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Annals of Anatomy 195 (2013) 581– 585

Contents lists available at ScienceDirect

Annals of Anatomy

jo ur nal ho mepage: www.elsev ier .de /aanat

esearch article

-kit positive cells and networks in tooth germs of human midtermetuses

ndreea Cristiana Didilescua,∗, Florinel Popb, Mugurel Constantin Rusuc,d

Division of Embryology, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, 8, Boulevard Eroilor Sanitari, 050474 Bucharest,omaniaDivision of Pathologic Anatomy, Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, RomaniaDivision of Anatomy, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, RomaniaMEDCENTER – Center of Excellence in Laboratory Medicine and Pathology, Bucharest, Romania

r t i c l e i n f o

rticle history:eceived 6 March 2013eceived in revised form 2 April 2013ccepted 4 June 2013

eywords:D117

s u m m a r y

Numerous studies have attempted to characterize the dental pulp stem cells. However, studies performedon prenatal human tissues have not been performed to evaluate the in situ characterization and topog-raphy of progenitor cells. We aimed to perform such a study using of antibodies for CD117/c-kit andmultiplex antibody for Ki67+ caspase 3. Antibodies were applied on samples dissected from five humanmidterm fetuses. Positive CD117/c-kit labeling was found in mesenchymal derived tissues, such as thedental follicle and the dental papilla. The epithelial tissues, that is, dental lamina, enamel organ andoral epithelia, also displayed isolated progenitor cells which were CD117/c-kit positive. Interestingly,

i67ental pulp stem cellsesenchymal stem cells

CD117/c-kit positive cells of mesenchymal derived tissues extended multiple prolongations buildingnetworks; the most consistent of such networks were those of the dental follicle and the perivascularnetworks of the dental papilla. However, the mantle of the dental papilla was also positive for CD117/c-kit positive stromal networks. The CD117/c-kit cell populations building networks appeared mostly witha Ki67 negative phenotype. The results suggest that CD117/c-kit progenitor cells of the prenatal toothgerm tissues might be involved in intercellular signaling.

. Introduction

Although the tooth is a unique organ from structural and func-ional points of view, the principles that guide its developmentre shared in common with other organs. The most importantevelopmental events are those guiding epithelial-mesenchymal

nteractions, which involve a molecular crosstalk between thectoderm and mesenchyme, two tissues with different originsD’Souza, 2002). The mechanisms controlling the development ofeeth are largely unknown. It has been suggested that incisorsre derived from cells having ectodermal characteristics, whereashe presumptive molar epithelium, despite being of ectoderm ori-in, has molecular commonality with pharyngeal endodermal cellsOhazama et al., 2010).

Constitutive activated tyrosine kinases (TKs) stimulate multi-le signaling pathways responsible for DNA repair, apoptosis, andell proliferation (Pytel et al., 2009). Stem cell factor (SCF) is the

∗ Corresponding author at: “Carol Davila” University of Medicine and Pharmacy,, Boulevard Eroilor Sanitari, 050474 Bucharest, Romania.

E-mail addresses: Andreea.Didilescu@gmail.com (A.C. Didilescu),natomon@gmail.com (M.C. Rusu).

940-9602/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.aanat.2013.06.002

© 2013 Elsevier GmbH. All rights reserved.

pleiotropic ligand for the TK receptor, which is c-kit (CD117). Bind-ing of SCF to c-kit promotes cell proliferation, differentiation, andrecruitment of progenitor cells in various biologic systems (Gagariet al., 2006). CD117/c-kit expression was demonstrated in humanadult dental pulp cells (Gagari et al., 2006; Graziano et al., 2008;Laino et al., 2005), which are mesenchymal derivatives (Laino et al.,2005).

There are no studies available to evaluate the specific prena-tal expression of CD117/c-kit during tooth development, neither in(ecto)mesenchymal-, nor in epithelial-derived tissues. Therefore,we aimed to perform such a study, targeting (ecto)mesenchymal-derived tissues; i.e. the dental follicle and dental papilla, andepithelial-derived tissues; i.e. the dental lamina, enamel organ andprimitive oral epithelium.

2. Materials and methods

Autopsy samples of tooth germs were dissected out in blocksfrom four human midterm fetuses with ages varying from 4 to

6 gestational months (g.m.). Samples were drawn immediatelypostabortion. The pregnancy interruption was due to miscarriage.No history of pathology that would adversely affect developmentwas recorded. Approval for the present study was granted by the

5 of Anatomy 195 (2013) 581– 585

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Fig. 1. Tooth germ of a 4.4 g.m. human fetus, immunolabeling with CD 117/c-kit. Theenamel organ (EO) and dental papilla (DP) are indicated. Numerous blood vessels of

dental laminae. Ectodermal–mesodermal interactions then lead todistinct stages, recognizable at the microscopic level. Once thetooth germ has developed, the neural crest cells differentiate intothe dental organ, dental papilla and dental follicle. Thus, dental

82 A.C. Didilescu et al. / Annals

thics Committee of the “Carol Davila” University of Medicine andharmacy, Bucharest, Romania.

Samples were fixed 24 h in buffered formalin (8%) and wererocessed with an automatic histoprocessor (Diapath, Marti-engo, BG, Italy) with paraffin embedding. Sections were cutanually at 3 micrometers, and were mounted on SuperFrost®

lectrostatic slides for immunohistochemistry (Thermo Scientific,enzel-Gläser, Braunschweig, Germany).The following primary antibodies were used: (1) CD117/c-kit

clone Y145, Biocare Medical-PME 296 AA, Concord, CA, USA, predi-uted RTU); (2) Ki 67+ caspase 3 (clone DVB-2, Biocare Medical-PPM40 DS AA, Concord, CA, USA, prediluted RTU).

For CD117/c-kit labeling, sections were incubated for 30 mint room temperature (RT) with a polymer, and then were incu-ated for 5 min at RT with Biocare’s DAB, and counterstained withematoxylin.

For Ki67+ caspase, sections were retrieved under pressure usingiocare’s Decloaking Chamber, followed by a wash in distilledater. Sections were incubated with the primary antibody for

0 min at RT; for double stain detection they were then incubatedor 30 min at RT using Biocare’s MACH 2 Double Stain 2. Chromogen:ections were incubated for 5 min at RT with Biocare’s BetazoidAB. Counterstaining was done with hematoxylin.

Positive controls: samples of gastrointestinal stromal tumorsnd colon cancer were used for CD117/c-kit, respectively Ki67+aspase 3 antibodies. Sections incubated with non-immune serumere used as negative controls.

The microscopic slides were analyzed and micrographs wereaken and scaled using a Zeiss work station described elsewhereRusu et al., 2013a).

. Results

All samples presented the same features, as described below.D117/c-kit labeling of dental papilla identified periendothelial

mmune positive cells (Fig. 1) sending processes that were coatinghe endothelial tubes; these cells were finally diagnosed as beingericytes, according to their topography, histological appearancend the periendothelial distribution.

CD117/c-kit diffuse interstitial positive labeling was found inhe dental papilla. Also, CD117/c-kit positive cells were found inhe proliferative and ameloblast layers of the enamel organ (Fig. 2).

Ki67 positive cells were scarcely observed within the dentalapilla, but not in periendothelial locations (Fig. 3). Ki67 labeling

dentified proliferative stages of cells of the enamel organ.CD117/c-kit positive cells of the dental follicle were found, being

i67 negative (Fig. 4). These were bipolar cells with prolongationsFigs. 4 and 5), serially linked, configuring a multilayered networkoating the outer adamantine epithelium (Fig. 5).

Multipolar progenitor cells, CD117/c-kit positive, were foundithin the secondary dental lamina, and were scarcely distributed

eneath the basal epithelial layer (Fig. 6).Ki67 positive, proliferative cells of the enamel organ were found

n the stellate reticulum and in the outer adamantine epithelium.carce Ki67 positive cells were also present in the dental follicle. Aigher density of Ki67 positive cells was observed in the secondaryental lamina, mostly in the basal epithelial layer (Fig. 7).

In the fetal oral epithelium CD117/c-kit positive cells were inasal or suprabasal locations, the later sending off prolongationsassing between the basal cells, toward the basal epithelial laminaFig. 8).

. Discussion

During the sixth week of human embryogenesis, the ectodermovering the stomodeum begins to proliferate, giving rise to the

the DP are identified (arrows) and present intense perivascular immune reactions.Inset: perivascular CD 117/c-kit positive cells (arrowheads) send prolongations withuneven caliber.

Fig. 2. Tooth germ of a 4.4 g.m. human fetus, immunolabeling with CD117/c-kit.Positive cells of the dental follicle are indicated (thin arrow). Positive progenitorcells (arrowhead) are identified at the border between the proliferative (1) andameloblasts (2) layers of the enamel organ. Within the dental papilla (3) immunepositive cells send processes and configure an interstitial network (thick arrow).

A.C. Didilescu et al. / Annals of Anatomy 195 (2013) 581– 585 583

Fig. 3. Tooth germ of a 4.4 g.m. human fetus, immune labeling with multiplex anti-bodies Ki 67+ caspase 3. Ki67 positive cells are scarcely observed within the dentalpapilla (1) where poor or absent proliferative activity is noticed in the vicinity ofbir

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lood vessels (arrows). The inner layers of the enamel organ, ameloblastic (2) andntermediate (3) mostly present Ki67 positive cells at their interface; (4) stellateeticulum.

ulp is made up of both ectodermal and mesenchymal components,ontaining neural crest cells that display plasticity and multipoten-ial capability. After crown mineralization, dental pulp tooth germemains entrapped within a hard structure that preserves it fromnvironmental differentiation stimuli (Erickson and Reedy, 1998;

raziano et al., 2008).

The stem cell niche is a specialized micro-environment neededor cells to retain their stemness (Schofield, 1978); it contains ele-

ents for the regulation of stem cell proliferation, control the

ig. 4. Dental follicle of a 4.4 g.m. human fetus, immunolabeling on successive slidesith CD117/c-kit (A) and Ki67/Caspase 3 (B). CD117/c-kit positive cells (arrows) of

he dental follicle are identified in (A) and detailed (inset) in (C). The respective cellsre Ki67 negative (B). Scale bars: (A) and (B): 50 �m; (C): 20 �m.

Fig. 5. Dental follicle of a 4.4 g.m. human fetus, immunolabeling with CD117/c-kit.

Fusiform immune positive cells send prolongations and are serially linked (arrows)in a network lying over the outer adamantine epithelium (*).

fate of stem cell progeny, and prevents the stem cells (SCs) fromexhaustion or death (Jones and Wagers, 2008; Scadden, 2006).There are two main types of SCs: embryonic (ESCs), and postna-tal/adult (ASCs). ESCs are blastocyst-derived. Interestingly, humannatal dental pulp SCs did not express CD117 in a previous study(Karaoz et al., 2010). This suggests that downregulation of CD117expression occurs temporarily after tooth formation, followed byupregulation in human adult teeth.

Although c-kit expression is often associated with cells ofneuroectodermal origin (Broudy, 1997), its presence in cells of mes-enchymal origin is equivocal (Gagari et al., 2006). The presence ofc-kit in dental pulp cells could be attributed to the ectomesenchy-mal origin of these cells (Laino et al., 2005). In the present study,dental pulp was highly positive to CD117/c-kit labeling, demon-strating its presence in pulp cells with pericytic characteristics,stemming from mesenchyme. This is in agreement with a previousstudy which suggested that pulp cells with pericytic characteris-tics could account for c-kit expression (Gagari et al., 2006). c-kitpositive pericytes were also found in human fetal endometrium(Rusu et al., 2013b). Pericytes are oligopotential cells (Hirschi andD‘Amore, 1996) which are able to influence endothelial permeabil-ity, survival, migration and maturation (Morikawa et al., 2002). Thediffuse CD117/c-kit positive phenotype of the dental pulp that wefound accounts for the heterogeneity in this stage of maturation ofthis tissue.

Interestingly, CD117/c-kit was found to label few cells in the

enamel organ, dental lamina and oral mucosa, which are knownto derive from ectoderm (Miletich and Sharpe, 2004). Early pat-terning of the oral ectoderm is clearly independent of the neural

584 A.C. Didilescu et al. / Annals of Anatomy 195 (2013) 581– 585

F it positive progenitor cells (arrows) scarcely distributed at the junction of the basal ands (inset).

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ig. 6. The secondary dental lamina of a 4.4 g.m. human fetus presents CD117/c-kuprabasal layers of the epithelial cord. Such a multipolar progenitor cell is detailed

rest (Miletich and Sharpe, 2004). More cells expressing CD117/c-it were building networks in the dental follicle, suggesting thathese act in a coordinated fashion. The dental follicle is ectomes-nchymally derived, being involved in tooth eruption coordinationTen Cate, 1994; Wise et al., 2002) and CD117/c-kit signaling. Panelsf specific markers(Huang et al., 2009; Lindroos et al., 2008; Yagyuut al., 2010) were used to assess the immune phenotype of dentalollicle progenitor cells able to differentiate into various cell typeshen grown in appropriate media(Yao et al., 2008); however, theserogenitor cells were not evaluated for their CD117/c-kit pheno-ype in the respective studies. In the current study, the close vicinityf the outer enamel epithelium suggests a possible inductive rolef this layer.

Caspase 3 was not identified, neither in the tissues of neuroec-odermal origin, nor in the ectomesenchymal tissues. This mayuggest that caspase 3 is not involved in apoptotic events occurringuring human odontogenesis, although caspase 3 was mentionedefore as being active during tooth morphogenesis of the murine

ower first molar (Shigemura et al., 2001).Numerous Ki67-immunoreactive proliferating cells in prena-

al rat molars were found distributed throughout the tooth germ

ig. 7. Tooth germ of a 4.4 g.m. human fetus, immune labeling with multiplex anti-odies Ki67+ caspase 3. Positive cells are identified: (a) within the stellate reticulum1) outermost layer (horizontal white arrows) and deeper (vertical white arrow);b) rarely (black arrowhead) in the outer adamantine epithelium (2); rarely (blackrrow) in the dental follicle (3); more numerous in the dental lamina (*).

Fig. 8. In a 4.4 g.m. fetus the basal epithelial layer of the oral mucosa presents basal(horizontal arrow) and suprabasal (vertical arrows) CD117/c-kit positive progenitor

cells. Inset: a multipolar suprabasal CD117/c-kit positive cell sends off dichoto-mously branching prolongations toward the basal epithelial membrane.

(Nakasone et al., 2006), and our results here support a similar dis-tribution in prenatal human tooth germs.

It appears that CD117/c-kit progenitor cells of the prenatal toothgerm tissues are involved in intercellular signaling. However, fur-ther studies are needed to evaluate the specific factors and cellswhich orchestrate the intrinsic signaling within the tooth germs.

Acknowledgement

All authors have equally contributed to the present study.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.aanat.2013.06.002.

References

Broudy, V.C., 1997. Stem cell factor and hematopoiesis. Blood 90, 1345–1364.D’Souza, R., 2002. Development of the pulpodentin complex. In: Hargreaves, K.M.,

Goodis, H.E., Tay, F.R. (Eds.), Seltzer and Bender’s Dental Pulp. QuintessencePublishing Co, Inc., IL.

of Ana

E

G

G

H

H

J

K

L

L

M

M

A.C. Didilescu et al. / Annals

rickson, C.A., Reedy, M.V., 1998. Neural crest development: the interplay betweenmorphogenesis and cell differentiation. Current Topics in Developmental Biol-ogy 40, 177–209.

agari, E., Rand, M.K., Tayari, L., Vastardis, H., Sharma, P., Hauschka, P.V., Damoulis,P.D., 2006. Expression of stem cell factor and its receptor, c-kit, in human oralmesenchymal cells. European Journal of Oral Sciences 114, 409–415.

raziano, A., d‘Aquino, R., Laino, G., Proto, A., Giuliano, M.T., Pirozzi, G., De Rosa, A.,Di Napoli, D., Papaccio, G., 2008. Human CD34+ stem cells produce bone nodulesin vivo. Cell Proliferation 41, 1–11.

irschi, K.K., D‘Amore, P.A., 1996. Pericytes in the microvasculature. CardiovascularResearch 32, 687–698.

uang, G.T., Gronthos, S., Shi, S., 2009. Mesenchymal stem cells derived from den-tal tissues vs. those from other sources: their biology and role in regenerativemedicine. Journal of Dental Research 88, 792–806.

ones, D.L., Wagers, A.J., 2008. No place like home: anatomy and function of the stemcell niche. Nature Reviews Molecular Cell Biology 9, 11–21.

araoz, E., Dogan, B.N., Aksoy, A., Gacar, G., Akyuz, S., Ayhan, S., Genc, Z.S., Yuruker,S., Duruksu, G., Demircan, P.C., Sariboyaci, A.E., 2010. Isolation and in vitro char-acterisation of dental pulp stem cells from natal teeth. Histochemistry and CellBiology 133, 95–112.

aino, G., d‘Aquino, R., Graziano, A., Lanza, V., Carinci, F., Naro, F., Pirozzi, G., Papac-cio, G., 2005. A new population of human adult dental pulp stem cells: a usefulsource of living autologous fibrous bone tissue (LAB). Journal of Bone and Min-eral Research: the Official Journal of the American Society for Bone and MineralResearch 20, 1394–1402.

indroos, B., Maenpaa, K., Ylikomi, T., Oja, H., Suuronen, R., Miettinen, S., 2008.Characterisation of human dental stem cells and buccal mucosa fibroblasts.Biochemical and Biophysical Research Communications 368, 329–335.

iletich, I., Sharpe, P.T., 2004. Neural crest contribution to mammalian tooth for-mation. Birth Defects Research Part C: Embryo Today: Reviews 72, 200–212.

orikawa, S., Baluk, P., Kaidoh, T., Haskell, A., Jain, R.K., McDonald, D.M., 2002.Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors.American Journal of Pathology 160, 985–1000.

tomy 195 (2013) 581– 585 585

Nakasone, N., Yoshie, H., Ohshima, H., 2006. The relationship between the termi-nation of cell proliferation and expression of heat-shock protein-25 in the ratdeveloping tooth germ. European Journal of Oral Sciences 114, 302–309.

Ohazama, A., Haworth, K.E., Ota, M.S., Khonsari, R.H., Sharpe, P.T., 2010. Ecto-derm, endoderm, and the evolution of heterodont dentitions. Genesis 48,382–389.

Pytel, D., Sliwinski, T., Poplawski, T., Ferriola, D., Majsterek, I., 2009. Tyrosine kinaseblockers: new hope for successful cancer therapy. Anti-Cancer Agents in Medic-inal Chemistry 9, 66–76.

Rusu, M.C., Hostiuc, S., Loreto, C., Paduraru, D., 2013a. Nestin immune labeling inhuman adult trigeminal ganglia. Acta Histochemica 115, 86–88.

Rusu, M.C., Motoc, A.G., Pop, F., Folescu, R., 2013b. Sprouting angiogenesis in humanmidterm uterus and fallopian tube is guided by endothelial tip cells. AnatomicalScience International 88, 25–30.

Scadden, D.T., 2006. The stem-cell niche as an entity of action. Nature 441,1075–1079.

Schofield, R., 1978. The relationship between the spleen colony-forming cell and thehaemopoietic stem cell. Blood cells 4, 7–25.

Shigemura, N., Kiyoshima, T., Sakai, T., Matsuo, K., Momoi, T., Yamaza, H., Kobayashi,I., Wada, H., Akamine, A., Sakai, H., 2001. Localization of activated caspase-3-positive and apoptotic cells in the developing tooth germ of the mouse lowerfirst molar. Histochemical Journal 33, 253–258.

Ten Cate, A.R., 1994. Oral Histology: Development, Structure and Function, 4th ed.Mosby, St. Louis, MO.

Wise, G.E., Frazier-Bowers, S., D‘Souza, R.N., 2002. Cellular, molecular, and geneticdeterminants of tooth eruption. Critical Reviews in Oral Biology and Medicine:an Official Publication of the American Association of Oral Biologists 13,323–334.

Yagyuu, T., Ikeda, E., Ohgushi, H., Tadokoro, M., Hirose, M., Maeda, M., Inagake, K.,Kirita, T., 2010. Hard tissue-forming potential of stem/progenitor cells in humandental follicle and dental papilla. Archives of Oral Biology 55, 68–76.

Yao, S., Pan, F., Prpic, V., Wise, G.E., 2008. Differentiation of stem cells in the dentalfollicle. Journal of Dental Research 87, 767–771.