTS

This work was supported by the Russian Foundationfor Basic Research (project no. 04-04-49336-a) and

grant of the Presidium of the Russian Academy of Sci-ences “Molecular and Cell Biology.”

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