Cytoskeletal elements of STRO-1⁺ DaMSCs and long-distance cell-to-cell connections (TNTs).

<p>(<b>a,b</b>) Different cell-to-cell connections between STRO-1⁺ cells. Intercellular connections are able to bridge long distances even across neighbouring cells (Phalloidin/Tubulin staining, merged images). (<b>b</b>) Thick tubes (diameter >0.4 µm, example is marked by red arrow) contain F-actin and α-tubulin, whereas thin tubes (diameter <0.4 µm, example is marked by green arrow) contain only F-actin. (<b>c</b>) Negative control, staining without Tubulin antibody. (<b>d–f</b>) Scanning electron microscope pictures (SEM) of a mixed culture of antlerogenic cells. Cells form long connections across neighbouring cells (<b>d,e</b>) and very high magnification (<b>f</b>) proves that the surface of TNTs exhibit small appendages; long-distance connections are also possible between morphologically distinguishable cell types (<b>d</b>). (<b>h–m</b>) Multichannel pictures of Phalloidin (<b>h,k</b>) and Tubulin (<b>i,l</b>) stained DaMSCs demonstrate that their intercellular connections continuously consist of F-actin but only partially of microtubules. The visible spindle apparatus (<b>i</b>, detail enlargement) point to an initiating cell division and provides evidence that the used α-tubulin antibody is also efficient in DaMSCs. (<b>j,m</b>). Merged Images.</p

Transport of Oct4 fusion protein via cytoplasmic connections.

<p>(<b>a–d</b>) Time series pictures of a transfected STRO-1⁺ DaMSC. The GFP fluorescence of the Oct4 fusion protein is visible within the cytoplasm as well as inside the membrane tubes. Membrane dilatations (gondolas) filled with Oct4 fusion proteins are moving away from the cellular body. Starting points are marked with white dashed lines. Scale bar = 20 µm.</p

Mixed culture of Oct4-GFP MEF cells and STRO-1⁺ DaMSCs.

<p>(<b>a</b>) Colony of Oct4-GFP MEF cells without any GFP expression. (<b>b</b>) Individual Oct4-GFP MEF cell after 24 hours of pre-cultivation in stem cell expansion medium on the eve of co-cultivation. (<b>c</b>) The same Oct4-GFP MEF cell after 24 hours of co-cultivation with STRO-1⁺ DaMSCs (interacting DaMSCs are outside of the display window). GFP expression (pseudo-coloured green) is visible within the cytoplasm. (<b>d</b>) Mixed culture after 96 hours of co-cultivation. More GFP⁺ cells interacting with GFP⁻ cells are visible. (<b>e</b>) Cell with distinct GFP expression after 120 hours of co-cultivation. At that time GFP⁺ cells interact continuously with GFP⁻ cells and are well integrated into the forming multilayer. (<b>f</b>) After 144 hours of co-cultivation some cells exhibit widely distributed intracellular GFP expression. (a,b,c,f = phase contrast pictures; d,e = varel contrast pictures; a–e = pictures of living cultures, f = fixed cells).</p

RT-PCR analyses of Oct4 expression in STRO-1⁺ cells.

<p>STRO-1⁺ cells derived from cultured pedicle periost (pp) of regenerating fallow deer antlers, growth zone (gz) of regenerating red deer antlers and human bone marrow (bm) from iliac crest biopsies were used as source for cDNA. PCR on these samples yielded bands representing the 3′-end of exon1 (335 bp). The bands were extracted and sequenced, demonstrating a very high homology of Oct4 of deer and human origin.</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

STRO-1⁺ cells in different locations of a velvet antler.

<p>(a-i) Paraffin embedded biopsy samples of velvet antler (main beam, cross-sections, samples taken about 1 cm below the tip), 9 yr-old fallow buck (<i>Dama dama</i>); samples were taken 74 days after onset of regeneration; scale bars: 100 µm. (a) Part of the cartilaginous zone, numerous blood vessels are located in the area between the cartilaginous trabeculae, white asterisks = vessels, white arrows = chondrogenic cells, Movat-staining. (b) Perivascular and endothelial cells staining positive for the STRO-1 antibody (fluorescence dye = FITC), phase-contrast picture. (c) Part of velvet skin containing hair follicles and sebaceous glands (black square), HE-staining. (d) STRO-1⁺ cells at the base of a sebaceous gland, red asterisk = sebaceous gland, varel-contrast picture. (e–i) Perivascular cells in the cartilaginous zone. (e) STRO-1⁺ cells, white asterisk = vessel, varel-contrast picture. (f) Same picture as (e), STRO-1⁺ fluorescence only. (g) CD271⁺ cells [CD271 antibody combined with an anti-mouse IgG secondary antibody conjugated with fluorescence dye (Alexa Fluor 546)], white asterisk = vessel, varel contrast picture. (h) Same picture as (g), CD271⁺ fluorescence only. (i) Merged image of (f) and (h).</p

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