Primary Mouse Keratinocyte Cultures Contain Hair Follicle Progenitor Cells with Multiple Differentiation Potential Jun Kamimura,*i" David Lee,* Howard P. Baden,* Janice Brissette,* and G. Paolo Dotto* *Cuwneous 13io logy ltese>1 rch Center, Massac husetts General Hospital and Harva rd Medica l School, C harl estown, M arylond, U .S. A.: and ·j·S hiseido R.esca rch Centre, Yokohama, Japan The interfollicular epidermis contains a single type of reassociation of cells. Instead, the reconstituted inter­ tenninally differentiated keratinocytes, whereas hair follicular epidennis contained distinct colum.nar units, follicles are composed of a minimum of six or seven comprising all the overlying layers and most likely derived distinct types. Whether or not these various populations from a single progenitor cell. In contrast, hair follicles of terminally differentiated keratinocytes originate from were found to be composed of cells of multiple origin, one or more progenitor cells has not been established. A with each population showing a striking localization related and in1portant question is whether keratinocyte to a separate concentric region. The vast majority of reconstituted progenitor cells with a pluripotent potential, able to follicles appeared to derive from a n1initnutn of two or, in a significant fraction of cases, form. not only epidern1is but also hair follicles, can be three progenitor cells, one for the generation of the shaft maintained i11 11itro for any period of time. We have (cuticle, cortex, and n1edulla), one for the inner root addressed these questions using skin reconstitution assays sheath, and the third for the outer root sheath. The with adrnixed populations of genetically labeled, cultured general implications of these findings for epidermis and keratinocytes. Examination of reconstituted epidennis hair follicle fonnation and for keratinocyte sten1 cell and hair follicles showed that neither was composed of cultivation are discussed. Key words: dermal papilla cells/ a random mixture of differently labeled keratinocytes, as J•etJ•olliral geue tmnsfer I stem cells I tissue reco ustitution. J bwest would be predicted if they originated from a random Dermatol 109:534-540, 1997 ike o th er terminall y differentiating ti ss ues, the epidermis is (medulla, cortex, and cuticle), three of th e inner r oot shea th (TR.S) likely to contain a compartment o F cells that are ca pable (H enlt! 's and Huxley's la yers, the c uticle), and the one or mo re of the of continuo us self-renewal and that can repl enish th e o mer root sheath (ORS). Whetb er or no t these va rious populations populations of keratinocytes that are los t dLI!;ng clifferenti­ of terminall y differentiated k eratinocytes Oti gin ate frotn one or more ati o n. Stem cells sho uld have two fundamental pro perties: progenitor cells has not been established. Finally, an important and as L(i) a n indefi nite life spa n, a11d (ii) t he ca pabili ty to give ri se to all o ther yet unsolved ques tion is w hether keratinocyte s tem ce!Js with a types of partia ll y committed and differentiated ·ells. The existence of pluripotent differentiation potential, able to form no t o nl y e pidermis keratin ocyte subpopul ations with increased proliferative po tential has but also hair follicles, can be maintained i11 tlitm for any length of time, been well establi shed, rtven if their precise locali zation and functional thus a!J owin g additional and mo n:: direct exp erimental evaluatio ns. role is still a matter of so me debate (Cotsarelis ct a/, '1990; Ya ng et a/, As stem cells of th e bo ne marrow, k eratinocyte progenitor cells a re 1993; Kobayas hi et a/, 1993; Jahoda and R eynold s, 1993; R oc!Jat et nl, th o ught to be tightly regula ted and dependent on signals produced b y 1994; Moll, 1995; Jones et a/, 1995). By contrast, evidence fo r closely juxtaposed stromal cells (Sengel, 1990). This is particularly keratinocyte progenitor cell s with a broad differentiation potential, evident in hair follicl es, yvhere cycles of keratinocyte growth (a nagen) able to reconstitute o n their own interfo Lli cul ar epidermis as well as and regression (ca tagen and tdogen) appear to be determined by th e hair folli cles, is stil.l lack in g. underl ying dermal papilla cells (Stenn el a/, 1991; Hardy, 1992) . The interfo lli cular epidermis contains a sin gle type of terminally .Epitheli al- mesenchymal interactions also play an essen tial role in th e differentiated ke ratin ocyres, whereas hair fo llicl es are composed of a inducti on of hair follicle formation during development. Three distinct minimum of seven distinct t ypes (S tenn et a/, 199 1; R.eynolds and sigmls are involved in this process (H ard y, 1992). A ftrst "dermal Jahoda, 1993). T hese include the three cell types of th e hair shaft message" fr om relatively undifre rentiated mesenchymal cell s induces a condensation of th e overl ying e pidermis. The epidermal condensa ti o n in turn produces an "epitheli al message" leading to dermal papil.l a cell formati on. Dermal papilla cells then release a "second dermal message" Manuscript received 1\pril 30, 1997; revised June 20, 1997; accep ted fo r that instructs th e overl ying epidermal condensation (hair bud) to fo rm pu bli cation June 27, 1997. a hair fo llicle. T his sequence of events c an be reproduced by a very P...eprint requests to: G .l'. Dotto, Bl"tC, MG H Eas t, 13th St r c~ t , elegant hair reconstitution C harl es town, MA 02 129. assay that bas been r ecently developed T hi s paper is dedicated to the mem01y of Dr. Jun l<a 1nimura who di ed in a (Weinberg e/ a/ , 1993; Lichti et a/ , 1993) . U sin g this assay, we now n1oumain cl imbing accident o n August 25, 1995. show that epidermis and hair follicl e progenitor cells with a broad but Abbreviations: AP, alka line phosphatase; EPU, cpid ennal pro liferati ve tlllit; di stin ct differentiation potential exist, and that th ey can be maintained in I R.S, inner root shea lth ; M 1-IC, major histocompatibility cornplex; OR.S, outer culture for at leas t a week. In particular, the reconstituted interfollicular root shca lth ; P13S, phosphate-buffered sa line. epidermis was found to contain distinct columnar units, each ori ginating 0022-202X/ 97/$ 10.50 · opyright © ·1997 by T he Society fo r Investi gative D ermatology, In c. 534 S VOL. 10'.1, NO. -1 OCT OUER 1997 KER.AT INOCYTE/ H A IR FO LLI C LE P I ~OGEN IT OR CELL 535 from a single cultured progenitor cell . [n contrast, hair fo Lli cl es appear in cubation w ith rh o daminL·-conjug3tcd goat anti- rabbit itnmunoglobulin G I 00 dilution in buffer A, Sigma, St. Louis, MO) and flu orescein -conjugated to derive fi·om a nunimum of two o r, in a significant fraction of cases, (I: strepra vidin ( I: I 00 in buffer A, Amershalll , Boston, MA) for I h in a li ght­ three progenitor cells, one for the generation of the shaft (cuticl e, right hlllnidificd box at room t e mp ~ ratur e. Fo ll owing in cubation with the a), o ne for the IRS, and th e third for the OR.S. cortex, and medull secondary antibodi es, secti o ns were washed exrensivcl y w ith PBS ~llld n1 0 U11ted fo lli cle T he general i.mplications of these findings for epidermis and hair in ·1% n-propylga ll are in glycerol. Comrol experim ems we re performed to formation and for keratinocyte stem cell cultiva tion are discussed . ve ri fY spec ificiry of antibody recognition of 13A LB /c versus C57BL/ 6 and nude (Swiss) mouse skins. MATEI:tiALS AND METHOD .RESULTS Cells and viruses Mouse prima1y keratinocytes we re iso lated fi·om 1-2-d­ old newborn Sencar, C57BL/6, or BALB/c mi ce and cultured in minimal Hair forming capability of cultured primary k eratinocytes essential n1 ediun1 at lovv ca lcium concentratio ns (0 .05 mN1; lo\v ca lciun1 The hai r reconstitution assay developed b y Li chti eta/ (1993; Weinberg ith 4% Chclex-treated feral calf serum and epid t!nnal medium) supplemented w et a/, 1993) is based on the grafting of hair buds (hair follicle precursors growth r.1ctor (10 ng per ml ; olbborative l:tesearch In c.) as previ ously present in newborn mo use skin as specif1 c ke ratinocyte aggregates) described (Hennings ct ril, 1980; Datto et nl, 1988) . Hair bud and interfolli cula r o nto n ude mice together with hair-inducing dermal papilla cell s keratin ocyte preparations were obtain ed as described by Weinberg el nl ( 1993) . mbinacion, Bri eAy, the epid ermis of '1 -2-d-old newborn mi ce was se parated fi·om th e (de ri ved fi·01n rat vibrissae) . These two components in co underl ying dermis by overni ght trypsin or dispase treatmellt at -l °C, fo ll owed but not eithe r one se parately, result in the formation of full y differe nti­ by sepa ration of the hair bud and interfo l.li cular fra ctions by two consecmive ated a nd o rga ni zed hair fo lli cl es. lmeres ti ngly, th ese authors reported Fi co ll gradi ent purifica tion steps.