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Keywords:

  • dermal skin;
  • fibroblast;
  • mesenchymal stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Abstract:  Dermal skin-derived fibroblasts from rodent and human have been found to exhibit mesenchymal surface antigen immunophenotype and differentiation potential along the three main mesenchymal-derived tissues: bone, cartilage and fat. Human dermal skin-derived mesenchymal stem cells constitute a promising cell source in clinical applications. Therefore, we isolated fibroblastic mesenchymal stem-cell-like cells from human dermis derived from juvenile foreskins, which share a mesenchymal stem cell phenotype and multi-lineage differentiation potential. We could show similar expression patterns for CD14(−), CD29(+), CD31(−), CD34(−), CD44(+), CD45(−), CD71(+), CD73/SH3-SH4(+), CD90/Thy-1(+), CD105/SH2(+), CD133(−) and CD166/ALCAM(+) in well-established adipose tissue derived-stem cells and fibroblastic mesenchymal stem-cell-like cells by flow cytometry. Immunostainings showed that fibroblastic mesenchymal stem-cell-like cells expressed vimentin, fibronectin and collagen; they were less positive for α-smooth muscle actin and nestin, while they were negative for epithelial cytokeratins. When cultured under appropriate inducible conditions, both cell types could differentiate along the adipogenic and osteogenic lineages. Additionally, fibroblastic mesenchymal stem-cell-like cells demonstrated a high proliferation potential. These findings are of particular importance, because skin or adipose tissues are easily accessible for autologous cell transplantations in regenerative medicine. In summary, these data indicate that dermal fibroblasts with multilineage differentiation potential are present in human dermis and they might play a key role in cutaneous wound healing.


Abbreviations:
ADSCs

adipose tissue derived stem cells

FmSCs

fibroblastic mesenchymal stem-cell-like cells

Mabs

monoclonal antibodies

MSCs

mesenchymal stem cells

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Mesenchymal stem cells (MSCs) can be isolated from many human tissues, including bone marrow, adipose tissue, human adult liver, peripheral blood, amniotic fluid, bronchial lung, articular synovium and other foetal tissues (1–6). These MSCs are a population possessing fibroblastic-like morphology, limited but long-term viability, self renewal capacity and multilineage potential (7,8). They are characterized by similar surface antigen expression patterns for CD14(−), CD31(−), CD34(−), CD45(−), CD71(+), CD73/SH3-SH4(+), CD90/Thy-1(+), CD105/SH2(+), CD133(−) and CD166/ALCAM(+) (1,2). These MSCs are able to differentiate along the adipogenic, osteogenic, myogenic, chondrogenic and neurogenic cell types (1,2,9–11).

In an effort to identify new sources of MSCs, dermal rodent fibroblast cell lines were examined for their mesenchymal potential (12–14). Toma et al. (2001) isolated skin-derived precursors (SKPs) from mice dermis and characterized them as nestin and fibronectin positive, but not as vimentin or cytokeratin expressing cells. These cells could differentiate into cells of both neural and mesodermal lineages, like neurons, glia, smooth muscle cells and adipocytes. Thus, SKPs could be passaged for at least 1 year without losing their differentiation potential (13). Crigler et al. (2007) found a murine dermal skin-derived subpopulation that had the capacity to differentiate into osteogenic, adipogenic, chondrogenic and myogenic cell lineages, and even into epidermal cell types (12).

These findings indicate that also adult mammalian dermis contains tissue derived-stem cells, and that even these fibroblastic MSCs are more plastic than previously appreciated. Dermis-derived fibroblastic MSCs are in focus for therapeutic applications like transplantation to support bone formation (15–17). There have been prior reports, for example by Toma et al. (2005) on the mesenchymal plasticity of primary human dermal fibroblasts in vitro with different approach regarding characterization and applications (18–20). Lysy et al. (2007) (18) compared the in vitro mesodermal and endodermal differentiation potential of human skin fibroblasts to human bone marrow MSCs (18). Chen et al. (2007) could demonstrate by clonal analysis that nestin vimentin+ human dermis-derived fibroblasts have multipotent differentiation potential. They also analysed the phenotypic characteristics of these fibroblasts and noted that the phenotype seems to be similar to that of adipose tissue derived stem cells (ADSCs) published by Zuk et al. (2002) (2,19,21).

Therefore, the objective of this study was to examine whether unselected human dermis fibroblastic mesenchymal cells possess stem cell-like characteristics and are phenotypical similar to ADSCs. In an effort to characterize fibroblastic mesenchymal stem-cell-like cells (FmSCs) in comparison to ADSCs, we analysed the cytoskeleton and extracellular matrix composition as well as the mesenchymal phenotype and differentiation properties of both. Our data show that primary human dermal fibroblasts in vitro share common characteristics for ADSCs, like the phenotype and differentiation potential.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Isolation and culture of ADSC and FmSC

Human adipose tissue was obtained from patients undergoing plastic surgery, and human juvenile foreskin samples (4 years old) were obtained from patients undergoing circumcision after written consent, with approval of the ethics commission of the Leipzig University and in accordance with the Declaration of Helsinki protocols.

Adipose tissue derived stem cells were isolated and expanded as previously described by Zuk et al. (2001) (21). Lipoaspirates were washed three times with sterile phosphate-buffered saline (PBS). The washed aspirates were incubated with 0.075% collagenase type A (Roche Diagnostics, Mannheim, Germany) for 30 min at 37°C with gentle agitation.

Epidermis and dermis were isolated by mechanical and enzymatic digestion as previously described by Ponec et al. (1988) (22). After removing the epidermis from the dermis, the tissue was cut into small pieces and washed three times with PBS at room temperature. Afterwards, the pieces were incubated with 0.075% collagenase for 12 h at 37°C with gentle agitation.

For ADSC and FmSC cell suspensions, the enzymatic reaction was inactivated with dulbecco's modified eagle's medium (DMEM)/10% foetal bovine serum (FBS) (Gibco/Invitrogen, Karlsruhe, Germany) and filtered through a 70-μm mesh. This cell suspension was centrifuged at 600 g for 5 min. Afterwards, the cell pellet was gently resuspended in DMEM/10% FBS, filtered through a 70-μm mesh, and plated onto conventional tissue culture flasks T75 (BD Falcon, Heidelberg, Germany). Cells were cultured in DMEM plus GlutaMAX-I with 4.5 g/l glucose and pyruvate (Gibco/ Invitrogen), supplemented with 10% FBS.

Cell immunophenotyping

The various monoclonal antibodies (Mabs) against surface antigens and flow cytometry analyses were as follows: fluorescein isothiocyanate or phycoerythrin (PE)-conjugated mouse anti human CD31 (Biozol Diagnostica, Munich, Germany), CD45 (Sigma-Aldrich, Seelze, Germany), CD14, CD34, CD71 (Dako Diagnostika, Hamburg, Germany), CD90, CD105 (BD Biosciences, Heidelberg, Germany), CD133 (R&D Systems, Wiesbaden, Germany) and CD166 (Acris Antibodies, Hiddenhausen, Germany). Mab mouse anti human CD73 (BD Bioscience) was unlabelled and combined with secondary antibody goat anti mouse PE labeled (Sigma-Aldrich). The incubation and flow cytometry analyses were performed according to conventional techniques (23). Isotype controls were equally concentrated labelled or unlabelled. The stained cells were analysed on a FACS Calibur (BD Biosience) using Cell Questpro (BD Biosience). The fluorescence intensity was determined by flow cytometry in a minimum of 1 × 104 cells.

For immunostaining of cytoskeleton and extra cellular matrix (ECM), Mabs were used as follows: mouse anti human vimentin (Sigma-Aldrich), nestin (Chemicon, Hampshire, UK) α-smooth muscle actin (α-SMA), collagen type I (Sigma-Aldrich) or rabbit anti human pan keratin (ICN Biomedicals, Irvine, CA, USA) and fibronectin (Sigma-Aldrich). These primary Mabs were either combined with goat anti mouse alexa 488 or goat anti rabbit alexa 555 (Invitrogen). The cells were grown on collagen 1 (BD Biosience, Bedford, UK) coated slides and fixed with 4% formaldehyde (Roth, Karlsruhe, Germany) and methanol, and processed according to protocols provided by Cell Signaling (Frankfurt am Main, Germany). The fluorescence staining was analysed on a fluorescence Axiovert 200 microscope and pictures were taken using an AxioCam camera and analysed with AxioVision software (Zeiss, Jena, Germany).

For detection of CD44 and CD29, the cells were stained with Mabs mouse anti human CD29 (Biozol Diagnostica) and CD44 (MEM85; Serva, Heidelberg, Germany) as well as with negative control Mabs mouse anti human CD31 (Biozol Diagnostica), CD34 (Dako Diagnostika), and compared with a secondary peroxidase-conjugated goat anti mouse antibody (Jackson ImmunoResearch, Dianova, Hamburg, Germany). Cell suspension was centrifuged onto poly-l-lysin-coated slides with a Shandon Cytospin 4 (Thermo Fisher Scientific, Langenselbold, Germany). Labelling of the positive cells was detected with aminoethylcarbazol (AEC) substrate solution containing aminoethylcarbazol, dimethylformamid and H2O2. The cell nucleus was counterstained with haematoxylin Lilie’s modification (DakoCytomation, Via Real, TX, USA) and analysed using microscopic analyses as described above.

Adipogenic and osteogenic differentiation assays

For induction of differentiation along the adipogenic and osteogenic lineages, the cells were cultured with specific induction media like previously described (1,21). Basal medium consisted of DMEM plus GlutaMAX-I with 4.5 g/l glucose and pyruvate, 1% gentamycin and 15% FBS; adipogenic differentiation medium was supplemented with 0.5 mm isobutyl-methylxanthine (IBMX) (Sigma-Aldrich), 0.1 μm dexamethason (Sigma-Aldrich) and 0.1% insulin transferrin selenium (ITS) 0.1 mm indomethacin (BD Biosience); osteogenic differentiation medium was supplemented with 0.1 μm dexamethason, 0.05 mm ascorbate-2-phosphate, 10 mmβ-glycerolphosphate (Sigma-Aldrich) and 1% ITS.

The adipogenic differentiation for ADSCs and FmSCs was assessed after 2 and 3 weeks respectively, using Oil Red O stain as an indicator of intracellular lipid accumulation (24). Cells were rinsed twice with PBS and fixed for 10 min with 4% formaldehyde (Roth), washed again with distilled water and rinsed with 50% ethanol. For staining, 2% Oil Red O (Sigma-Aldrich) solution was added and incubated for 15 min. Afterwards, the cells were washed with 50% ethanol, followed by several changes with distilled water. Osteogenic differentiation was induced for 4 weeks and examined by ECM calcification by van Kossa staining. Cells were washed with PBS without calcium and fixed for 10 min with 10% formaldehyde (Roth). After washing them with PBS and distilled water, the cells were overlaid with 5% silver nitrate solution (Riedel de Haen, Seelze, Germany) for 30 min. Cells were washed again with distilled water and incubated for 3 min with 1% pyrogallol (Merck, Darmstadt, Germany), washed again two times, and incubated for 3 min with 5% natriumthiosulphate (50 mg/ml; Sigma-Aldrich), washed with distilled water, and analysed by microscope as described above.

Gene expression analyses

Total cellular RNA was isolated and reverse transcribed using standard protocols. Target gene products like peroxisome proliferator activated receptor gamma (PPARγ) gene bank accession number NM_138712, lipoprotein lipase (LPL) gene bank accession number NM_000237, osteopontin (OPN) gene bank accession number NM_001040058.1, osteocalcin (OC) gene bank accession number NM_199173 and house keeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene bank accession number NM_002046 were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using following intron spanning primers: PPARγ forward 5′-CCG AGA AGG AGA AGC TGT TG-3′ and reverse 5′-TCG GAT ATG AGA ACC CCA TC-3′ (product length 466 bp, 30 cycles), LPL forward 5′-CCA TAC CAA TCA GGC CTT TG-3′ and reverse 5′-GAT CTT CTG AAT GGC GAA GC-3′ (product length 226 bp, 40 cycles), OPN forward 5′-ACA GCC AGG ACT CCA TTG AC-3′ and reverse 5′-CAT TCA ACT CCT CGC TTT CC-3′ (product length 306 bp, 30 cycles), OCN forward 5′-TCA CAC TCC TCG CCC TAT TG-3′ and reverse 5′-TCA GCC AAC TCG TCA CAG TC-3′ (product length 244 bp, 40 cycles), and GAPDH forward 5′-CTG CAC CAC CAA CTG CTT AG-3′ and reverse 5′-AGC TCA GGG ATG ACC TTG C-3′ (product length 219 bp, 30 cycles).

Statistical analyses

Fluorescence activated cell sorting (FACS) analyses and differentiation assays were performed in five independent experiments. Immunophenotyping and PCR were analysed in three independent experiments. Representative figures are given for fluorescence stainings.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Phenotypic characterization of ADSC and FmSC

To characterize the human dermal skin-derived fibroblast FmSCs, surface antigens were examined and compared with well-established human ADSCs (Fig. 1) by flow cytometry and immunostaining (Fig. 2). FmSCs expressed homogenously CD90, a fibroblast specific antigen epitope (25). Additionally, they were positive for specific antigen markers of MSCs like CD29, CD44, CD71, CD73, CD105 and CD166 (1,26) (Figs 1 and 2). In contrast, expression of the endothelial cell surface markers like CD31 and CD34 could not be detected in either analyses (Fig. 2e–h). In addition, haematopoietic cell subpopulations positive for surface antigens like CD45 (Fig. 1), CD14, and CD133 (data not shown) could not be observed. The analysed cell populations appeared homogenous; no subpopulation could be identified in any of the cell types by analysing cell size and granularity (Fig. 3e,f), and the expression level of CD90 and CD105 as demonstrated in Fig. 3g–j. This homogeneity was confirmed by flow cytometry and of cell culture observations in passage 0 and passage 6 (Fig. 3a–d).

image

Figure 1.  Analyses of mesenchymal stem cells (MSCs) surface antigen marker expressions in human fibroblastic mesenchymal stem-cell-like cells (FmSCs) (left column) in comparison to human adipose tissue-derived stem cells (ADSCs) (right column) by flow cytometry. Cell suspensions were stained with specific mouse anti human monoclonal antibodies (Mabs) (red line) as indicated in histograms. The green line is the respective IgG isotype control.

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image

Figure 2.  Immunostaining of cytospins with monoclonal antibodies (Mabs) mouse anti human as indicated. Primary antibodies were labelled with peroxidase-conjugated goat anti mouse antibody and stained with AEC-substrate. Cell nuclei were stained with haematoxylin (Lilies Modification). Fibroblastic mesenchymal stem-cell-like cells (FmSCs) (left column) were compared with adipose tissue-derived stem cells (ADSCs) (right column) (100× magnifications).

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image

Figure 3.  The homogeneity of fibroblastic mesenchymal stem-cell-like cells (FmSCs) and adipose tissue-derived stem cells (ADSCs) populations were examined during cell culture. Cell culture observations: FmSCs (left) and ADSCs (right) after isolation in passage 0 (a, b) and after passage 6 (c, d). Images were taken with 100× magnification. In addition, homogeneity of FmSCs (left column) and ADSCs (right column) cell populations were verified with FACS analyses. Representative diagrams are given for: cell size Forward Scatter (FSC) versus cell granularity Side Scatter (SSC) (e, f); CD90 expression (g, h); and CD105 expression (i, j).

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We could observe that the cell doubling rate of FmSCs was about twofold higher than that of ADSCs.

Cytoskeleton and ECM composition of FmSCs and ADSCs

To examine the composition of cytoskeleton and the expression profiles of ECM for FmSCs and ADSCs, we stained cells for vimentin and nestin, the intermediate filaments of MSC cytoskeleton. FmSCs as well as ADSCs were positive for vimentin. The staining for nestin was weaker than that for vimentin, but was present in all cells as shown in Fig. 4a–d. While all cells were negative for epithelial and epidermal cytokeratins, we could detect some positive cells for α-SMA (data not shown). The ECM compositions of FmSCs and ADSCs were quite similar to that of MSCs and included collagen type I and fibronectin (Fig. 4e–h).

image

Figure 4.  Cytoskeleton and extracellular matrix compositions in fibroblastic mesenchymal stem-cell-like cells (FmSCs) (left) and adipose tissue-derived stem cells (ADSCs) (right) were examined by immunfluorescence staining. Cells were stained as indicated using monoclonal antibodies (Mabs) (a) and (b): anti human vimentin; (c) and (d): anti human nestin; (e) and (f): anti human collagen type I and (g) and (h): anti human fibronectin. Images were analysed with fluorescent microscopy. Original magnification 400×.

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Adipogenic and osteogentic differentiation

To determine if FmSCs are cells with mesenchymal differentiation potential, we cultured them under appropriate conditions. We used ADSCs, known as inducible stem cell type, as positive reference for mesenchymal differentiation potential.

Adipogenic differentiation in ADSCs was observed after 2 weeks in culture (Fig. 5d) using inductive conditions as described in Materials and Methods. In FmSCs, fat vesicles could be detected after 3 weeks of culture under inducible conditions (Fig. 5a), and intracellular lipid accumulation could be verified with Oil Red Staining in all adipogenic stimulated cells (Fig. 5c,f). mRNA expression analysis showed a distinct up regulation of specific fat cell-related genes like PPARγ and LPL (Fig. 5g).

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Figure 5.  Induction of adipogenic differentiation in passage 6 monolayers of FmSCs (top row) and ADSCs (bottom row), respectively. (a) and (d): Morphology of FmSCs and ADSCs during culture with adipogenic media. (b) and (e): Oil Red staining of unstimulated control cultures. (c) and (f): Oil Red staining of adipogenic stimulated cultures. Images were taken with light microscopy and original magnification of 400×. (g): mRNA expressions profile for adipogenic (PPARγ, LPL) related genes in FmSCs vs ADSCs. Left panel: unstimulated cells; Right panel: stimulated cells.

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Osteogenic differentiation was analysed after 4 weeks of cultivation with osteogenic differentiation media. Calcium deposition could be observed in all of the analysed cell cultures (Fig. 6a,d) of FmSCs as well as ADSCs and verified by van Kossa staining (Fig. 6c,f). Gene expression analyses for markers related to osteogenic cell types revealed a pronounced upregulation of osteonectin and osteocalcin (Fig. 6g).

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Figure 6.  Induction of osteogenic differentiation in passage 6 monolayers of fibroblastic mesenchymal stem-cell-like cells (FmSCs) (top row) and adipose tissue derived stem cells (ADSCs) (bottom row) respectively. (a) and (d): calcium accumulation in cell culture supernatant; (b) and (e): von Kossa staining of unstimulated control cultures; (c) and (f): von Kossa staining of osteogenic stimulated cultures. Images were taken with light microscopy and original magnification of 400×. (g): mRNA expressions profile for osteogenic (ON, OCN) related genes in FmSCs versus ADSCs. Left panel: unstimulated cells; right panel: stimulated cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Adult MSCs have been found to differentiate along a wide range of cell types, (1,2,9–11,27) including non-mesenchymal lineages like epidermal cells or neurogenic cell types (20,28,29). The most apparent therapeutic use of such adult multipotent stem cells is tissue repair through cell transplantation or tissue replacement. MSCs represent also a new tool for delivery of therapeutic agents to tumor cells (30). For the last years, a run on to various tissues able for isolation of adult MSCs has been started.

The human hair follicle as a source of stem cells is already a subject of a great deal of investigation (31,32). Recent studies show that adult hair follicle dermal papilla and dermal sheath cells were capable of being directed to lipid and bone differentiation (33). But also the dermis itself contains tissue-based stem cells with mesodermal and endodermal differentiation potential (12,14,18–20).

The MSCs are characterized by the expression of a specific surface antigen phenotype. In our studies, we were able to assign a specific CD-Marker profile of the cell surface immunophenotype of human FmSCs. This profile is quite similar to MSCs and consists of CD14(−), CD29(+) CD31(−), CD34(-), CD44(+), CD45(−), CD71(+), CD73/SH3-SH4(+), CD90/Thy-1(+), CD105/SH2(+), CD133(−) and CD166/ALCAM(+) antigen expression.

As described by Wagner et al. (2005) (34), MSCs should not only be characterized by their non-specific surface antigens but also by their differentiation potential (34). In accordance to Wagner et al. we could demonstrate that the surface antigen profile of dermis fibroblastic mesodermal stem cells is quite similar to that of MSCs as well as ADSCs (2,21). Interestingly, the human foreskin-derived dermal fibroblasts studied by Wagner et al. could not differentiate into adipogenic or osteteogenic cell lineages (34). It is worth noting that the mesenchymal differentiation potential of fibroblasts was decreased in later passages (18,19). By comparing FACS data, we recognize that fibroblasts analysed by Wagner et al. were only weak positive for CD105, while MSCs, responsible for induction of differentiation, were mostly positive for CD105. Lysy et al. (2007) also found only 30% positive dermal-derived fibroblasts with positive expression of CD105. Fibroblasts of this study were not able to differentiate into mesodermal cell lineages beyond three passages (18). This interestingly data correlation supports the presumption that variations in isolation and cell culture conditions have a significant impact on the developmental and differentiation potential of the cell populations generated. Lysy et al. (2007) demonstrated that human dermal-derived fibroblasts have mesodermal stem cell characteristics. By comparing the dermal fibroblasts with bone marrow-derived stem cells, they demonstrated the osteocytic, adipocytic and hepatocytic differentiation potential of these cells (18). In contrast to their findings, we could show that 93.42% of FmSCs are positive for CD105, and additionally express CD166, but not CD133 or CD31. Additionally, we could verify that FmSCs had differentiation potential in passage 4 and even in passage 6 (data not shown).

Fibroblastic mesenchymal stem-cell-like cells expressed vimentin and nestin as intermediate filaments of the cytoskeleton. Vimentin is a cytoskeleton element of all cells of mesenchymal origin. Therefore, in combination with negative staining for cytokeratin, we could proof that FmSCs originate from the mesenchyme. Nestin is also a marker for multilineage progenitor cells and is mainly restricted to areas of regeneration in adult tissues and it has been shown to be re-expressed in mesenchymal progenitor cells after tissue injury (35–37). Skin-derived SKPs from human neonatal foreskins have been isolated by Toma et al. (2005) by clonal culture selection (20). SKPs were characterized, although no surface marker expression studies were performed. They could show that the clonal SKPs were able to differentiate along the neurogenic cell lineage and into smooth muscle cells.

Recent studies by Chen et al. (2007) (19) demonstrated that human dermis contains subpopulations of nestin vimentin+ multipotent fibroblasts. Single cell-derived clonal analyses were performed to obtain homogeneous populations. Additionally, we could show that FmSCs also expressed common mesenchymal ECM elements like fibronectin and collagen type I. This expression profile is quite similar to adult MSCs isolated from many other adult tissues (2,8,20,23,38,39).

Examination of cell size and granularity, as shown by FACS analyses, indicates the homogeneity of the FmSC population. Furthermore, we could not detect a subpopulation by analysing CD90 expression (Fig. 3). As shown in Fig. 1, this homogenous expression is also true for all MSC markers, with the exception of CD73 (ecto-5′-nucleotidase). The surface-bound CD73 is a membrane-bound glycoprotein that functions to hydrolyse extracellular nucleotides into bioactive nucleoside intermediates (40), e.g. it converts AMP to adenosine (41,42). CD73, a surface antigen of leucocytes (43) and MSCs (6,44), is controlled by many factors regulated by cell metabolism leading into higher expression of CD73 (45,46). Because of this regulation, we believe that the heterogenous expression of CD73 is a result of the metabolic indifferent cell cycle status of proliferative active FmSCs. We could observe CD71 (transferrin receptor) expression at higher levels in FmSCs than ADSCs. CD71 is mainly expressed on cycling cells, means on proliferative active cells (47,48). We could also confirm this more active proliferation of FmSCs versus ADSCs by cell culture observations.

Interestingly, we could observe that FmSCs are able to differentiate to adipogenic and osteogenic lineages. Given the similarities of FmSCs to ADSCs and their comparable mesenchymal differentiation potential in vitro, we suggest that human dermal skin contains FmSCs not only as single cells but also as dominant cell population. Fathke et al. (2004) could show that mice skin is a target for bone marrow derived cells (49). They showed that haematopoietic as well as MSCs are present in uninjured dermis. After injury, the bone marrow derived stem cells are contributing mainly to the cutaneous wound repair and collagen deposition (49).

Resident MSCs in adult tissues are involved in tissue maintenance and wound repair. We could demonstrate that human dermis is composed of FmSCs, which are assumed to be quiet until tissue injury. The weak expression of nestin we have observed in the FmSCs supports this assumption. For complete dermal function, it is also necessary that wound contraction is ensured by α-SMA positive fibroblasts, the so called myofibroblasts (50,51). It can be assumed that by the isolation method itself a tissue repair process is induced and the FmSCs become activated. This mechanism could explain the few α-SMA cells and weak nestin-positive cells, we have observed.

In summary, we could demonstrate that FmSCs fulfil the three main characteristics of MSCs: They express homogenously all MSC-related surface antigens; their cytoskeleton and matrix composition is quite similar to that of MSCs; they differentiate along the adipogenic and osteogenic cell lineages.

In addition, we aimed to compare the unselected FmSC population and ADSC population with each other. Both mesenchymal cell types could be isolated easily, and expansion in cell culture is very convenient, thus, making them ideal candidates in autologous cell transplantation.

As a tool for tissue engineering and autologous transplantation, MSCs are already in application.

Skin is the largest human organ and it is dissected daily in plastic or cosmetic surgery. Skin-derived FmSCs provide a valuable tool to support stem cell research and are a useful cell source regenerative medicine.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

We thank Sabine Ebert for excellent technical assistance. This work was supported by the Bundesministerium fuer Bildung und Wissenschaft, Foerderkennzeichen: 01GN0121.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References