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The dose effect of human bone marrow-derived mesenchymal stem cells on epidermal development in organotypic co-culture
Journal of Dermatological Science 55 (2009) 150–160
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The dose effect of human bone marrow-derived mesenchymal stem cells on epidermal development in organotypic co-culture Filip Laco a,d,*, Ma Kun b, Hans Joachim Weber a, S. Ramakrishna c,d, Casey K. Chan e,f a
Department of Medical Technology and Technomathematics, University of Applied Sciences, Aachen, Germany Graduate Program in Bioengineering, National University of Singapore, Singapore 117576, Singapore c Department of Mechanical Engineering, National University of Singapore, Singapore 117576, Singapore d Nanoscience and Nanotechnology Initiative, National University of Singapore, Singapore 117576, Singapore e Department of Bioengineering, National University of Singapore, Singapore 117576, Singapore f Department of Orthopedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074, Singapore b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 January 2009 Received in revised form 12 May 2009 Accepted 29 May 2009
Background: A wealth of evidences have shown the participation and benefits of bone marrow-derived mesenchymal stem cells (BM-MSCs) in wound healing and skin tissue repair in vivo. However, their role in epidermal development and reconstitution is not clearly investigated. Objective: Here we examine the quantitative effect of human BM-MSCs on epidermal regeneration in vitro. Method: Human keratinocytes and BM-MSCs are cultured at ratios from 0% to 100% on top of a fibroblast-embedded collagen gel in a three-dimensional organotypic co-culture model at an air–liquid interface up to 20 days and analyzed by histochemical and immunochemical staining of filaggrin, involucrin and keratin 10 on days 14 and 20. Human BM-MSCs were tracked with quantum dots in organotypic co-cultures. Results: It was found that epidermal development is strongly influenced by the percentage of cocultured BM-MSCs. A strong chemotactic effect between keratinocytes and MSCs was seen in the group with 50% of BM-MSCs, which resulted in an impaired epidermal development, whereas at a low percentage of BM-MSCs (10%), a stratified epidermal structure resembling native skin was established on day 14 of culture. Moreover, the immunostaining studies revealed that BM-MSCs in the low percentage (10%) participated in the basal periphery of reconstructed epidermis and a similar pattern characteristic of native epidermis was demonstrated in this experimental group, which was superior to all other experimental groups in terms of the thickness of stratum corneum and the expression profile of epidermal differentiation markers. Conclusion: This study indicates the advantage of using a new skin equivalent model incorporating a small fraction of MSCs to develop biologically useful tissues for maintaining homeostasis during skin regeneration and wound healing process. ß 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Mesenchymal stem cells Organotypic Epidermis Skin regeneration
1. Introduction After skin tissue injury, multipotent progenitor cells like mesenchymal stem cells (MSCs) are mobilized from bone marrow into the pool of circulating cells. These cells then home to the sites of injury, where they differentiate into keratinocytes (KERs) and
Abbreviations: BM, bone marrow; MSCs, mesenchymal stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells; KERs, keratinocytes; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; OC, organotypic co-culture; QDs, quantum dots; EGF, epidermal growth factor; VD3, 1,25 dihydroxyvitamin D3; 3D, three-dimensional; H&E, hematoxylin and eosin. * Corresponding author at: University of Strathclyde, Wolfson Center, 106 Rottenrow East, Glasgow G1 0NW, United Kingdom. Tel.: +44 7553093121. E-mail address: fi[email protected] (F. Laco).
regulate the proliferation and migration of cutaneous cells to reconstitute epidermis and facilitate wound healing during the early inflammatory phase [1–3]. Previous works suggest that there are several mechanisms in which BM-MSCs could participate in cutaneous wound healing of the epidermis. They could either differentiate into phenotypes of various damaged cells [4] and/or enhance repair by creating a microenvironment that promotes the local regeneration of cells endogenous to the tissue [5]. In recent studies, it was found that after homing to the injury sites, BM-MSCs are incorporated in the epidermis especially in the region of hair follicle suggesting they may be involved in skin repair and regeneration [6], and it was demonstrated that BM-MSCs are able to trans-differentiate into epithelia lineage cells [7,8]. Another important fact is that in response to injury, BM-MSCs are stimulated to increase the
0923-1811/$36.00 ß 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2009.05.009
F. Laco et al. / Journal of Dermatological Science 55 (2009) 150–160
expression of several crucial growth factors (e.g. transforming growth factor) for epidermal development to promote the healing process of the damaged tissue [9]. Furthermore, the strong interactions between MSCs and KERs within the epidermis indicate BM-MSCs have the potential to regenerate epidermis and facilitate skin repair under appropriate conditions [9,10]. However, the biological significance of BM-MSCs in skin regeneration has not been clearly defined. A simple way to investigate the mesenchymal–epidermal interactions is to use an organotypic co-culture (OC) model to mimic the morphological and functional properties of native skin [11]. In conventional OC model, the KERs are cultivated on a dermal substitute, which consists of a porous membrane that is seeded with dermal fibroblast-embedded collagen I matrix. The upper epidermal layers are then exposed to air by lowering down the culture medium to facilitate epidermalization and stratification [12]. In earlier studies, the OC model was used to investigate the crosstalk between KERs and fibroblasts in regulating KERs growth and differentiation [13]. Recently the influence of BM-MSCs in the dermal equivalent was investigated and they were found to demonstrate functions resembling those of dermal fibroblasts during wound healing process [14]. However, an investigation of BM-MSCs in epidermal development in vitro has not been done so far. In spite of its biomimetic construction and functions, the conventional OC model does not feature all aspects of the native skin and some abnormal patterns of protein expression in the neoepidermis were observed [15]. This lead to the assumption by Nobert E. Fusening in 1994 that homeostasis is not (yet) achieved in OCs [16]. To our knowledge, up to now, there is no OC model which demonstrates an expression profile of epidermal differ-
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entiation markers with a close similarity to native epidermis. Our aim was to investigate the interactions between KERs and cocultured BM-MSCs in the biomimetic OC model using morphometric and indirect immunodetection analysis. Variations of coculture ratios were used in this study to compare their expression patterns of several epidermal differentiation markers including involucrin, filaggrin and keratin 10. This study not only provides a better understanding of the role of BM-MSCs in regulating skin regeneration, but also put insights on the design of an artificially engineered microenvironment for skin graft development. 2. Results 2.1. Surface appearance of organotypic co-culture The OCs with KERs and MSCs ratios from 0% to 100% are shown in Fig. 1. The MSCs were visible due to their QDs labeling. The orange stain showed the endocytosed QDs within MSCs. Cells were evenly distributed over the whole dermal substitute in all groups, except the 50% MSCs group (Fig. 1b), which displayed a dotted surface on day 7. However, this dotted surface was not observed on the initial 3 days after cell seeding, indicating an active cell migration occurred and resulted in the formation of cell clusters on the dermal substitute. 2.2. Histological analysis On days 14 and 20, epidermal development of cellular stratification were seen in the experimental groups with 0% MSCs, 10% MSCs and 50% MSCs (Fig. 2), while no epidermal structures
Fig. 1. Organotypic co-culture with a mounting titanium ring showing KERs with MSCs on dermal substitute. Appearance of organotypic co-cultures groups on day 14 with different fractions of quantum dots-labeled MSCs (orange stain): (a) 10% MSCs, (b) 50% MSCs, (c) 90% MSCs, (d) 100% MSCs, and (e) 0% MSCs. The organotypic co-cultures with 50% MSC illustrates an orange dotted surface, which demonstrates the assembly of MSCs into cell clusters (b).
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Fig. 2. Stratification of different organotypic co-culture groups stained with hematoxylin and eosin on day 14 (a, c and e) and day 20 (b, d and f). a and b: group with 0% MSCs. c and d: group with 10% MSCs. e and f: group with 50% MSCs. Dark blue: cell nucleus. Purple: cytoplasm of neo-epidermis. White: dermal substitute. Arrow: cell cluster. Scale bars: a–b 50 mm; c–f 20 mm.
were demonstrated in 90% MSCs and 100% MSCs groups (data not shown). On day 14 no difference was observed between the groups of 0% MSCs and 10% MSCs (Fig. 2a–d), both of which showed the stratified epidermal structures with the distinctive morphology; the basal layer shows columnar arranged cells with their long axis at right angles to the dermo-epidermal junction. These cells have prominent elongated nucleus. The upward moving KERs form the spinous layer and show morphological changes as cell become flattened and decrease in size of the nucleus. Moreover the KERs appear prickly (‘spiny’) by the shrinkage of adherence at their desmosomal junctions. The granular layer is characterized by the loss of cell nucleus, and by the appearance of keratohyalin granules. This layer of somewhat flattened cells is lying just between the prickle cell layer and the stratum corneum of the epidermis. The stratum corneum (‘horny layer’) is the top layer of the epidermis resembling cornified (‘keratinized’) KERs. Cells here are flat and scale-like (‘squamous’) in shape. These cells are dead, contain a lot of keratin and are arranged in overlapping layers that impart a tough and waterproof character to the skin’s surface. The structure of the stratum corneum is described as a thin cornified envelope of a loose basket wave structure (Fig. 2b). The size of
these envelopes showed no significant difference in thickness (KERs: 130 mm 25 mm; 10% MSCs: 120 mm 25 mm, p > 0.05) (Fig. 2a and c). However, on day 20, the formation of stratum corneum and cornified envelope was enhanced in the KERs group compared with 10% MSCs group (Fig. 2b and d). And a statistic difference was detected in the thickness of stratum corneum between the two groups (KERs: 190 mm 25 mm; 10% MSCs: 140 mm 20 mm, p < 0.05), indicating the development of hypertrophic epidermis in the KERs group. On day 14, 50% MSCs group was characterized with an unequal distribution of cells over the surface of the dermal substitute, as represented by the formation of cell clusters (Fig. 2e). On day 20, a multilayer of neo-epidermis with stratified structures were developed to cover the dermal equivalent (Fig. 2f), however degenerated cellular clusters could still be seen either above the stratified layers (Fig. 5f) or between the neo-epidermis and dermal substitute (Fig. 6e–f). 2.3. Mesenchymal stem cell location within the neo-epidermis In the 10% MSCs group, accumulations of MSCs labeled by QDs were predominantly found at the stratum basale with a few single
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Fig. 3. The brightfield images of organotypic co-culture cross sections overlaid with the fluorescence image of the quantum dot-labeled MSCs (red stain) on day 14; (a) 10% MSCs group. It showed the incorporation of MSCs in neo-epidermis; (b) 50% MSCs group. It showed cell cluster formation of MSCs (red) and KERs and an overgrowth by keratinocytes. Scale bar: 20 mm.
MSCs being incorporated within the spinous and granular layers of neo-epidermal structure on day 14 (Fig. 3a). In contrast, the 50% MSCs group showed the QDs-labeled MSCs clumped together with KERs on day 14, which indicates both KERs and MSCs migrate towards each other, resulted in the formation of cell clusters or colonies (Fig. 3b). On day 20, the emerging neo-epidermal structure on top or beneath these cell clusters showed predominantly KERs with a few incorporated MSCs labeled by QDs (Fig. 5f).
cornified envelope from day 14 (Fig. 6c and Table 1). The 50% group shows no involucrin expression of the cell cluster (Fig. 6e), but in the neo-epidermis involucrin seems to be over-expressed (Fig. 6e– f). Altogether it suggested a neo-epidermis more characteristic of the native skin was better developed in OCs with the incorporation of MSCs at a low concentration (10%) than the conventional OCs culture with 0% MSCs (Table 1). 3. Discussion
2.4. Immunostaining analyses 3.1. Organotypic co-culture model for skin regeneration Three specific markers for epidermal differentiation stages: early (keratin 10), intermediate (filaggrin) and late (involucrin) were used to characterize the orchestrated process of skin regeneration temporally and spatially. The immunostaining analyses showed that keratin 10, filaggrin and involucrin were only expressed in the experimental groups with a stratified neoepidermis: groups of 0% MSCs, 10% MSCs and 50% MSCs (Figs. 4–6). The expression of these epidermalization markers was not seen in groups with 90% MSCs and 100% MSCs (data not shown). All experimental groups of OCs differ from the native skin in the expression location of epidermalization markers (Table 1). The early differentiation marker, keratin 10 was expressed in the suprabasal layers within the groups with 0% MSCs, 10% MSCs and 50% MSCs on days 14 and 20 (Fig. 4c–f). However, the 0% MSCs group showed a delayed expression of keratin 10 in the spinous layers until day 20 (Fig. 4a and b), while 10% MSCs group demonstrated a normal expression pattern of keratin 10 in all suprabasal layers resembling the native epidermis from day 14 (Fig. 4c and d). In OC cultures with 10% MSCs and 50% MSCs, a premature expression of filaggrin, an intermediate epidermal differentiation marker, was observed on days 14 and 20 compared with the 0% MSCs group, which shows an expression pattern close to the granular layer (Fig. 5). The co-localization of the expression of keratin 10 and filaggrin with QDs-labeled MSCs were observed in groups with 10% MSCs and 50% MSCs suggests under culture conditions on day 14, MSCs were able to express the early and intermediate epidermal differentiation markers (Figs. 4c and e, and 5c and e). Whereas, involucrin, the terminal epidermal differentiation marker, expressed in the cornified layers in 10% MSCs group on day 14, was very rarely co-localized with the QDs-labeled MSCs (Fig. 6c). Similarly, no involucrin was seen to be expressed by QDslabeled MSCs in 50% MSCs group (Fig. 6e). Furthermore, compared with native epidermis, involucrin was over-expressed in the suprabasal layers in the groups of 0% MSCs on days 14 and 20 (Fig. 6a and b), whereas 10% MSCs group demonstrated a normal expression profile of involucrin in the stratum corneum and
The OC is a well-established model to mimic the natural epidermal development in vitro [12,17]. It has continuously improved and contributed to the understanding of epithelial– mesenchymal interactions during epidermal regeneration. It was shown that a dystrophic epithelium was developed in OCs with KERs cultured alone on acellular dermal substitutes [18,19]. However, incorporation of fibroblasts in the dermal substitutes facilitate the formation of stratified epidermis in vitro [20,21], which suggests the crucial role of mesenchymal-interactions in epidermal development. Later it was found that dermal fibroblasts could be substituted with MSCs or preadipocytes to achieve a superior epidermal stratification [14,22,23]. In further OC experiments pure MSCs were cultured on top of fibroblast-embedded dermal substitutes showing the potential of MSCs to transdifferentiate into KERs lineage under specific conditions. However, only a pseudo epidermis can be achieved so far [24,25]. In this study, apical co-cultures of BM-MSCs and KERs at different ratios on conventional dermal substitutes were used to investigate the dose effect of MSCs on the epidermal development and homeostasis. It was found that a stratified epidermis with morphology and expression profile mimicking the native skin was established only if a small fraction of MSCs (10%) were incorporated in the epidermal layer. Contribution of epidermal regeneration from MSCs is also supported by another in vitro study where BM-MSCs were co-cultured with airway epithelial cells. It showed that MSCs adopted an airway epithelial phenotype whenever they are cocultured in low concentration (<25%) [26], which is consistent with our result. 3.2. Epidermal development and homeostasis regulated by MSCs The mature of epidermal regeneration and development is commonly shown by the expression of different types of keratin and collagen (IV and VII), involucrin, filaggrin, transglutaminase and loricrin. Expression of these markers in native skin is
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Fig. 4. Organotypic co-culture cross sections with indirect immunodetection of keratin 10 expression on day 14 (a, c and e) and day 20 (b, d and f). a and b: group with 0% MSCs. c and d: group with 10% MSCs. e and f: group with 50% MSCs. Green: keratin 10 expression. Red: quantum dot-labeled MSCs. MSCs were labeled by quantum dots for day 14. Scale bars: 50 mm.
temporally and spatially determined, indicating epidermal homeostasis of the skin. However, in OCs several markers are not expressed due to the simplicity of the model. For example, collagen VII is a marker of the basal membrane, but is rarely demonstrated in OCs [15,19]. Keratin 10 is an early differentiation marker expressed in the suprabasal layers and its expression was found to be delayed in OCs model [15,27]. Collagen IV is a marker expressed in dermo-epidermal junction [28]. Filaggrin is an intermediate marker of epidermal differentiation. It is dephosphorylated and synthesized in the stratum granulosum [11,27]. Involucrin is a
component of the cornified envelope and found in the cytoplasm. The cornified envelope is formed by cross-linking of precursor molecules such as involucrin and loricrin to membrane proteins by transglutaminase. As cells differentiate in culture, there is a successive increase in involucrin, transglutaminase, and cornified envelope formation [29]. These are the markers of KER terminal differentiation and involucrin mostly expressed in the cornified layers of stratified squamous epithelium [27,30,31]. It has been shown that undifferentiated MSCs express collagen IV [32] and several epidermal keratin transcripts like keratin 8, 18 and 10
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Fig. 5. Organotypic co-culture cross sections with indirect immunodetection of filaggrin expression on day 14 (a, c and e) and day 20 (b, d and f). a and b: group with 0% MSCs. c and d: group with 10% MSCs. e and f: group with 50% MSCs. Green: filaggrin expression. Red: quantum dot-labeled MSCs. MSCs were labeled by quantum dots for day 14. Scale bars: 50 mm.
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Fig. 6. Organotypic co-culture cross sections with indirect immunodetection of involucrin expression on day 14 (a, c and e) and day 20 (b, d and f). a and b: group with 0% MSCs. c and d: group with 10% MSCs. e and f: group with 50% MSCs. Green: involucrin expression. Red: quantum dot-labeled MSCs. MSCs were labeled by quantum dots for day 14. Scale bars: 50 mm.
[5,33] even though the expression of keratin 8 and 10 at a protein level has not been found in undifferentiated MSCs [5], which indicates under certain specific conditions, MSCs may enter a developmental program that would commit it to an epidermal cell fate. Previous study has shown that under certain biomimic conditions in OC model, human MSCs were able to transdifferentiate into epidermis-like cells and express keratin 10 and filaggrin [24], whereas keratin 10, filaggrin and involucrin were not expressed in undifferentiated human MSCs [24,25]. Therefore, in this study, we use the three temporally and spatially
specific markers—keratin 10, filaggrin and involucrin to characterize the KER differentiation stages and morphogenesis in different experimental groups. The potential of co-cultured group with 10% MSCs showed improved epidermal homeostasis in OC conditions after 20 days, compared with conventional 0% MSCs culture. This was demonstrated by the expression location of epidermal differentiation markers—keratin 10 and involucrin on days 14 and 20 (Table 1). We showed the abnormal expression pattern of keratin 10 and involucrin in 0% MSCs group, which is consistent with the results
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Table 1 The expression location of the neo-epidermis with keratin 10 (K), filaggrin (F), involucrin (I) and epidermal organization with H&E staining in different groups: native skin, 0% MSCs, 10% MSCs, 50% MSCs. Strong similarities were seen between the native skin and 10% MSCs group on days 14 and 20. Blue: Abnormal expression location of the epidermal differentiation markers compared with native skin; Green: Normal expression location of the epidermal differentiation markers compared with native skin.
observed by other groups [11,12,15,34]. Compared with native epidermis [15], the expression of keratin 10 was found to be delayed in 0% MSCs OCs and not expressed within the basal periphery of the epidermis after 14 days. Filaggrin as a differentiation marker for the granular layer [11,27], expressed in the upper layers of the granulosum in 0% MSCs OCs group, was found to be over-expressed in the suprabasal layers within MSCsincorporated OCs groups. Involucrin is abnormally found in all suprabasal layers in 0% MSCs group, but in native skin, it was expressed only in the cornified layers [11]. Altogether traditional OCs with 0% MSCs showed abnormal expression pattern compared to native skin in homeostasis. This lead to the assumption that homeostasis is not (yet) achieved in OCs by Fusenig [16]. In our experimental group with 10% MSCs, it was clearly shown that the expression patterns of several differentiation markers were similar to those of native skin; only filaggrin expression displayed a premature expression. The superiority of MSCs-incorporated OCs is shown in the keratin 10 expression on day 14. Keratin 10 was not observed in the spinous layer within 0% MSCs group, while it was clearly seen in 10% MSCs group, similar to its expression pattern in native skin of homeostasis. Moreover it was demonstrated that the over expression of the cornified envelope was reduced in the OC if KERs were cocultured with 10% MSCs. What is commonly found in 0% MSCs OCs models is an accumulation of corneocyte layers, the cornified envelope which is thought to be linked to a dysregulated lipid metabolism with impaired organization of lamellar bodies that demonstrate a reduced barrier function of the skin in vitro [15]. The mechanism that BM-MSCs regulate the skin regeneration and homeostasis in vitro can be addressed on different levels. First, It was shown that MSCs are able to trans-differentiate into epidermal lineage both in vitro and in vivo [2,3,7,24] within a skin-specific environment or the so-called ‘stem cell niche’ [2,35]. Second, MSCs was found to enhance skin repair by creating a microenvironment that promotes or contributes to the local regeneration of cells endogenous to the tissue [5]. It was shown that during wound healing process, BM-MSCs transdifferentiated into multiple cell types including preadipocytes, endothelial and mesenchymal cell types besides the KER lineage. This is explained as natural wound area is a complex environment with various signals that can trigger MSCs differentiation into many lineages [4]. Further those preadipocytes, endothelial and mesenchymal cells were shown to benefit epidermal constitution in OCs model with paracrine interactions by cytokine and growth factor release [9,18,22]. Therefore, in our OC model which includes media supplements for adipogenesis [36] and osteogenesis [37], we do not rule out the possibilities that MSCs improve epidermal development by adopting other
phenotypes and excreting some growth factors for skin regeneration. Last, it was found that BM-MSCs could be transdifferentiated into cells with the phenotype of its co-cultured cells via juxtacrine interactions [38]. Another study also showed that the direct contact between KERs and BM-MSCs is required for the skin-specific morphogenesis, like the formation of rete ridge structure through a mechanism that differ from IL-1a/c-Jun pathway [22]. Although it is not clear whether BM-MSCs exclusively trans-differentiate into epidermal lineage, the OCs in our experiment demonstrate a superior structure and protein expression profile compared with the conventional OCs which do lack of the juxtacrine interactions [11,13,15,18–23,34]. Therefore our findings suggest that the direct ‘‘cell–cell’’ mesenchymal– epidermal interactions of KERs with MSCs at a small concentration (10%) helps to regulate the development and organization of the neo-epidermis and lead to an improved epidermal homeostasis in vitro. Additionally, this study also suggests that it may be an alternative approach to design novel skin grafts by including a low quantity of BM-MSCs for future clinical application. 3.3. Mesenchymal–epidermal interactions The MSCs benefit for epidermal development and homeostasis is dose-dependent. One reason for impaired epidermal development in higher MSCs concentration (e.g. 50% MSCs) is the strong chemotactic interaction between each other. Akino et al. [10] showed MSCs would migrate with a significantly higher affinity towards KERs than towards fibroblasts and endothelia cells. In monolayer co-culture, MSCs was found to be elongated and adhere to human epidermal KERs through a basement membrane-like structure built between each other [10]. In our experiment, these chemotactic effects were demonstrated by the formation of cell clusters or colonies in co-cultured group with 50% MSCs on day 14. It suggests that the attractions between MSCs and KERs were so strong that MSCs could aggregate KERs from the whole surface of the dermal equivalent, and therefore prevented a proper epidermalization. However, we did not observe an initial cell cluster formation on day 1 (data not shown), which indicates an active migration of KERs and MSCs towards each other occurred over time. Further observations on day 20 showed an outgrowth of an epidermal layer out of this colony structure towards a stratified neo-epidermis. It could be either KERs migrating out of the cellcolonies or a single non-colonial KER forming a neo-epidermis. However, with a very high MSCs concentration (90%), a similar epidermal outgrowth could not be observed, which suggests that KERs were anchored by such a strong interaction with a rich population of MSCs that an impaired epidermalization was resulted. Hence the role of MSCs in epidermal development is
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dose-depended. KERs incorporated with a low fraction of MSCs displayed different cellular interactions, which led to the development of a proper neo-epidermis, whereas KERs co-cultured with a higher concentration of MSCs resulted the formation of mixed cell-colonies with blocked epidermalization. 3.4. Quantum dots indicate MSCs location and role in the epidermal structure Quantum dots (QDs) are semiconductor nanoparticles, which showed a narrow band emission and broad band excitation with a high quantum yield, high photostability, luminescence and resistance to chemical and metabolically degeneration. QDs are endocytosed by MSCs and distributed in the cytosol and aggregates are found in the endosomal vesicles around the cell nucleus. The QDs are inherited by daughter cells for at least 6 generations (15days), and show limited cytotoxicity over a 10 nM concentration [39]. QDs are not exocytosed from the endosomal vesicles in live cells, and phagocytosis of dead QDs-labeled MSCs by other co-cultured cells was not found in mixed culture studies. However during cell passaging or mechanical disturbance, QD-positive dead cells can disintegrate and free QD will be re-endocytosed by the surrounding cells, which was shown as single QD events (supplement 1). Therefore, in our experiment, single QD events in cells were not considered as an identification marker for MSC. Only multiple QD events in cells are representative markers for locating QD-labeled MSCs. Furthermore, cell-fusion is an extremely rare event for BM-MSCs to adopt epidermal phenotype. Many studies using the Cre/lox system together with b-galactosidase and fluorescence in situ hybridization (FISH) analysis, found that BM-MSCs differentiate into epithelial cells in the skin, liver, and lungs in vivo without cell-fusion [1,4,8]. Therefore, in this study, we use QD to label and locate the co-cultured MSCs in OC model. The QDs-labeled MSCs clearly showed the participation of MSCs within different epidermal layers on day 14. A high concentration of QDs can be found at the basal periphery of the 10% MSCs group (Figs. 4c, 5c and 6c), and single QDs are located at the upper granular layers of this group (Figs. 4c, 5c and 6c). Although it showed single QDs events accumulated in the upper granular layers, it is not representative for a true QD-labeled MSCs, thus hardly to confirm the contribution of MSCs to this upper granular layer. The involucrin-expressed cells in the upper granular and cornified layers are responsible for the barrier function of the skin. However, the expression of involucrin by MSCs was not shown in this study (Fig. 6c and e), which is coherent with our previous findings [24]. This indicates a strong participation of MSCs in the basal periphery only and no involvement in the barrier function of the epidermis. Moreover, we observed a few concentrated MSCs in the uppermost cornified envelope in the 10% MSCs group (Figs. 4c, 5c and 6c). These QDs-labeled MSCs represented most likely dead cells accumulated after seeding and air-lift. One possible reason is the direct contact of the uppermost MSCs to the external dry environment induces apoptosis in many MSCs. Therefore, during the epidermal development from day 14 to day 20, dead cells were pushed out and accumulated in the uppermost cornified envelope (Figs. 4c, 5c and 6c), whereas only the population of MSCs near the basal layer were able to survive within the wet–dry environment at the air–liquid interface.
impaired epidermalization. KERs cultured with a low concentration of MSCs (10%) demonstrated a proper epidermal structure organization and expression pattern of keratin 10 and involucrin resembling those of the native skin in homeostasis. To our knowledge, this OCs group with 10% MSCs developed a tissue architecture and distribution of differentiation markers recapitulating the situation closely to native epidermis and demonstrated a superior performance compared with any previously reported epidermal OC models. Even though the role of MSCs in epidermal development remains unclear, the co-localization of MSCs with KER and expression of distinctive KER makers in these co-cultured groups strongly supports a regulatory role of MSCs in epidermal tissue and a possible differentiation potential of MSCs into epidermal lineage. 5. Materials and methods 5.1. Materials Human BM-MSCs were ordered from Cambrex Bio Science Walkersville, Inc., USA. Human dermal fibroblasts, Human KERs, keratinocytes medium with defined growth supplements, fetal bovine serum (FBS), low glucose Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12 medium, Quantum Dots (QDs)Qtracker655 Cell labeling Kit, penicillin–streptomycin solution, amphotericin, 40 ,6-diamidino-2-phenylindole, dihydrochloride (DAPI) were all purchased from Invitrogen Corporation, USA. PureColTM collagen was ordered from INAMED Corporation, USA. LGlutamine, ascorbic acid, epidermal growth factor (EGF), 1,25dihydroxyvitamin D3 (VD3), hydrocortisone, insulin, 3,30 ,5-triiodoL-thyronine sodium (T3), mouse anti-human pan-keratin antibody (Ab), mouse anti-human invulocrin Ab and goat anti-mouse IgG FITC-conjugated secondary Ab were purchased from Sigma– Aldrich Pte Ltd., Singapore. Mouse anti-human keratin 10 and filaggrin antibodies were ordered from Thermo Fisher Scientific Inc, USA. Organotypic co-culture devices (12 mm Transwell1) were purchased from Corning Incorporated, USA. 5.2. Cell culture Human dermal fibroblasts and BM-MSCs were cultured in low glucose DMEM supplemented with 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin. Human KERs were grown in keratinocyte medium with defined growth supplements, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin. Human dermal fibroblasts, MSCs and KERs were all grown at standard culture conditions of 5% CO2 at 37 8C in a sterile humidified incubator. Media were changed every 2–3 days and cells were passaged at 70% confluent. 5.3. MSCs labeling with quantum dots MSCs of passage 6–8 at 90% confluence in T-175 cell culture flasks were incubated with 10 nmol/l quantum dots (QDs) in 2 ml cell culture medium for 1 h. Thereafter cells were washed with phosphate-buffered saline. Adherent cells were detached by trypsin, counted with a Thoma hematocytometer and seeded onto the dermal substitute.
4. Conclusion
5.4. Organotypic co-culture
The benefit of maintaining homeostasis during epidermal development from human BM-MSCs is in a dose-dependent matter. Organotypic co-cultures (OCs) of MSCs and KERs at 1:1 ratio had a strong chemotactic interaction to form separate cellular colonies, which impeded the KERs spreading and resulted in an
The dermal substitute was generated according to Vaccariello et al., 1994. Briefly, cold collagen type I solution was mixed with 10 DMEM solution, 10% FBS, 200 mM L-glutamine solution and 72 g/l sodium bicarbonate solution on ice. The pH was adjusted with a 1 M NaOH solution to 7.4. The solution was poured into filter
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inserts of the OC device forming the acellular gel layer. For gelation, the collagen solution in filter inserts was incubated for 1–2 h at 37 8C in a humidified incubator. Thereafter the cellular layer was prepared. The previous solution composition was blended with fibroblasts (100,000 per well). The solution was then poured onto the acellular layer. For gelation, the collagen solution in filter inserts was incubated for 2–4 h at 37 8C in a humidified incubator. The dermal substitute of acellular and cellular layer was cultured within DMEM supplemented with 10% FBS and 50 mg/ml ascorbic acid, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin under culture conditions. After 7 days, a titanium ring was placed onto the dermal substitute and gently pushed down by sterile forceps in order to prevent further contraction and confine the area for KERs and MSCs seeding. Thereafter the medium was aspirated and KERs as well as MSCs were seeded inside the titanium ring at the following concentrations: (a) 5 105 KERs (group with 0% MSCs); (b) 0.5 105 MSCs with 4.5 105 KERs (group with 10% MSCs); (c) 2.5 105 MSCs with 2.5 105 KERs; (group with 50% MSCs); (d) 4.5 105 MSCs with 0.5 105 KERs (group with 90% MSCs); (e) 5 105 MSCs (group with 100% MSCs). The OC were cultured within DMEM and Ham’s F12 (3:1) supplemented with 10% FBS, 10 ng/ml EGF, 5 mg/ml insulin, 0.4 mg/ml hydrocortisone, 1 nM triiodo-L-thyronine and 1 ng/ml VD3, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin under standard culture conditions. After 24 h, KERs and/or MSCs were exposed to the air by lowering the culture medium down to the dermal substitute surface level. The OC was then maintained under standard culture conditions with daily medium change. On days 14 and 20, samples were then fixed and processed for histoimmunological analyses. 5.5. Histological and immunological analyses For morphometric and histoimmunological analysis, the OC were first fixed in 2.5% formaldehyde over night and covered by a drop of 2% hand-warm agarose to prevent dislodgement of the epithelium during further processing procedures. Then the whole specimen was processed for paraffin embedding following standard protocols. Serial 10 mm paraffin sections were cut with a rotating microtome (MICROM), followed by hematoxylin and eosin (H&E) staining according to routine histology protocols. For indirect immunostaining analysis, the antigen retrieval was performed by pretreatment of the samples in 10 mM Citrate Buffer (pH 6.0) at 95 8C for 20 min and cooling down the samples at room temperature for 20 min. The primary antibodies against keratin 10, filaggrin and invulocrin were then incubated, followed by the incubation of FITC-conjugated secondary Ab. The H&E stained samples were observed under a phase-contrast microscope (Leica DM IRB, ebq 100). Samples with Ab incubation and QDs labeling were observed under a confocal laser microscope (FV 500, Olympus). 5.6. Statistical analysis Values (at least triplicate) were averaged and expressed as means standard deviation (SD). Each experiment was repeated five times. Statistical differences were determined by Student two-sample t test. Differences were considered statistically significant at p < 0.05. Acknowledgements This work was performed at the Healthcare and Energy Materials Laboratory, National University of Singapore, Singapore 117576. This work was supported by R39700036112, National University of Singapore, Ministry of Education, and MINDEF-NUSJPP/07/09, Singapore.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jdermsci.2009.05.009.
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Journal of Dermatological Science journal homepage: www.intl.elsevierhealth.com/journals/jods
The dose effect of human bone marrow-derived mesenchymal stem cells on epidermal development in organotypic co-culture Filip Laco a,d,*, Ma Kun b, Hans Joachim Weber a, S. Ramakrishna c,d, Casey K. Chan e,f a
Department of Medical Technology and Technomathematics, University of Applied Sciences, Aachen, Germany Graduate Program in Bioengineering, National University of Singapore, Singapore 117576, Singapore c Department of Mechanical Engineering, National University of Singapore, Singapore 117576, Singapore d Nanoscience and Nanotechnology Initiative, National University of Singapore, Singapore 117576, Singapore e Department of Bioengineering, National University of Singapore, Singapore 117576, Singapore f Department of Orthopedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074, Singapore b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 January 2009 Received in revised form 12 May 2009 Accepted 29 May 2009
Background: A wealth of evidences have shown the participation and benefits of bone marrow-derived mesenchymal stem cells (BM-MSCs) in wound healing and skin tissue repair in vivo. However, their role in epidermal development and reconstitution is not clearly investigated. Objective: Here we examine the quantitative effect of human BM-MSCs on epidermal regeneration in vitro. Method: Human keratinocytes and BM-MSCs are cultured at ratios from 0% to 100% on top of a fibroblast-embedded collagen gel in a three-dimensional organotypic co-culture model at an air–liquid interface up to 20 days and analyzed by histochemical and immunochemical staining of filaggrin, involucrin and keratin 10 on days 14 and 20. Human BM-MSCs were tracked with quantum dots in organotypic co-cultures. Results: It was found that epidermal development is strongly influenced by the percentage of cocultured BM-MSCs. A strong chemotactic effect between keratinocytes and MSCs was seen in the group with 50% of BM-MSCs, which resulted in an impaired epidermal development, whereas at a low percentage of BM-MSCs (10%), a stratified epidermal structure resembling native skin was established on day 14 of culture. Moreover, the immunostaining studies revealed that BM-MSCs in the low percentage (10%) participated in the basal periphery of reconstructed epidermis and a similar pattern characteristic of native epidermis was demonstrated in this experimental group, which was superior to all other experimental groups in terms of the thickness of stratum corneum and the expression profile of epidermal differentiation markers. Conclusion: This study indicates the advantage of using a new skin equivalent model incorporating a small fraction of MSCs to develop biologically useful tissues for maintaining homeostasis during skin regeneration and wound healing process. ß 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Mesenchymal stem cells Organotypic Epidermis Skin regeneration
1. Introduction After skin tissue injury, multipotent progenitor cells like mesenchymal stem cells (MSCs) are mobilized from bone marrow into the pool of circulating cells. These cells then home to the sites of injury, where they differentiate into keratinocytes (KERs) and
Abbreviations: BM, bone marrow; MSCs, mesenchymal stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells; KERs, keratinocytes; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; OC, organotypic co-culture; QDs, quantum dots; EGF, epidermal growth factor; VD3, 1,25 dihydroxyvitamin D3; 3D, three-dimensional; H&E, hematoxylin and eosin. * Corresponding author at: University of Strathclyde, Wolfson Center, 106 Rottenrow East, Glasgow G1 0NW, United Kingdom. Tel.: +44 7553093121. E-mail address: fi[email protected] (F. Laco).
regulate the proliferation and migration of cutaneous cells to reconstitute epidermis and facilitate wound healing during the early inflammatory phase [1–3]. Previous works suggest that there are several mechanisms in which BM-MSCs could participate in cutaneous wound healing of the epidermis. They could either differentiate into phenotypes of various damaged cells [4] and/or enhance repair by creating a microenvironment that promotes the local regeneration of cells endogenous to the tissue [5]. In recent studies, it was found that after homing to the injury sites, BM-MSCs are incorporated in the epidermis especially in the region of hair follicle suggesting they may be involved in skin repair and regeneration [6], and it was demonstrated that BM-MSCs are able to trans-differentiate into epithelia lineage cells [7,8]. Another important fact is that in response to injury, BM-MSCs are stimulated to increase the
0923-1811/$36.00 ß 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2009.05.009
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expression of several crucial growth factors (e.g. transforming growth factor) for epidermal development to promote the healing process of the damaged tissue [9]. Furthermore, the strong interactions between MSCs and KERs within the epidermis indicate BM-MSCs have the potential to regenerate epidermis and facilitate skin repair under appropriate conditions [9,10]. However, the biological significance of BM-MSCs in skin regeneration has not been clearly defined. A simple way to investigate the mesenchymal–epidermal interactions is to use an organotypic co-culture (OC) model to mimic the morphological and functional properties of native skin [11]. In conventional OC model, the KERs are cultivated on a dermal substitute, which consists of a porous membrane that is seeded with dermal fibroblast-embedded collagen I matrix. The upper epidermal layers are then exposed to air by lowering down the culture medium to facilitate epidermalization and stratification [12]. In earlier studies, the OC model was used to investigate the crosstalk between KERs and fibroblasts in regulating KERs growth and differentiation [13]. Recently the influence of BM-MSCs in the dermal equivalent was investigated and they were found to demonstrate functions resembling those of dermal fibroblasts during wound healing process [14]. However, an investigation of BM-MSCs in epidermal development in vitro has not been done so far. In spite of its biomimetic construction and functions, the conventional OC model does not feature all aspects of the native skin and some abnormal patterns of protein expression in the neoepidermis were observed [15]. This lead to the assumption by Nobert E. Fusening in 1994 that homeostasis is not (yet) achieved in OCs [16]. To our knowledge, up to now, there is no OC model which demonstrates an expression profile of epidermal differ-
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entiation markers with a close similarity to native epidermis. Our aim was to investigate the interactions between KERs and cocultured BM-MSCs in the biomimetic OC model using morphometric and indirect immunodetection analysis. Variations of coculture ratios were used in this study to compare their expression patterns of several epidermal differentiation markers including involucrin, filaggrin and keratin 10. This study not only provides a better understanding of the role of BM-MSCs in regulating skin regeneration, but also put insights on the design of an artificially engineered microenvironment for skin graft development. 2. Results 2.1. Surface appearance of organotypic co-culture The OCs with KERs and MSCs ratios from 0% to 100% are shown in Fig. 1. The MSCs were visible due to their QDs labeling. The orange stain showed the endocytosed QDs within MSCs. Cells were evenly distributed over the whole dermal substitute in all groups, except the 50% MSCs group (Fig. 1b), which displayed a dotted surface on day 7. However, this dotted surface was not observed on the initial 3 days after cell seeding, indicating an active cell migration occurred and resulted in the formation of cell clusters on the dermal substitute. 2.2. Histological analysis On days 14 and 20, epidermal development of cellular stratification were seen in the experimental groups with 0% MSCs, 10% MSCs and 50% MSCs (Fig. 2), while no epidermal structures
Fig. 1. Organotypic co-culture with a mounting titanium ring showing KERs with MSCs on dermal substitute. Appearance of organotypic co-cultures groups on day 14 with different fractions of quantum dots-labeled MSCs (orange stain): (a) 10% MSCs, (b) 50% MSCs, (c) 90% MSCs, (d) 100% MSCs, and (e) 0% MSCs. The organotypic co-cultures with 50% MSC illustrates an orange dotted surface, which demonstrates the assembly of MSCs into cell clusters (b).
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Fig. 2. Stratification of different organotypic co-culture groups stained with hematoxylin and eosin on day 14 (a, c and e) and day 20 (b, d and f). a and b: group with 0% MSCs. c and d: group with 10% MSCs. e and f: group with 50% MSCs. Dark blue: cell nucleus. Purple: cytoplasm of neo-epidermis. White: dermal substitute. Arrow: cell cluster. Scale bars: a–b 50 mm; c–f 20 mm.
were demonstrated in 90% MSCs and 100% MSCs groups (data not shown). On day 14 no difference was observed between the groups of 0% MSCs and 10% MSCs (Fig. 2a–d), both of which showed the stratified epidermal structures with the distinctive morphology; the basal layer shows columnar arranged cells with their long axis at right angles to the dermo-epidermal junction. These cells have prominent elongated nucleus. The upward moving KERs form the spinous layer and show morphological changes as cell become flattened and decrease in size of the nucleus. Moreover the KERs appear prickly (‘spiny’) by the shrinkage of adherence at their desmosomal junctions. The granular layer is characterized by the loss of cell nucleus, and by the appearance of keratohyalin granules. This layer of somewhat flattened cells is lying just between the prickle cell layer and the stratum corneum of the epidermis. The stratum corneum (‘horny layer’) is the top layer of the epidermis resembling cornified (‘keratinized’) KERs. Cells here are flat and scale-like (‘squamous’) in shape. These cells are dead, contain a lot of keratin and are arranged in overlapping layers that impart a tough and waterproof character to the skin’s surface. The structure of the stratum corneum is described as a thin cornified envelope of a loose basket wave structure (Fig. 2b). The size of
these envelopes showed no significant difference in thickness (KERs: 130 mm 25 mm; 10% MSCs: 120 mm 25 mm, p > 0.05) (Fig. 2a and c). However, on day 20, the formation of stratum corneum and cornified envelope was enhanced in the KERs group compared with 10% MSCs group (Fig. 2b and d). And a statistic difference was detected in the thickness of stratum corneum between the two groups (KERs: 190 mm 25 mm; 10% MSCs: 140 mm 20 mm, p < 0.05), indicating the development of hypertrophic epidermis in the KERs group. On day 14, 50% MSCs group was characterized with an unequal distribution of cells over the surface of the dermal substitute, as represented by the formation of cell clusters (Fig. 2e). On day 20, a multilayer of neo-epidermis with stratified structures were developed to cover the dermal equivalent (Fig. 2f), however degenerated cellular clusters could still be seen either above the stratified layers (Fig. 5f) or between the neo-epidermis and dermal substitute (Fig. 6e–f). 2.3. Mesenchymal stem cell location within the neo-epidermis In the 10% MSCs group, accumulations of MSCs labeled by QDs were predominantly found at the stratum basale with a few single
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Fig. 3. The brightfield images of organotypic co-culture cross sections overlaid with the fluorescence image of the quantum dot-labeled MSCs (red stain) on day 14; (a) 10% MSCs group. It showed the incorporation of MSCs in neo-epidermis; (b) 50% MSCs group. It showed cell cluster formation of MSCs (red) and KERs and an overgrowth by keratinocytes. Scale bar: 20 mm.
MSCs being incorporated within the spinous and granular layers of neo-epidermal structure on day 14 (Fig. 3a). In contrast, the 50% MSCs group showed the QDs-labeled MSCs clumped together with KERs on day 14, which indicates both KERs and MSCs migrate towards each other, resulted in the formation of cell clusters or colonies (Fig. 3b). On day 20, the emerging neo-epidermal structure on top or beneath these cell clusters showed predominantly KERs with a few incorporated MSCs labeled by QDs (Fig. 5f).
cornified envelope from day 14 (Fig. 6c and Table 1). The 50% group shows no involucrin expression of the cell cluster (Fig. 6e), but in the neo-epidermis involucrin seems to be over-expressed (Fig. 6e– f). Altogether it suggested a neo-epidermis more characteristic of the native skin was better developed in OCs with the incorporation of MSCs at a low concentration (10%) than the conventional OCs culture with 0% MSCs (Table 1). 3. Discussion
2.4. Immunostaining analyses 3.1. Organotypic co-culture model for skin regeneration Three specific markers for epidermal differentiation stages: early (keratin 10), intermediate (filaggrin) and late (involucrin) were used to characterize the orchestrated process of skin regeneration temporally and spatially. The immunostaining analyses showed that keratin 10, filaggrin and involucrin were only expressed in the experimental groups with a stratified neoepidermis: groups of 0% MSCs, 10% MSCs and 50% MSCs (Figs. 4–6). The expression of these epidermalization markers was not seen in groups with 90% MSCs and 100% MSCs (data not shown). All experimental groups of OCs differ from the native skin in the expression location of epidermalization markers (Table 1). The early differentiation marker, keratin 10 was expressed in the suprabasal layers within the groups with 0% MSCs, 10% MSCs and 50% MSCs on days 14 and 20 (Fig. 4c–f). However, the 0% MSCs group showed a delayed expression of keratin 10 in the spinous layers until day 20 (Fig. 4a and b), while 10% MSCs group demonstrated a normal expression pattern of keratin 10 in all suprabasal layers resembling the native epidermis from day 14 (Fig. 4c and d). In OC cultures with 10% MSCs and 50% MSCs, a premature expression of filaggrin, an intermediate epidermal differentiation marker, was observed on days 14 and 20 compared with the 0% MSCs group, which shows an expression pattern close to the granular layer (Fig. 5). The co-localization of the expression of keratin 10 and filaggrin with QDs-labeled MSCs were observed in groups with 10% MSCs and 50% MSCs suggests under culture conditions on day 14, MSCs were able to express the early and intermediate epidermal differentiation markers (Figs. 4c and e, and 5c and e). Whereas, involucrin, the terminal epidermal differentiation marker, expressed in the cornified layers in 10% MSCs group on day 14, was very rarely co-localized with the QDs-labeled MSCs (Fig. 6c). Similarly, no involucrin was seen to be expressed by QDslabeled MSCs in 50% MSCs group (Fig. 6e). Furthermore, compared with native epidermis, involucrin was over-expressed in the suprabasal layers in the groups of 0% MSCs on days 14 and 20 (Fig. 6a and b), whereas 10% MSCs group demonstrated a normal expression profile of involucrin in the stratum corneum and
The OC is a well-established model to mimic the natural epidermal development in vitro [12,17]. It has continuously improved and contributed to the understanding of epithelial– mesenchymal interactions during epidermal regeneration. It was shown that a dystrophic epithelium was developed in OCs with KERs cultured alone on acellular dermal substitutes [18,19]. However, incorporation of fibroblasts in the dermal substitutes facilitate the formation of stratified epidermis in vitro [20,21], which suggests the crucial role of mesenchymal-interactions in epidermal development. Later it was found that dermal fibroblasts could be substituted with MSCs or preadipocytes to achieve a superior epidermal stratification [14,22,23]. In further OC experiments pure MSCs were cultured on top of fibroblast-embedded dermal substitutes showing the potential of MSCs to transdifferentiate into KERs lineage under specific conditions. However, only a pseudo epidermis can be achieved so far [24,25]. In this study, apical co-cultures of BM-MSCs and KERs at different ratios on conventional dermal substitutes were used to investigate the dose effect of MSCs on the epidermal development and homeostasis. It was found that a stratified epidermis with morphology and expression profile mimicking the native skin was established only if a small fraction of MSCs (10%) were incorporated in the epidermal layer. Contribution of epidermal regeneration from MSCs is also supported by another in vitro study where BM-MSCs were co-cultured with airway epithelial cells. It showed that MSCs adopted an airway epithelial phenotype whenever they are cocultured in low concentration (<25%) [26], which is consistent with our result. 3.2. Epidermal development and homeostasis regulated by MSCs The mature of epidermal regeneration and development is commonly shown by the expression of different types of keratin and collagen (IV and VII), involucrin, filaggrin, transglutaminase and loricrin. Expression of these markers in native skin is
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Fig. 4. Organotypic co-culture cross sections with indirect immunodetection of keratin 10 expression on day 14 (a, c and e) and day 20 (b, d and f). a and b: group with 0% MSCs. c and d: group with 10% MSCs. e and f: group with 50% MSCs. Green: keratin 10 expression. Red: quantum dot-labeled MSCs. MSCs were labeled by quantum dots for day 14. Scale bars: 50 mm.
temporally and spatially determined, indicating epidermal homeostasis of the skin. However, in OCs several markers are not expressed due to the simplicity of the model. For example, collagen VII is a marker of the basal membrane, but is rarely demonstrated in OCs [15,19]. Keratin 10 is an early differentiation marker expressed in the suprabasal layers and its expression was found to be delayed in OCs model [15,27]. Collagen IV is a marker expressed in dermo-epidermal junction [28]. Filaggrin is an intermediate marker of epidermal differentiation. It is dephosphorylated and synthesized in the stratum granulosum [11,27]. Involucrin is a
component of the cornified envelope and found in the cytoplasm. The cornified envelope is formed by cross-linking of precursor molecules such as involucrin and loricrin to membrane proteins by transglutaminase. As cells differentiate in culture, there is a successive increase in involucrin, transglutaminase, and cornified envelope formation [29]. These are the markers of KER terminal differentiation and involucrin mostly expressed in the cornified layers of stratified squamous epithelium [27,30,31]. It has been shown that undifferentiated MSCs express collagen IV [32] and several epidermal keratin transcripts like keratin 8, 18 and 10
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Fig. 5. Organotypic co-culture cross sections with indirect immunodetection of filaggrin expression on day 14 (a, c and e) and day 20 (b, d and f). a and b: group with 0% MSCs. c and d: group with 10% MSCs. e and f: group with 50% MSCs. Green: filaggrin expression. Red: quantum dot-labeled MSCs. MSCs were labeled by quantum dots for day 14. Scale bars: 50 mm.
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Fig. 6. Organotypic co-culture cross sections with indirect immunodetection of involucrin expression on day 14 (a, c and e) and day 20 (b, d and f). a and b: group with 0% MSCs. c and d: group with 10% MSCs. e and f: group with 50% MSCs. Green: involucrin expression. Red: quantum dot-labeled MSCs. MSCs were labeled by quantum dots for day 14. Scale bars: 50 mm.
[5,33] even though the expression of keratin 8 and 10 at a protein level has not been found in undifferentiated MSCs [5], which indicates under certain specific conditions, MSCs may enter a developmental program that would commit it to an epidermal cell fate. Previous study has shown that under certain biomimic conditions in OC model, human MSCs were able to transdifferentiate into epidermis-like cells and express keratin 10 and filaggrin [24], whereas keratin 10, filaggrin and involucrin were not expressed in undifferentiated human MSCs [24,25]. Therefore, in this study, we use the three temporally and spatially
specific markers—keratin 10, filaggrin and involucrin to characterize the KER differentiation stages and morphogenesis in different experimental groups. The potential of co-cultured group with 10% MSCs showed improved epidermal homeostasis in OC conditions after 20 days, compared with conventional 0% MSCs culture. This was demonstrated by the expression location of epidermal differentiation markers—keratin 10 and involucrin on days 14 and 20 (Table 1). We showed the abnormal expression pattern of keratin 10 and involucrin in 0% MSCs group, which is consistent with the results
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Table 1 The expression location of the neo-epidermis with keratin 10 (K), filaggrin (F), involucrin (I) and epidermal organization with H&E staining in different groups: native skin, 0% MSCs, 10% MSCs, 50% MSCs. Strong similarities were seen between the native skin and 10% MSCs group on days 14 and 20. Blue: Abnormal expression location of the epidermal differentiation markers compared with native skin; Green: Normal expression location of the epidermal differentiation markers compared with native skin.
observed by other groups [11,12,15,34]. Compared with native epidermis [15], the expression of keratin 10 was found to be delayed in 0% MSCs OCs and not expressed within the basal periphery of the epidermis after 14 days. Filaggrin as a differentiation marker for the granular layer [11,27], expressed in the upper layers of the granulosum in 0% MSCs OCs group, was found to be over-expressed in the suprabasal layers within MSCsincorporated OCs groups. Involucrin is abnormally found in all suprabasal layers in 0% MSCs group, but in native skin, it was expressed only in the cornified layers [11]. Altogether traditional OCs with 0% MSCs showed abnormal expression pattern compared to native skin in homeostasis. This lead to the assumption that homeostasis is not (yet) achieved in OCs by Fusenig [16]. In our experimental group with 10% MSCs, it was clearly shown that the expression patterns of several differentiation markers were similar to those of native skin; only filaggrin expression displayed a premature expression. The superiority of MSCs-incorporated OCs is shown in the keratin 10 expression on day 14. Keratin 10 was not observed in the spinous layer within 0% MSCs group, while it was clearly seen in 10% MSCs group, similar to its expression pattern in native skin of homeostasis. Moreover it was demonstrated that the over expression of the cornified envelope was reduced in the OC if KERs were cocultured with 10% MSCs. What is commonly found in 0% MSCs OCs models is an accumulation of corneocyte layers, the cornified envelope which is thought to be linked to a dysregulated lipid metabolism with impaired organization of lamellar bodies that demonstrate a reduced barrier function of the skin in vitro [15]. The mechanism that BM-MSCs regulate the skin regeneration and homeostasis in vitro can be addressed on different levels. First, It was shown that MSCs are able to trans-differentiate into epidermal lineage both in vitro and in vivo [2,3,7,24] within a skin-specific environment or the so-called ‘stem cell niche’ [2,35]. Second, MSCs was found to enhance skin repair by creating a microenvironment that promotes or contributes to the local regeneration of cells endogenous to the tissue [5]. It was shown that during wound healing process, BM-MSCs transdifferentiated into multiple cell types including preadipocytes, endothelial and mesenchymal cell types besides the KER lineage. This is explained as natural wound area is a complex environment with various signals that can trigger MSCs differentiation into many lineages [4]. Further those preadipocytes, endothelial and mesenchymal cells were shown to benefit epidermal constitution in OCs model with paracrine interactions by cytokine and growth factor release [9,18,22]. Therefore, in our OC model which includes media supplements for adipogenesis [36] and osteogenesis [37], we do not rule out the possibilities that MSCs improve epidermal development by adopting other
phenotypes and excreting some growth factors for skin regeneration. Last, it was found that BM-MSCs could be transdifferentiated into cells with the phenotype of its co-cultured cells via juxtacrine interactions [38]. Another study also showed that the direct contact between KERs and BM-MSCs is required for the skin-specific morphogenesis, like the formation of rete ridge structure through a mechanism that differ from IL-1a/c-Jun pathway [22]. Although it is not clear whether BM-MSCs exclusively trans-differentiate into epidermal lineage, the OCs in our experiment demonstrate a superior structure and protein expression profile compared with the conventional OCs which do lack of the juxtacrine interactions [11,13,15,18–23,34]. Therefore our findings suggest that the direct ‘‘cell–cell’’ mesenchymal– epidermal interactions of KERs with MSCs at a small concentration (10%) helps to regulate the development and organization of the neo-epidermis and lead to an improved epidermal homeostasis in vitro. Additionally, this study also suggests that it may be an alternative approach to design novel skin grafts by including a low quantity of BM-MSCs for future clinical application. 3.3. Mesenchymal–epidermal interactions The MSCs benefit for epidermal development and homeostasis is dose-dependent. One reason for impaired epidermal development in higher MSCs concentration (e.g. 50% MSCs) is the strong chemotactic interaction between each other. Akino et al. [10] showed MSCs would migrate with a significantly higher affinity towards KERs than towards fibroblasts and endothelia cells. In monolayer co-culture, MSCs was found to be elongated and adhere to human epidermal KERs through a basement membrane-like structure built between each other [10]. In our experiment, these chemotactic effects were demonstrated by the formation of cell clusters or colonies in co-cultured group with 50% MSCs on day 14. It suggests that the attractions between MSCs and KERs were so strong that MSCs could aggregate KERs from the whole surface of the dermal equivalent, and therefore prevented a proper epidermalization. However, we did not observe an initial cell cluster formation on day 1 (data not shown), which indicates an active migration of KERs and MSCs towards each other occurred over time. Further observations on day 20 showed an outgrowth of an epidermal layer out of this colony structure towards a stratified neo-epidermis. It could be either KERs migrating out of the cellcolonies or a single non-colonial KER forming a neo-epidermis. However, with a very high MSCs concentration (90%), a similar epidermal outgrowth could not be observed, which suggests that KERs were anchored by such a strong interaction with a rich population of MSCs that an impaired epidermalization was resulted. Hence the role of MSCs in epidermal development is
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dose-depended. KERs incorporated with a low fraction of MSCs displayed different cellular interactions, which led to the development of a proper neo-epidermis, whereas KERs co-cultured with a higher concentration of MSCs resulted the formation of mixed cell-colonies with blocked epidermalization. 3.4. Quantum dots indicate MSCs location and role in the epidermal structure Quantum dots (QDs) are semiconductor nanoparticles, which showed a narrow band emission and broad band excitation with a high quantum yield, high photostability, luminescence and resistance to chemical and metabolically degeneration. QDs are endocytosed by MSCs and distributed in the cytosol and aggregates are found in the endosomal vesicles around the cell nucleus. The QDs are inherited by daughter cells for at least 6 generations (15days), and show limited cytotoxicity over a 10 nM concentration [39]. QDs are not exocytosed from the endosomal vesicles in live cells, and phagocytosis of dead QDs-labeled MSCs by other co-cultured cells was not found in mixed culture studies. However during cell passaging or mechanical disturbance, QD-positive dead cells can disintegrate and free QD will be re-endocytosed by the surrounding cells, which was shown as single QD events (supplement 1). Therefore, in our experiment, single QD events in cells were not considered as an identification marker for MSC. Only multiple QD events in cells are representative markers for locating QD-labeled MSCs. Furthermore, cell-fusion is an extremely rare event for BM-MSCs to adopt epidermal phenotype. Many studies using the Cre/lox system together with b-galactosidase and fluorescence in situ hybridization (FISH) analysis, found that BM-MSCs differentiate into epithelial cells in the skin, liver, and lungs in vivo without cell-fusion [1,4,8]. Therefore, in this study, we use QD to label and locate the co-cultured MSCs in OC model. The QDs-labeled MSCs clearly showed the participation of MSCs within different epidermal layers on day 14. A high concentration of QDs can be found at the basal periphery of the 10% MSCs group (Figs. 4c, 5c and 6c), and single QDs are located at the upper granular layers of this group (Figs. 4c, 5c and 6c). Although it showed single QDs events accumulated in the upper granular layers, it is not representative for a true QD-labeled MSCs, thus hardly to confirm the contribution of MSCs to this upper granular layer. The involucrin-expressed cells in the upper granular and cornified layers are responsible for the barrier function of the skin. However, the expression of involucrin by MSCs was not shown in this study (Fig. 6c and e), which is coherent with our previous findings [24]. This indicates a strong participation of MSCs in the basal periphery only and no involvement in the barrier function of the epidermis. Moreover, we observed a few concentrated MSCs in the uppermost cornified envelope in the 10% MSCs group (Figs. 4c, 5c and 6c). These QDs-labeled MSCs represented most likely dead cells accumulated after seeding and air-lift. One possible reason is the direct contact of the uppermost MSCs to the external dry environment induces apoptosis in many MSCs. Therefore, during the epidermal development from day 14 to day 20, dead cells were pushed out and accumulated in the uppermost cornified envelope (Figs. 4c, 5c and 6c), whereas only the population of MSCs near the basal layer were able to survive within the wet–dry environment at the air–liquid interface.
impaired epidermalization. KERs cultured with a low concentration of MSCs (10%) demonstrated a proper epidermal structure organization and expression pattern of keratin 10 and involucrin resembling those of the native skin in homeostasis. To our knowledge, this OCs group with 10% MSCs developed a tissue architecture and distribution of differentiation markers recapitulating the situation closely to native epidermis and demonstrated a superior performance compared with any previously reported epidermal OC models. Even though the role of MSCs in epidermal development remains unclear, the co-localization of MSCs with KER and expression of distinctive KER makers in these co-cultured groups strongly supports a regulatory role of MSCs in epidermal tissue and a possible differentiation potential of MSCs into epidermal lineage. 5. Materials and methods 5.1. Materials Human BM-MSCs were ordered from Cambrex Bio Science Walkersville, Inc., USA. Human dermal fibroblasts, Human KERs, keratinocytes medium with defined growth supplements, fetal bovine serum (FBS), low glucose Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12 medium, Quantum Dots (QDs)Qtracker655 Cell labeling Kit, penicillin–streptomycin solution, amphotericin, 40 ,6-diamidino-2-phenylindole, dihydrochloride (DAPI) were all purchased from Invitrogen Corporation, USA. PureColTM collagen was ordered from INAMED Corporation, USA. LGlutamine, ascorbic acid, epidermal growth factor (EGF), 1,25dihydroxyvitamin D3 (VD3), hydrocortisone, insulin, 3,30 ,5-triiodoL-thyronine sodium (T3), mouse anti-human pan-keratin antibody (Ab), mouse anti-human invulocrin Ab and goat anti-mouse IgG FITC-conjugated secondary Ab were purchased from Sigma– Aldrich Pte Ltd., Singapore. Mouse anti-human keratin 10 and filaggrin antibodies were ordered from Thermo Fisher Scientific Inc, USA. Organotypic co-culture devices (12 mm Transwell1) were purchased from Corning Incorporated, USA. 5.2. Cell culture Human dermal fibroblasts and BM-MSCs were cultured in low glucose DMEM supplemented with 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin. Human KERs were grown in keratinocyte medium with defined growth supplements, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin. Human dermal fibroblasts, MSCs and KERs were all grown at standard culture conditions of 5% CO2 at 37 8C in a sterile humidified incubator. Media were changed every 2–3 days and cells were passaged at 70% confluent. 5.3. MSCs labeling with quantum dots MSCs of passage 6–8 at 90% confluence in T-175 cell culture flasks were incubated with 10 nmol/l quantum dots (QDs) in 2 ml cell culture medium for 1 h. Thereafter cells were washed with phosphate-buffered saline. Adherent cells were detached by trypsin, counted with a Thoma hematocytometer and seeded onto the dermal substitute.
4. Conclusion
5.4. Organotypic co-culture
The benefit of maintaining homeostasis during epidermal development from human BM-MSCs is in a dose-dependent matter. Organotypic co-cultures (OCs) of MSCs and KERs at 1:1 ratio had a strong chemotactic interaction to form separate cellular colonies, which impeded the KERs spreading and resulted in an
The dermal substitute was generated according to Vaccariello et al., 1994. Briefly, cold collagen type I solution was mixed with 10 DMEM solution, 10% FBS, 200 mM L-glutamine solution and 72 g/l sodium bicarbonate solution on ice. The pH was adjusted with a 1 M NaOH solution to 7.4. The solution was poured into filter
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inserts of the OC device forming the acellular gel layer. For gelation, the collagen solution in filter inserts was incubated for 1–2 h at 37 8C in a humidified incubator. Thereafter the cellular layer was prepared. The previous solution composition was blended with fibroblasts (100,000 per well). The solution was then poured onto the acellular layer. For gelation, the collagen solution in filter inserts was incubated for 2–4 h at 37 8C in a humidified incubator. The dermal substitute of acellular and cellular layer was cultured within DMEM supplemented with 10% FBS and 50 mg/ml ascorbic acid, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin under culture conditions. After 7 days, a titanium ring was placed onto the dermal substitute and gently pushed down by sterile forceps in order to prevent further contraction and confine the area for KERs and MSCs seeding. Thereafter the medium was aspirated and KERs as well as MSCs were seeded inside the titanium ring at the following concentrations: (a) 5 105 KERs (group with 0% MSCs); (b) 0.5 105 MSCs with 4.5 105 KERs (group with 10% MSCs); (c) 2.5 105 MSCs with 2.5 105 KERs; (group with 50% MSCs); (d) 4.5 105 MSCs with 0.5 105 KERs (group with 90% MSCs); (e) 5 105 MSCs (group with 100% MSCs). The OC were cultured within DMEM and Ham’s F12 (3:1) supplemented with 10% FBS, 10 ng/ml EGF, 5 mg/ml insulin, 0.4 mg/ml hydrocortisone, 1 nM triiodo-L-thyronine and 1 ng/ml VD3, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin under standard culture conditions. After 24 h, KERs and/or MSCs were exposed to the air by lowering the culture medium down to the dermal substitute surface level. The OC was then maintained under standard culture conditions with daily medium change. On days 14 and 20, samples were then fixed and processed for histoimmunological analyses. 5.5. Histological and immunological analyses For morphometric and histoimmunological analysis, the OC were first fixed in 2.5% formaldehyde over night and covered by a drop of 2% hand-warm agarose to prevent dislodgement of the epithelium during further processing procedures. Then the whole specimen was processed for paraffin embedding following standard protocols. Serial 10 mm paraffin sections were cut with a rotating microtome (MICROM), followed by hematoxylin and eosin (H&E) staining according to routine histology protocols. For indirect immunostaining analysis, the antigen retrieval was performed by pretreatment of the samples in 10 mM Citrate Buffer (pH 6.0) at 95 8C for 20 min and cooling down the samples at room temperature for 20 min. The primary antibodies against keratin 10, filaggrin and invulocrin were then incubated, followed by the incubation of FITC-conjugated secondary Ab. The H&E stained samples were observed under a phase-contrast microscope (Leica DM IRB, ebq 100). Samples with Ab incubation and QDs labeling were observed under a confocal laser microscope (FV 500, Olympus). 5.6. Statistical analysis Values (at least triplicate) were averaged and expressed as means standard deviation (SD). Each experiment was repeated five times. Statistical differences were determined by Student two-sample t test. Differences were considered statistically significant at p < 0.05. Acknowledgements This work was performed at the Healthcare and Energy Materials Laboratory, National University of Singapore, Singapore 117576. This work was supported by R39700036112, National University of Singapore, Ministry of Education, and MINDEF-NUSJPP/07/09, Singapore.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jdermsci.2009.05.009.
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