Localization and Characterization of STRO-1+ Cells in the Deer Pedicle and Regenerating Antler

The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper insight into the mechanisms that prevent limb regeneration in humans and other mammals, and, with regard to medical treatments, may possibly even show ways how to overcome these limitations. Traditionally, antler regeneration has been characterized as a process involving the formation of a blastema from de-differentiated cells. More recently it has, however, been hypothesized that antler regeneration is a stem cell-based process. Thus far, direct evidence for the presence of stem cells in primary or regenerating antlers was lacking. Here we demonstrate the presence of cells positive for the mesenchymal stem cell marker STRO-1 in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama). In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR) were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. We found evidence that STRO-1+ cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages. Our results support the view that the annual process of antler regeneration might depend on the periodic activation of mesenchymal progenitor cells located in the pedicle periosteum. The findings of the present study indicate that not only limited tissue regeneration, but also extensive appendage regeneration in a postnatal mammal can occur as a stem cell-based process

Localization, proliferation and differentiation of STRO-1-positiv cells of antlers of fallow deer (Dama dama)

Geweihe sind komplexe Gebilde aus Haut, Nerven, Blutgefäßen, Knorpel und Knochen. Als einziges Beispiel eines Anhangsorgans bei Säugetieren besitzen diese die Fähigkeit, jährlich vollständig zu regenerieren. Zahlreiche Studien zeigen den Einfluss von Tageslichtveränderungen und Schwankungen des Sexualhormonspiegels auf diesen zyklischen Prozess. Die Zellen des Hirschgeweihs weisen eine hohe Wachstumsrate auf und bieten hiermit eine gute Grundlage zur Erforschung der Osteogenese in vitro. Transplantationsversuche konnten zeigen, dass die Regeneration des Geweihs vom Periost des Rosenstockes ausgeht. Welche Prozesse dieser Regeneration zugrunde liegen, blieb bisher unklar. Es wird vermutet, dass im Periost Vorläuferzellen existieren, von denen diese ausgeht. Im Rahmen dieser Arbeit ist es gelungen, Zellen aus dem regenerierenden Geweihknochen zu isolieren, welche sich mit dem mesenchymalen Stammzellmarker STRO-1 markieren ließen. Die isolierten Zellen entwickelten unter dem Einfluss verschiedener Wachstumsmedien (Osteoblasten-, Neurogenes oder Mininalmedium) unterschiedliche Proliferationsraten. Mit zunehmender Kulturzeit bildeten die zunächst homologen Zellen unterschiedliche Morphologien. Die Zellen im Osteoblastenmedium zeigten den höchsten Umsatz an Calcein, was auf eine gesteigerte Osteoblastenaktivität hinweist. In allen Medien konnte die Expression knochenspezifischer Gene nachgewiesen werden. Dies kann auf eine mögliche Vorprägung der STRO-1+-Zellen hindeuten. Insgesamt besteht die Möglichkeit, die STRO-1+-Damhirschgeweihzellen durch Anzucht in verschiedenen Medien und unter Zugabe von Wachstumsfaktoren in ihrem Wachstums- und Differenzierungsverhalten zu beeinflussen. Die Ergebnisse der vorliegenden Arbeit geben neue Hinweise darauf, dass die Regeneration des Geweihs nicht auf einer Dedifferenzierung entsprechender Zellen, sondern eher auf einer Aktivierung von Stammzellnischen beruht

Isolation of STRO-1⁺, CD271⁺ and CD133⁺ cells derived from regenerating deer antler and pedicle periosteum.

<p>The mixed cell populations were analysed by flow cytometry (FACS). (a,b) Mixed population of cells derived from the antler growth zone (b) Percentage of STRO-1⁺ cells within the gated population (R1). (c) Scanning electron microscopy (SEM) picture of a mixed antler cell population, scale bar: 20 µm (×500). (d) SEM picture of a pure STRO-1⁺ cell population, scale bar: 50 µm (×200). Samples shown at pictures (c) and (d) were prepared after cell cultures had reached confluence. (e–m) Mixed cell population derived from the pedicle periosteum; (e,h,k) Global mixed populations (FSC/SSC); (f,i,l) Gated populations (unstained), cells of gate R1 (FSC/SSC) plotted as FL2 as a function of FL1; (g) Double staining (CD34/STRO-1), FL1 = STRO-1, FL2 = CD34; (j) Double staining (CD34/CD271), FL1 = CD271, FL2 = CD34; (m) Double staining (CD34/CD133), FL1 = CD133, FL2 = CD34.</p

Expression profiles and morphology of isolated STRO-1⁺ cells.

<p>(a) Expression profiles of STRO-1 negative versus STRO-1⁺ cells. RT-PCR was used to detect the mRNA of specific markers for the osteogenic [Collagen 1, cbfa 1, osteocalcin (OCN)] and the chondrogenic lineages (chondroadherin). Expression of deer ß-actin was used for standardization. (+) = STRO-1⁺ cells, (−) = STRO-1 negative cells, (M₁) = Marker: 500 bp DNA ladder, (M₂) = Marker: 100 bp DNA ladder. (b,c) Typical morphology of STRO-1⁺ cells isolated from fallow deer antler cell cultures [STRO-1 antibody combined with fluorescence dye (FITC), nuclei counter-stained with Hoechst 33342], scale bar: 100 µm. (d,e) STRO-1⁺ stem cells with three nuclei, (d) phase contrast picture; (e) same staining as shown in (b) and (c); scale bars: 100 µm.</p

STRO-1⁺ cells in different areas of the pedicle.

<p>(a) Methylmetacrylate (Technovit® 9100 New) embedded sample of the pedicle shown in (b) and (c); cross-section, overview, HE-staining. (E) epidermis, (D) dermis, (SC) subcutaneous tissue with superficial muscle (asterisk), (Mf) Part of the frontoscutular muscle, (Fa) fascia (<i>tissue slightly lacerated during histological processing</i>) , (CP) cambial layer of the periosteum, (B) pedicle bone; white asterisk = bony trabeculae, scale bar: 500 µm. (b) Left pedicle and primary velvet antler of a 1 yr-old fallow buck (<i>Dama dama</i>), the antler was cut below the coronet (dashed line) to obtain a cross-section of the distal pedicle, scale bar: 10 cm. (c) Cross-section of the distal pedicle shown in (b); white rectangle marks the area shown in (a); scale bar:1 cm. <u>For all pictures (d-m):</u> [STRO-1 antibody was combined with an anti-mouse IgM secondary antibody conjugated with fluorescence dye (FITC), nuclei were counter-stained with Hoechst 33342]. (d,e) STRO-1⁺ cells within the reticular layer of the dermis, located between thick collagen fibres; (d) STRO-1⁺ fluorescence only, same area as (e); (e) Fluorescence combined with varel-contrast picture; (f) Negative control; similar area as shown in (e); the small green dots are erythrocytes marked by the fluorescence dyes; identical exposure times for pictures (e) and (f), scale bars: 100 µm. (g) Vascular associated STRO-1⁺ cells within the subcutaneous tissue, varel-contrast picture, scale bar: 100 µm. (h) Negative control; same area as shown in (g); identical exposure times for pictures (g) and (h), varel-contrast picture, scale bar: 100 µm. (i–k) STRO-1⁺ cells between fibres of the frontoscutular muscle, scale bars: 100 µm; (i) Fluorescence combined with varel-contrast picture; (j) STRO-1⁺ fluorescence only, same area as (i); (k) Negative control, similar area as shown in (i); varel-contrast picture, identical exposure times for pictures (i) and (k); the bright green dots in picture (k) are erythrocytes marked by the fluorescence dyes. (l) STRO-1⁺ cells within the cambial layer of the periosteum; scale bar: 100 µm. (m) Negative control, similar area as shown in (l); scale bar: 100 µm, identical exposure times for pictures (l) and (m); the bright dots in pictures (l) and (m) are erythrocytes marked by the fluorescence dyes.</p

STRO-1⁺ cells in the cambial layer of the perichondrium and the cartilaginous zone of an antler.

<p>Paraffin embedded biopsy samples of a velvet antler from a 4 yr-old fallow buck (<i>Dama dama</i>); samples were taken 46 days after onset of regeneration. (a) Cross section of brow tine about 1 cm below the tip, overview, (E) epidermis, (D) dermis, (CP) cambial layer of the perichondrium, (CZ) cartilaginous zone, white arrows = vessels, black arrows = sebaceous glands; HE-staining, scale bar: 500 µm. (b) STRO-1⁺ cells in the cambial layer of the perichondrium [STRO-1 antibody combined with an anti-mouse IgM secondary antibody conjugated with fluorescence dye (FITC), nuclei counter-stained with Hoechst 33342], scale bar: 100 µm. (c) Negative control, cambial layer of the perichondrium, same staining as (b) without STRO-1 antibody, scale bar: 100 µm, identical exposure times for pictures (b) and (c). (d) Cross section of part of a main beam, cartilaginous zone, HE-staining, scale bar: 100 µm. (e) STRO-1⁺ cells within the cartilaginous zone [same staining as (b)], scale bar: 100 µm. (f) Negative control, comparable area of the cartilaginous zone, same staining as (e) without STRO-1 antibody, scale bar: 100 µm, identical exposure times for pictures (e) and (f).</p

Primer sequences used in RT–PCR analyses

<p>Primer sequences used in RT–PCR analyses</p

Growth and differentiation of STRO-1⁺ cells in different culture media.

<p>(a) Time –dependent increase in cell numbers (ΔN/Δt) in Dulbecco's Minimal Eagle Medium (DMEM), osteoblast proliferation medium (OB), and NeuroBasal medium containing 50 ng/ml nerve growth factor (NB). The peak values of the curves coincide with the time when the cells reached confluence (t_k), culture well area = 2 cm². (b) Expression of osteocalcin in isolated STRO-1⁺ cells cultured for several weeks in DMEM and OB-medium. RT-PCR was used to detect the mRNA of osteocalcin (OCN); expression was investigated at culture days 7, 14 and 21. (c,d) STRO-1⁺ cells after four days of induced adipogenic differentiation in adipocyte differentiation medium starting with intracellular lipid formation (white arrows), (c) phase contrast, (d) varel contrast; scale bars: 100 µm. (e,f) STRO-1⁺ cells after 10 days culture in adipocyte differentiation medium. Cells were fixed, stained for lipid accumulation (Oil Red O) and observed under a light microscope; scale bars: 100 µm.</p

MACS-Analyses of “mixed” cell cultures derived from regenerating antlers.

<p>Percentages of cells positive for different surface markers</p>*<p>Single analysis/Second passage of cells derived from the antler growth zone of an adult fallow deer.</p>**<p>Values obtained from different culture analyses (analysed were primary cultures till third passages)</p