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Smola, Thiekotter, Fusenig - 1993 - Mutual Induction of Growth Factor Gene Expression by Epidermal - Dermal Cell Interaction
Smola, Thiekotter, Fusenig - 1993 - Mutual Induction of Growth Factor Gene Expression by Epidermal - Dermal Cell Interaction
Smola, Thiekotter, Fusenig - 1993 - Mutual Induction of Growth Factor Gene Expression by Epidermal - Dermal Cell Interaction
Abstract. Epithelial-mesenchymal interactions control on collagen gels (repopulated with dermal cells), a
epidermal growth and differentiation, but little is virtually normal epidermis was formed within 7 to
known about the mechanisms of this interaction. We 10 d. Keratinocyte proliferation was drastically stimu-
have examined the effects of human dermal microvas- lated by dermal cells (histone 3 mRNA expression and
cular endothelial cells (DMEC) and fibroblasts on BrdU labeling) which continued to proliferate as well
keratinocytes in conventional (feeder layer) and in the gel. Expression of all typical differentiation
HERE is accumulating evidence that epithelial-mes- pared to the in vivo situation (9, 24). Improvement of tissue
tum reticulare. Thus, the small cell clusters and single endo- Growth Stimulation of Keratinocytes by DMEC and
thelial cells released by gentle squeezing originated from up- Fibroblasts in Monolayer Cultures
permost dermal microvessels (see also reference 25). After The positive impact on growth of normal human keratino-
settling and spreading on fibronectin-coated culture dishes, cytes was demonstrated by cloning experiments on feeder
small colonies of endothelial cells with typical cobblestone layers. As seen with dermal fibroblasts, irradiation with 70
morphology and a distinct pericellular halo formed. With ex- Gy completely inhibited growth of both dermal cells. While
pansion endothelial cells gradually acquired a more elon- keratinocytes when seeded at clonal densities (up to 104
gated form, but remained positive for specific endothelial cells/60-mm dish) were unable to grow to colonies (not
cell markers, usually tested at second or third passage. Anti- shown), these postmitotic mesenchymal cells actively sup-
bodies to factor VIII related antigen (VIllr:Ag), strongly la- ported their clonal growth (Fig. 2). A mixture (1:1) of irradi-
beled blood vessels in the dermis (not shown) and revealed ated DMEC and fibroblasts (with the same total number of
the typical perinuclear granular staining by IIF irrespective feeder cells) did not demonstrate additional cooperative
of the actual cell shape (Fig. 1, a and b). As a further en- effects of both types of dermal cells on keratinocyte prolifer-
dothelial cell marker uptake of acetylated low density lipo- ation (Table 1I). In general, colony size and morphology
protein (fluorescent labeled DiI-Ac-LDL) was analyzed, were not grossly influenced by the type of feeder cells used.
which was internalized by DMEC and intracellularly depos- Keratinocyte colonies appeared mitotically active, were
ited in vesicles (Fig. I, c and d). Cells were also positive in composed of small polygonal cells and showed virtually no
IIF for collagen type IV and laminin while being negative for signs of cornification after 10 d. These feeder effects of
smooth muscle actin (not shown), thus, demonstrating ab- DMEC and fibroblasts, respectively, were consistently ob-
sence of smooth muscle cells and pericytes. Throughout the served when the cells were tested between passage 3 and 7.
whole culture period DMEC appeared as a rather homoge- On the other hand and in agreement with earlier literature
nous cell population although minor contamination by data (42, 54), conditioned media of mesenchymal feeder
fibroblasts or other DMEC-marker negative ceils cannot be cells and keratinocyte-fibroblast cocultures, respectively,
excluded. Also, cells derived and expanded from frozen were unable to induce clonal growth of keratinocytes (data
stocks were essentially unchanged in their reactivity for fac- not shown).
tor Vl/Ir:Ag. Cells could be propagated up to passage 10
consistently maintaining their elongated shape (with a split
ratio of 1:3). Then a marked reduction of growth rate was Regulation of KGF, IL 3, GM-CSF,
noted. IL 6 and Collagenase mRNA Levels in
Typical fibroblast cultures on plastic were negative for fac- Epidermal-Dermal Cocultures
tor VllIr:Ag, DiI-Ac-LDL uptake and mostly though not al- We further analyzed possible mechanisms which might be
ways for collagen type IV and laminin, whereas •10% were involved in the stimulation of keratinocyte proliferation by
positive for smooth muscle actin. Due to the absence of any feeder layer cells and studied the regulation of known growth
positive and unique fibroblast marker, these cells were stimulating factors in epidermal-dermal cocultures versus
defined by the absence of DMEC-markers and presence of dermal cells alone. For this purpose, mRNA levels for KGE
vimentin cytoskeleton. IL 3, GM-CSF, IL 6 and interstitial collagenase were ana-
lyzed in cocultures of keratinocytes with irradiated DMEC (17). Irradiation of DMEC and fibroblasts with 70 Gy did not
and fibroblasts versus irradiated dermal cells (DMEC and substantially change the level of KGF message in dermal
fibroblasts) as well as nonirradiated fibroblasts. Dermal cells cells. However, basal KGF mRNA levels were significantly
were selectively removed from the culture dish leaving ker- stimulated when keratinocytes were cocultured with dermal
atinocytes still attached. From each cell compartment total feeder cells and grown to 'x,50% confluence. A marked and
RNA was prepared, blotted and hybridized with the indi- similar induction of KGF mRNA levels could be demon-
cated probes (Fig. 3). In controls, i.e., proliferating fibro- strated in all dermal feeder cells (DMEC, a 1:1 mixture of
blast and keratinocyte cultures (c), basal levels of KGF DMEC and HDF). In contrast, dermal feeder cells cultured
mRNA transcripts were faintly detected in fibroblasts without keratinocytes expressed only low amounts of KGF
whereas keratinocytes were negative as previously described mRNA, whereas keratinocytes cultured with and without
dermal feeder cells were always negative for KGF message.
For IL-3 no signals could be detected in total RNA of ei-
ther keratinocytes or dermal feeder cells (not shown). How-
Table IL Colony Forming Efficiency of Keratinocytes on ever, GM-CSF transcripts (although at a low level) were in-
Irradiated Feeder Layers
duced in HDF feeder cells by keratinocytes but not in DMEC
DMEC + HDF (Fig. 3). Hybridization signals for IL-6 mRNA were noticed
Keratinocytes DMEC HDF inoculated in all feeder cell populations (DMEC, HDF and the 1:1
5 x 102 89,2 :t: 2,8 42,6 :t: 2,7 65 5:2,7 DMEC/HDF mixture) cocultured with keratinocytes. This
(209%) (100%) (152%) was particularly remarkable, because cultures were grown in
1 × 103 128 + 5,0 66,4 -t- 3,1 102 + 6,2 the presence of hydrocortisone (0.4 #g/ml), a known inhibi-
(192%) (100%) (153%) tor of interleukin expression (56). In dermal feeder cells ir-
radiation alone was sufficient to cause a detectable IL,6
1 x 104 NC NC NC mRNA expression in HDF but not DMEC. Interstitial col-
Keratinocyte numbers as indicated were inoculated onto x-ray-irradiated feeder lagenase was used as a positive control for epidei'mal/dermal
layers of either 1 × 105 DMEC, or 1 x l 0 s flbroblasts (HDF), or a mixture cell interactions (55). Collagenase mRNA expression was
of 5 x llY DMEC and 5 x 10~ HDF/6 cm dish and cultivated for 10 d. Stained
colonies were counted from five replicate dishes and standard deviations were strongly induced in dermal cells by keratinocytes, obviously
calculated. In parentheses, percentages of colony forming eflieiences are given more in DMEC than in HDF. In comparison, irradiated and
relative to colony numbers on fibroblast feeder layer ( 100 % ); (NC, not counted nonirradiated dermal cells cultured without keratinocytes as
due to high colony density). Control keratinocyte cultures (without feeder cells)
did not form colonies when plated at 1 x 103 and 1 × 104/60 ram dish (not well as keratinoytes themselves expressed no visible col-
listed). lagenase transcripts under these conditions.
Mesenchymal Cell Induced Epidermal Proliferation when cocultured with dermal cells, labeling was maximal at
and Morphogenesis in Organotypic Cocultures 7 d with up to 50% positive basal and, to a lesser extent, im-
mediate suprabasal cells (Fig. 5, a and b). At 10 d, the label-
Comparable growth stimulatory effects on keratinocytes by
ing index had dropped but still 10% of the basal cells were
cocultured dermal cells were observed in the organotypic
labeled. Although there was inhomogeneity in the distribu-
culture assay. In contrast to control cultures of keratinocytes
tion of labeled cells, with distinct clustering, both the num-
on lifted cell-free collagen gels (Fig. 4 a), epithelial growth
ber of labeled cells and the length of the proliferative period
and morphogenesis improved dramatically, when keratino-
were always drastically higher in cocultures than in control
cytes were cocultured with DMEC and fibroblasts, respec-
(mesenchyme-cell free) organotypic cultures.
tively, in such organotypic cultures (dermal equivalent type).
The analysis of histone 3 mRNA levels (as marker for cell
Within 7 d, DMEC incorporated in the collagen gel induced
replication) further underlined the effect of dermal cells to
a multilayered well structured epithelium. Cells in the lower
stimulate keratinocyte proliferation under these organotypic
layers appeared more cuboidal, and a typical stratum spino-
culture conditions (Fig. 6). Thus, in controls (organotypic
sum and granulosum were discernible after 10 to 14 d cov-
cultures without dermal cells)-both at early (2 d, lane 1 )
ered by several layers of cornified cells resembling a typical
and late (I0 d, lane 5) stages-there was virtually no histone
orthokeratotic stratum corneum (Fig. 4 b). The effects of
3 expression by keratinocytes indicating absence of DNA
DMEC in this system were generally comparable to those
synthesis. In contrast, keratinocytes in cocultures (here ex-
of dermal fibroblasts (Fig. 4 c). Further, the combination of
emplified with fibroblasts) continued to proliferate even after
both dermal cell types did not cause consistent or pro-
i0 d (lane 6), although there was a decrease in the histone
nounced morphological changes in the developing epithe-
3 mRNA levels as compared to day 2 (lane 2). This decrease
lium (Fig. 4 d). In contrast, keratinocytes without DMEC
reflects at least in part an increase of terminally differentiat-
or fibroblasts ceased to proliferate after 2-3 d, started termi-
ing cells at later stages, leading to a relative dilution of his-
nal differentiation and were mostly atrophic after one week
tone 3 transcripts in the RNA of the whole tissue.
(Fig. 4 a). While this restricted proliferative activity of ker-
atinocytes on lifted cell-free collagen gels was clearly evi-
dent by morphological observation, it could be further sub- Proliferation of Mesenchymal Cells in Collagen Gels
stantiated by detailed analysis. BrdU labeling (for 24 h) Similar to keratinocytes growing at the surface of collagen
revealed only a few labeled cells at day 2 (maximum 5 per gels, the mesenchymal cells, both fibroblasts and DMEC, in-
section with &,350 basal cells), a number which dropped corporated into the gel continued to proliferate and this even
within the following days to zero (not shown). However, in the absence of cocultured keratinocytes. This is in contrast
to what had been reported for contracting collagen gels (34, For DMEC, cell counting studies clearly proved both the
37, 48) but could be clearly demonstrated by different continued proliferative activity of DMEC in noncontracted
methods. gels as well as a smaller increase in coculture with keratino-
Histone 3 mRNA expression revealed already rather high cytes (Fig. 7). Both in control dermal equivalents and in
basic levels in fibroblasts in noncontracted collagen gels in cocultures with keratinocytes, the number of endothelial
the absence of keratinocytes both at 2 and 10 d after embed- cells in the gel increases steadily with a steeper increase in
ding (Fig. 6). This was even further stimulated by cocultured controls as compared to cocultures. The reduction in relative
keratinocytes (Fig. 6, lanes 3 and 7 vs. lanes 4 and 8). In DMEC number in cocultures with keratinocytes coincided
BrdU labeling studies, labeled nuclei were observed in both with the onset of gel contraction and partial digestion,
control dermal equivalents (without cocultured keratino- changes which were always less or absent in control cultures
cytes) and cocultures with keratinocytes (see Fig. 5a) al- without keratinocytes. Whether the slower increase in
though quantitation was difficult due to uneven distribution DMEC in contracting cocultures is only due to the con-
of cells in the gel. traction-induced inhibition of cell proliferation as demon-
Expressionand l.a~calizarionofEp~lermal
Differentiation Markers
Differentiation and functional organization of the epithelium
formed under the influence of DMEC and HDF, were ana-
lyzed by UF to identify and localize typical differentiation
markers (Fig. 8). While keratin 14 (K14) is expressed and
synthesized exclusively in basal cells in normal epidermis
the protein persists in upper epidermal layers. Certain con-
Figure 6. Detection of histone 3 (H3) specific mRNA transcripts formation specific antibodies (as LH8 used here [40]) exclu-
in total RNA preparations of keratinocytes cultured on cell-free sively decorate the basal layer of epidermis in situ (Fig. 8 a)
delayed (Fig. 9, c-f). Usually 5-8 lower cell layers remained tion assay (Fig. 9 g; see reference 7). Within 7-10 d after
unstained irrespective of the cocultured dermal cell type. transplantation both staining for keratin 14 was confined to
There was only a very minor delay in the appearance of K10 the basal layer and keratin 10 (as well as K1, not shown here)
as compared to K1 (occurring also in normal epidermis) in- localization started in the immediate suprabasal layer. This
dicating a rather regular coregulation of both keratins (al- normalization was achieved long before the collagen gel
though starting late in this reconstructed epidermis). The (with cultured dermal cells) was dissolved and keratinocytes
somewhat earlier disappearance of K10 staining towards got in contact with the mouse mesenchyme.
horny layers reflects a rather normal modification or loss of
epitopes (8, 9).
This retardation in the onset of K1 and K10 synthesis was
Discussion
rapidly reversed to normality when such cocultures were Extensive studies have documented the importance of der-
transplanted onto nude mice using our surface transplanta- mal or mesenchymal elements for keratinocyte growth and