The handle http://hdl.handle.net/1887/47933 holds various files of this Leiden University dissertation
Author: Janson, David
Title: Development of human skin equivalents mimicking skin aging : contrast between papillary and reticular fibroblasts as a lead
Issue Date: 2017-04-19
Discussion
The aim of this thesis was to contribute to the development of a human skin equivalent (HSE) that mimics skin aging characteristics. For this purpose we have used the fibroblast- derived matrix (FDM)-HSEs that completely consist of human cells and investigated the role of different fibroblast sub-populations on structural dermal aging.
In our first study we showed that FDM-HSEs can indeed be used for skin-aging research. Earlier in vivo studies describe positive effects of a commercially available anti- aging product on extracellular matrix (ECM) production and stimulation of epidermal cell proliferation. Similar positive effects of this anti-aging compound were shown on the development of the dermal matrix and epidermal morphogenesis in the FDM-HSE model.
In subsequent chapters, different types of fibroblasts were used for the generation of FDM-HSEs to study the effects on the morphogenesis of HSEs and investigate whether the use of different fibroblast types can give rise to characteristics of aged skin in the HSEs. The types of fibroblasts that were investigated can be broadly divided in three categories: i) fibroblasts different by donor age, ii) early- and late passage fibroblasts and iii) fibroblasts of papillary and reticular origin. The latter proved the most successful in introducing skin aging characteristics in HSEs. The majority of data in this thesis describes the in vitro characterisation of these two dermal fibroblast populations and how differentiation and loss of papillary fibroblasts could lead to skin aging.
Validation of fibroblast derived matrix type skin equivalents for (anti) skin aging research
Chapter 2 of this thesis describes the validation of the fibroblast derived matrix (FDM) HSE and its use in skin aging research (1). In this type of HSE the dermal layer is generated by stimulating fibroblasts to produce ECM. This allows for the study of the ECM compo- sition and morphology, and it allows for the study of effects of experimental interventions on ECM homeostasis. Because dermal atrophy is an important process in skin aging, the presence of a representative dermal layer is an important advantage of FDM HSEs over traditional full-thickness HSEs, which usually have an artificial matrix as dermal layer (2- 4).To demonstrate that FDM HSEs can contribute to skin aging research, we have treated FDM HSEs with Bio Marine Complex (BMC), a known anti-aging compound.
BMC is a constituent of the Imedeen cosmeceutical, which is already on the market and has been proven to be effective in vivo (5, 6). The major cosmetic improvements of Imedeen are increased collagen production and increased epidermal proliferation. These effects were also detected in FDM HSEs treated with BMC. These findings indicate that FDM HSEs are a useful tool to study characteristics of skin aging in vitro that cannot or less effectively be studied in traditional full-thickness HSEs. This may be explained by the dermal layer of the FDM HSE, which better mimics the in vivo dermis.
Generation of an aged FDM skin equivalent
The effects of aging change many characteristics of the skin. Examples of these changes include: altered collagen composition, loss of rete ridges, increased secretion of MMP1, increased epidermal stress and loss of basement membrane proteins. Reproducing such characteristics of aged skin in HSEs would add to the validity of a skin aging model in HSEs and would allow for experimental studies on processes that contribute to skin aging.
Furthermore, testing of potential anti-aging compounds is likely more relevant in an aged HSE than in standard HSEs.
Therefore, the next step was to demonstrate that characteristics of skin aging can be introduced in the HSE by manipulating the fibroblasts. Three approaches were taken to mimic characteristics of aged skin in HSEs, as mentioned before: i) fibroblasts different by donor age, ii) early- and late passage fibroblasts and iii) fibroblasts of papillary and reticular origin.
Donor age does not affect the morphogenesis of FDM equivalents
The first approach was to use cells isolated from aged individuals to generate FDM HSEs.
Both fibroblasts and keratinocytes isolated from aged individuals (50 – 70 years) were used. The resulting HSEs were compared to FDM HSEs generated with cells from young individuals (20 – 30 years). No differences were found between HSEs generated with cells from aged individuals and young individuals.
Earlier studies describe in vitro effects of donor age of fibroblasts in monolayer cultures and HSEs. For example, it has been shown that monolayer cultures of fibroblasts isolated from aged and young donors display different stress-induced responses (7-10).
However, these effects are small and hard to replicate (11). In addition, the studies with HSEs have low numbers of donors and highly variable results between donors, regardless of age.
There are several explanations for why it proves difficult to detect effects of donor age in vitro. The first explanation is that perhaps the aged donors used for the generation of FDM HSEs in this thesis were not aged enough and even higher donor ages are required before clear effects occur in vitro. As shown by Giangreco et al, there are some changes in (photoprotected) skin from individuals aged 50+ compared with skin from individuals aged 20 – 30, most notably flattening of rete ridges (12). However, several changes do not become apparent (or significant) until after age 60, such as a reduction in basal cell density (12). Hence, it is advisable to use cells from donors of at least 60 years (and not 50 years, as used in the experiments for this thesis) for future studies on donor age in HSEs.
Another explanation for the lack of effects of donor age in vitro is clonal selection.
During the isolation of the cells, a selection takes place of the most rapidly growing,
healthy cells. The out-competed cells that could contribute to skin aging are lost and not incorporated in the eventual HSE. Finally, accumulation of damaged, degradation- resistant matrix molecules is one of the suspected, contributive factors in skin aging (13, 14). However, during isolation of cells from the skin all ECM is discarded. For generation of HSEs the fibroblasts synthesize the ECM anew. This is not representative of in vivo aged skin; as with clonal selection, the disturbances that could lead to skin aging are not incorporated in the HSE.
Prolonged fibroblast passaging has little effect on the morphogenesis of FDM equiva- lents
The next approach taken to introduce skin aging characteristics in the HSE was to use senescent fibroblasts. Senescence is a cellular state that is triggered by genotoxic stressors (15, 16, 17). It is described as an irreversible cell-cycle arrest, usually in combination with the secretion of pro-inflammatory, matrix degrading substances (18, 19). This makes it a likely candidate for causing aging. And indeed, the number of senescent cells is often increased in aged tissues compared with young tissues, including in skin (8, 20, 21).
The use of senescent fibroblasts in the generation of HSEs is described in chapter 3.
Senescence was induced by prolonged in vitro culture of the fibroblasts. Prolonged culture causes cells to enter a senescent state and is commonly used as an in vitro model of aging (22, 23). Fibroblast monolayer populations that contained approximately 15 % senescent cells, based onβ-galactosidase staining at pH6, were used for generation of HSEs. The effects of these long-cultured fibroblasts on the HSE were compared with early passage fibroblasts, which contained less than 1 % senescent fibroblasts. This led to some changes that resemble aged skin: increased expression of keratin 16 in the epidermis, a thinner FDM dermal part and increased secretion of MMP1. However, some changes were notably absent, such as changes in epidermal turnover and epidermal differentiation.
One of the problems in these studies was the difficulty in identifying senescent cells.
We have used two markers,β-galactosidase staining at pH6 and tumor suppressor p16.
Both markers have been used to identify senescent cells in in vivo skin (8, 20, 21). However, both markers showed high variation and little colocalisation in the experiments described in this thesis.
The combination of experimental difficulties and limited results with senescent fi- broblasts shifted the attention away to the next approach. The third and final approach focused on fibroblasts from the papillary and reticular dermis.
Definition of reticular and papillary fibroblasts
The in vivo dermis can be divided in two morphologically different parts. The upper, papillary dermis has a loose ECM and high cell density and the deeper, reticular dermis has a dense ECM and low cell density (24). It was already known that fibroblasts isolated separately from these two layers look and behave differently in vitro (25, 26). However, no markers were known that could discriminate between the fibroblast populations.
Accordingly, the first step described in this thesis was the definition of reticular and papillary fibroblasts in terms of biomarkers in vitro, as described in chapter 4. This led to several useful in vitro markers that could discriminate between reticular and papillary fibroblast populations on both RNA and protein level. Except for matrix gla protein (MGP), the markers could not be validated in vivo by immunohistochemistry; the staining of the markers was not clearly confined to one of the dermal layers. In chapter 7, another marker was validated that could distinguish between the papillary and the reticular dermis in vivo:
collagen type VIα2. Like MGP, collagen type VIα2 is a matrix component of the ECM in the dermis. Therefore, it cannot distinguish between individual cells; only between the papillary and reticular dermis.
Regardless of the lack of in vivo validation, the identified markers proved useful in the next step. Because the markers allowed for discrimination between papillary and reticular fibroblast populations in vitro, they could be used to investigate if papillary fibroblasts can differentiate into reticular fibroblasts.
Papillary fibroblasts can differentiate into reticular fibroblasts in vitro
The hypothesis that papillary fibroblasts differentiate to reticular fibroblasts was based on a publication by Mine et al., in which it was shown that the papillary dermis is more affected by aging than the reticular dermis (27). This leads to a thinning of the papillary dermis during skin aging and a relative increase of reticular fibroblasts over papillary fibroblasts. This process would influence skin physiology in such a way that it leads to skin aging. Fibroblast differentiation was hypothesized to be a potential mechanism underlying this process.
Two methods were used to demonstrate that papillary fibroblasts can indeed differen- tiate into reticular fibroblasts; by prolonged culture in chapter 5 and by TGF-β1 treatment in chapter 6.
Prolonged culture induces differentiation
In chapter 5 papillary fibroblasts were cultured for several months. After this period the papillary fibroblasts resembled reticular fibroblasts. This was based on their morphology,
expression of markers and behaviour in HSEs, which were all more similar to (low- passage) reticular fibroblasts than papillary fibroblasts.
Both in chapter 3 and chapter 5 prolonged culture is used to mimic skin aging, although the starting populations were different. In chapter 3 all dermal fibroblasts were used and in chapter 5 only papillary fibroblasts were used. The effects of prolonged culture were different in both situations. An example of this is the generation of the dermal matrix of the HSE by late-passage fibroblasts: in chapter 3 the dermal matrix becomes thinner and in chapter 5 becomes denser. Interestingly, the papillary fibroblasts in chapter 5 did show signs of senescence after prolonged culture (β-galactosidase staining at pH6). This questions whether the results in chapter 3 are directly caused by senescence, or through some other process, such as differentiation. It has been discussed before that senescence in vitro is often a culture artifact and not related to aging processes relevant for in vivo tissues (28). Rather, it might be an end stage of an in vitro differentiation process (29, 30). This suggests that senescence, at least in vitro, is more of a consequence of aging rather than a cause.
TGF-β1 induces differentiation
In chapter 6 it was shown that TGF-β1 can also induce differentiation of papillary fibroblasts into reticular fibroblasts in monolayer culture. TGF-β1 is also known to cause differentiation into myofibroblasts (31). Myofibroblasts are a type of fibroblast that produce a large amount of ECM and show a high contraction of its environment (32).
Reticular fibroblasts share a number of properties with myofibroblasts. For example, several reticular markers described in chapter 4 are related to contraction and reticular fibroblasts show strong contraction in HSEs (33). However, there are also factors that are different between reticular fibroblasts and myofibroblasts. For example, the levels of myofibroblast marker α-smooth muscle actin (αSMA) are relatively low in in vitro reticular fibroblasts; only up to five percent of fibroblasts in early passage populations are positive, as described in chapter 6. In healthy skin in vivo there are noαSMA positive fibroblasts (34). Furthermore, reticular fibroblasts still show proliferation in vitro, whereas fully differentiated myofibroblasts do not (35).
The definition that best describes reticular fibroblasts is the proto-myofibroblast (36).
A proto-myofibroblast contains the beginnings of a myofibroblast, but lacks the full blown machinery of a myofibroblast. An important factor in the differentiation to a proto- myofibroblast is mechanical tension (37). One of the hallmarks of the reticular dermis is high matrix density, which leads to increased stiffness (resistance to deformation) (38).
This could explain why reticular fibroblasts resemble proto-myofibroblasts. Complete differentiation to a myofibroblast requires TGF-β1 stimulation (37).
The results in this thesis on fibroblast differentiation are summarized in figure 1.
Several questions remain with regard to papillary to reticular fibroblast differentiation. For example, what mechanism causes the differentiation process during prolonged culture, as in chapter 5? There is no tension in monolayer culture and no TGF-βadded to the culture medium. However, TGF-βcan be found in the Fetal Bovine Serum that is added to the culture medium (39). As such, the culture medium used during cell culture could have driven the differentiation process. Repeating the experiments in serum free culture conditions could shed some light on the effects of the serum on the differentiation process.
Another remaining question is what prevents fibroblast differentiation in healthy skin in vivo, while differentiation can be easily induced in vitro? In chapter 6 a potential answer is given to this last question: papillary fibroblasts in FDM HSEs do not differentiate when stimulated with TGF-β1. This may be explained by the presence of ECM molecules that block the differentiation effects of TGF-β1. For example, it has been shown that Decorin, an ECM molecule specific for the papillary dermis, can inhibit TGF-β(40, 41).
An additional answer may be the biphasic nature of TGF-β. The effects of TGF-βcan vary significantly in a tissue and depend on, amongst others, its concentration and expression of cofactors, such as epidermal growth factor (35, 42, 43). It appears as if a concentration of more than 1 ng/mL of TGF-β1 is required for myofibroblast differentiation (44). The concentration of TGF-β1 used to stimulate the papillary HSEs was 2 ng/mL; enough to induce differentiation. However, the local concentration of TGF-β1 in HSEs could be lower than expected. There are several “diluting” factors present in HSEs, such as the inert filter on which the HSE is cultured and the presence of ECM. A clarification of what stimuli are involved in differentiation of papillary fibroblasts to reticular fibroblasts in vitro is required.
TGF-β1 induced fibroblast differentiation is an important step in wound healing. An interesting observation is that wound depth is an important factor in determining the wound healing response, the effectiveness of wound closure and scar formation (45).
It appears that the depth of the wound and the amount of damage determine which fibroblast population (papillary, reticular or even adipose derived in case of extensive wounds) is activated, which in turn influences the healing reaction (46). Reticular fibroblasts seem more capable of wound healing, based on the proto-myofibroblast phenotype; if the reticular dermis is damaged a more severe healing response seems appropriate. However, recently it was shown that papillary fibroblasts also play an important role in wound healing: they are required for closure of the wound (keratinocyte migration) and the formation of new hair follicles (47). This agrees with another result of this thesis: keratinocytes prefer to grow and migrate on a papillary dermis.
Figure 1: A summary of the results of fibroblast differentiation in monolayers as described in this thesis. The markers for papillary fibroblasts, reticular fibroblasts and myofibrob- lasts used in this thesis are shown, divided by whether the markers were used in vitro or in vivo. The black arrows denote the route of differentiation. Both for papillary to reticular differentiation and reticular to myofibroblast differentiation TGF-β1 is an important stimulus, making reticular fibroblasts appear as an intermediate state. However, TGF-β1 is not essential for papillary to reticular differentiation, but is essential for myofibroblast differentiation. It is not yet known whether reticular fibroblasts can “de-differentiate” into papillary fibroblasts. Abbreviations:α-SMA: alpha smooth muscle actin, CNN1: calponin 1, Col VI: Collagen type VI, DCN: Decorin, MGP: Matrix Gla Protein, PDPN: podoplanin, TGF-β: transforming growth factor.
Papillary matrix effects on keratinocytes
In chapters 5, 6 and 7 it is shown that the use of reticular fibroblasts in HSEs leads to several characteristics of aged epidermis, such as decreased proliferation and increased expression of epidermal-activation-associated markers such as keratin 16 (48, 49). In chapter 7 it is shown that the different effects of reticular and papillary fibroblasts on the epidermis are likely caused by the different ECM the fibroblasts generate. What causes the different growth of keratinocytes on papillary or reticular matrix is not known, but two important aspects could play a role: ECM constituents and ECM density.
The specific ECM constituents of the papillary dermis may be better for the attachment and migration of keratinocytes. One of the ECM constituents that is specific for the papillary dermis is collagen type VI α2. It has been shown that collagen type VI can bind integrins (50, 51). Progenitor keratinocytes, which have high proliferative capacity, express high levels of integrins (52). Binding of these integrins to any substrate can prevent differentiation of keratinocytes (53). It appears that papillary ECM components, like collagen type VIα2, can better bind to integrins and as such prevent keratinocytes from differentiating. This would allow keratinocytes to remain proliferative and thus would explain why the epidermis shows more sustained proliferation and a longer lifespan on papillary HSEs than reticular HSEs, as shown in chapter 7.
Keratinocytes grow differently depending on the density of and tension in the substrate matrix (38). Again, a cue can be taken from wound healing. In the first phases of wound healing, contraction of the tissue is very important. However, for wound healing to succeed a final relaxation of the newly formed (ECM) tissue needs to occur to allow wound closure (54). It is not clear if papillary fibroblasts play a role in this process, but based on the findings mentioned above it is likely (47). It is not clear how density, tension and other mechanical properties exactly differ between the papillary and reticular dermis. Yet since it seems to play a role both in fibroblast differentiation and keratinocyte growth, it is an interesting field of future research. The use of more advanced cell culture equipment that can impose mechanical stress on cells or HSEs will be crucial.
Conclusion and future perspectives
The main goal of this thesis was to contribute to the development of an in vitro model that mimics skin aging characteristics in HSEs. Three approaches were taken to include these characteristics. Two of these approaches, using cells from aged donors and using long cultured (senescent) fibroblasts, did not lead to a robust model with characteristics of aged skin. The third approach, the use of papillary and reticular fibroblasts, did lead to such a model. The results from the three approaches are summarized in table 1.
The focus on papillary and reticular fibroblasts in skin aging is a relatively new
Aged skin characteristic HSE generated with cells from aged
donors
HSE generated with senescent fibroblasts
HSE generated with reticular fibroblasts
Dermal atrophy - + -
Increased MMP1 expression
- + ni
Reduced epidermal turnover
- - +
Epidermal activation (K6, K16, K17)
- + +
Reduced expression terminal differentiation markers (Inv, Fil)
- - +
Table 1: Summary of characteristics of aged skin investigated in this thesis and whether they were found in the FDM-HSEs generated by the three approaches described in this thesis. Symbols: - characteristic not present in FDM-HSE, + characteristic present in FDM-HSE, ni.: not investigated in FDM-HSE. Abbreviations: K6, K16, K17: Keratin 6, 16, 17; Inv: Involucrin; Fil: Filaggrin.
development and the exact roles of these fibroblasts during in vivo skin aging are not yet clear. Nonetheless, the distinction between these fibroblast populations is a promising subject in skin aging. Figure 2 shows an integration of the (in vitro) results of this thesis into the (in vivo) model proposed by Mine et al. (27).
One hypothesis emerging from this thesis is that loss of the papillary dermis is a cause of intrinsic aging. This would correspond with the actually observed in vivo thinning of the papillary dermis in aging skin. Immunhistochemical analyses in chapter 4 confirmed a loss of markers of the papillary ECM in aged skin in vivo. However, a more comprehensive analysis on the details of the process will be required. The work in this thesis provides two markers that can be used for showing that the papillary dermis shrinks with age: collagen type VIα2 and MGP, which are specific for, respectively, the papillary dermis and the reticular dermis.
Another hypothesis from this thesis is that the composition of the ECM plays a crucial role in the differentiation of fibroblasts (chapter 6). During skin aging, ECM is damaged.
Damaged ECM plays an important role in cellular aging (13, 55-57). It will be interesting to test whether damaged ECM can induce differentiation of papillary fibroblasts to reticular fibroblasts. Damaged ECM can be mimicked in vitro by, for example, inducing advanced glycation end-products (56). As discussed above, the lack of damaged ECM is a deficiency of current skin aging models in HSEs. The use of damaged ECM in HSEs could therefore improve the validity of this skin aging model in general as well.
Figure 2: Hypothesized model for the effects of different fibroblast populations on skin aging. During in vivo skin aging the papillary dermis gets thinner. This thesis shows that papillary fibroblasts can differentiate into reticular fibroblasts, which could be the underlying mechanism of the thinning of the papillary dermis. Because the papillary dermis gets thinner, the relative number of reticular fibroblasts increases.
This has consequences for the generation of ECM, which will have a higher tension and different composition when generated predominantly by reticular fibroblasts. In addition, keratinocytes grow better on papillary ECM than on reticular ECM. As the dermis becomes more reticular during skin aging, keratinocytes will grow less effectively.
This could explain several age-related skin changes such as the loss of rete ridges and slower closing of wounds.
Based on the HSEs made with reticular fibroblasts, it was concluded that keratinocytes do not grow well on ECM produced by reticular fibroblasts. While this recreates some effects of skin aging in vivo, it can be questioned whether in vivo keratinocytes will ever grow solely on the reticular dermis; this would imply the complete loss of the papillary dermis. A potential improvement of the current HSEs is the incorporation of both a reticular and papillary dermal layer. This will make the HSEs more similar to in vivo skin and allow for better predictions on the role of both layers during skin aging. During the experiments for this thesis, first steps were made to develop HSEs that contained two dermal layers. However, the handling of these FDM layers proved difficult and may not be the optimal approach for the generation of this type of FDM-HSE. Future experiments with dermal equivalents generated with scaffolds (e.g. collagen, chitosan) may be more successful, since they are easy to handle and modulate (58).
We have shown that papillary fibroblasts differentiate into reticular fibroblasts under specific conditions. However, the question remains whether a possibility exists to re- differentiate the reticular fibroblasts into a papillary fibroblast? This new anti-aging concept has already gained interest from the cosmetic industry and compound-screening projects have already been initiated where cell morphology and gene-signature are used as readouts. If successful, this could be a promising approach in the treatment of other skin related conditions, such as scars and delayed wound healing.
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