标题: Stem/Progenitor Cell-Like Properties of Desmoglein 3dim Cells in Primary and Imm [打印本页] 作者: 江边孤钓 时间: 2009-3-5 00:54 标题: Stem/Progenitor Cell-Like Properties of Desmoglein 3dim Cells in Primary and Imm
作者：Hong Wana, Ming Yuanb, Cathy Simpsonc, Kirsty Allenc, Felicity N.E. Gavinsd, Mohammed S. Ikramb, Subham Basub, Nuzhat Bakshe, Edel A. O 7 E, S4 R+ ~5 S" w! I
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【摘要】& T1 ~9 Y0 }2 R! Z+ b( z: z D0 Q
We showed previously that primary keratinocytes selected for low desmoglein 3 (Dsg3) expression levels exhibited increased colony-forming efficiency and heightened proliferative potential relative to cells with higher Dsg3 expression levels, characteristics consistent with a more "stem/progenitor cell-like" phenotype. Here, we have confirmed that Dsg3dim cells derived from cultured primary human adult keratinocytes have comparability with 6bri/CD71dim stem cells in terms of colony-forming efficiency. Moreover, these Dsg3dim cells exhibit increased reconstituting ability in in vitro organotypic culture on de-epidermalized dermis (DED); they are small, actively cycling cells, and they express elevated levels of various p63 isoforms. In parallel, using the two immortalized keratinocyte cell lines HaCaT and NTERT, we obtained essentially similar though occasionally different findings. Thus, reduced colony-forming efficiency by Dsg3bri cells consistently was observed in both cell lines even though the cell cycle profile and levels of p63 isoforms in the bri and dim populations differed between these two cell lines. Dsg3dim cells from both immortalized lines produced thicker and better ordered hierarchical structural organization of reconstituted epidermis relative to Dsg3bri and sorted control cells. Dsg3dim HaCaT cells also show sebocyte-like differentiation in the basal compartment of skin reconstituted after a 4-week organotypic culture. No differences in percentages of side population cells (also a putative marker of stem cells) were detected between Dsg3dim and Dsg3bri populations. Taken together our data indicate that Dsg3dim populations from primary human adult keratinocytes and long-term established keratinocyte lines possess certain stem/progenitor cell-like properties, although the side population characteristic is not one of these features.( l' e: t7 d1 _& k2 X: d
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Disclosure of potential conflicts of interest is found at the end of this article. 3 ?) Y1 l/ |- ~
【关键词】 Desmoglein Desmosomes Keratinocytes Stem cells- q5 Z, O- D3 w3 X, `% q1 ~% p7 P
INTRODUCTION 2 `: L- n! u/ b, q9 k9 r / _4 `" D$ }/ v7 Q$ x; zDesmosomes are specialized cell-cell adhesion structures enriched in tissues, such as epidermis, that undergo substantial mechanical stress . * h% I' n' H' R $ w, A+ A+ `. C. G2 QEpidermis undergoes constant regeneration and cell turnover, with the epidermal stem cells, (also known as label-retaining cells , stem cells are clustered exclusively at the tips of the epithelial rete ridges." J5 C1 t8 Z; z/ @9 e
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Selection for stem cells often has been based on differential expression of specific cell surface markers. Thus, high levels of ¦Â1 integrin were observed in populations enriched for keratinocyte stem cells . 0 o$ f9 Y2 \! T 0 L! n2 Z% y S# QPreviously, we showed that the basal keratinocytes in the epidermis of human palm skin, which are heterogeneous for desmosome expression, demonstrated a differential proliferative capacity based on different levels of Dsg3 expression . We show that FACS-based selection for low Dsg3 expression levels using these lines results in isolation of cells with generally similar stem/progenitor cell characteristics¡ªthat is, the potential to generate enhanced epidermal thickness and a better ordered epidermal structure in vitro. " g& u. t- g: ]; u: x- }( @6 k4 [0 z# V1 I/ x
MATERIALS AND METHODS 1 K6 A' _" q9 e. g% Q. t5 e6 y & t5 b6 X9 o8 G# x9 M* o; q+ s: kAntibodies s8 j, J. U% t3 e/ W2 s( o4 O' _2 l7 A" G- w. {& \- {' u' H" D
Mouse monoclonal and rabbit polyclonal antibodies (Abs) used were as follows: 5H10, mouse Ab to the extracellular domain of Dsg3 (gift from Dr. M. Amagai); purified rat anti-human CD49f (BD Pharmingen, San Diego, http://www.bdbiosciences.com); mouse Ab to human CD71 fluorescein isothiocyanate (FITC) conjugate (RDI, Concord, MA, http://www.researchd.com); Dsg1-P124, mouse Ab to Dsg1 (Progen, Heidelberg, Germany, http://www.progen.de); 10G11, mouse Ab to Dsg2 (Progen); Dsc1-U100 and Dsc3-U114, mouse anti-human Dsc1 and Dsc3 (Progen); Pab to Dsc2, rabbit Ab to Dsc2a and Dsc2b (Progen); AHP320, rabbit Ab against Dp (Serotec, Oxford, U.K., http://www.serotec.com/asp/advancedsearch.asp); mouse anti-K14 (Cancer Research UK); ¦Â-actin Ab ab6276 (Abcam, Cambridge, U.K., http://www.abcam.com); and 4A4, mouse Ab to p63 (Abcam), PCNA , anti-beta actin ab6276 (Abcam). Secondary Abs used were Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Molecular Probes via Cambridge BioSciences, Cambridge, U.K., http://www.bioscience.co.uk); mouse IgG:(R)-phycoerythrin (RPE) (DAKO, Glostrup, Denmark, http://www.dako.com); and rat IgG:RPE (Jackson ImmunoResearch, http://www.jacksonimmuno.com).. d, i; N3 A* H# q
% s. h6 \& b: \; K/ [Human Keratinocyte Culture/ s5 n. O7 ]$ z- p6 }3 N; s
' r6 v* \4 Y+ y# K% b% D. UPrimary adult keratinocytes from human breast and foreskin routinely were maintained on a 3T3 feeder layer, lethally treated with 4 µg/ml mitomycin C (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), in keratinocyte culture medium (Dulbecco's modified Eagle's medium and Ham's F12 medium in a ratio of 3:1 (vol/vol; Gibco, Paisley, U.K., http://www.invitrogen.com) supplemented with 10% fetal bovine serum (FBS; Biosera, East Sussex, U.K., http://www.biosera.com), 0.4 µg/ml hydrocortisone (Sigma-Aldrich), 10¨C10 M cholera toxin (Sigma-Aldrich), 10 ng/ml epidermal growth factor (Gibco), 5 µg/ml insulin (Sigma-Aldrich), and 1.8 x 10¨C4 M adenine (Calbiochem, Darmstadt, Germany, http://www.emdbiosciences.com) and incubated in an incubator with an humidified atmosphere containing 8% CO2. At approximately 60%¨C70% confluence, the cells were passaged with 0.25% trypsin plus 0.02% EDTA after removal of 3T3 feeders with 0.02% EDTA, and only a passage number below 3 was used for the experiments. The immortalized NTERT cells were cultured in the same keratinocyte culture medium as described herein without the feeder layer support, whereas HaCaT cells were routinely cultured in DMEM supplemented with 10% FBS (Biosera).9 ^3 `; x& J4 h: W. |
* o7 A* i5 S( S% P. @7 rImmunofluorescent Labeling, Flow Cytometry, and Cell Sorting/ J. s, K2 S' h4 C+ m
9 J5 c P8 G0 I8 wFor Dsg3 labeling, the adult primary keratinocytes, or HaCaT and NTERT cells, were routinely suspended with 1:1 of trypsin/EDTA and 0.25% trypsin-containing 1 mM Ca2 . Approximately 2 x 105 cells were incubated with primary antibody for 10¨C20 minutes on ice, washed in FACS buffer (phosphate-buffered saline or seeded into the metal ring for organotypic culture on de-epidermalized dermis (described later herein). For direct comparison of CFE by 6bri/CD71dim cells and Dsg3dim cells, the same source of primary adult breast keratinocytes at passage number 1¨C2 were labeled separately for either 6 and CD71, using indirect immunofluorescence for 6 (rat anti-human 6 integrin and rat IgG:RPE) and then direct immunofluorescence for CD71 (CD71:FITC), or indirect immunofluorescence for Dsg3 (5H10 and Alexa Fluor 488 IgG conjugate) together with DAPI before sorting. DAPI was used for exclusion of dead cells (approximately 20%).! t& K3 J7 R4 x: g& M5 s! r
$ L W. x0 p2 ^, R, Q( B, w8 lFor bromodeoxyuridine (BrdU) incorporation assays on the sorted Dsg3dim and Dsg3bri HaCaT cells, cells were pulse labeled with 10 µM BrdU in normal medium for 45 minutes before harvesting and cell surface labeling for Dsg3. Ten percent of Dsg3dim and 10% of Dsg3bri cells were collected separately on a Moflo (2 x 105 in each group) and spun down before processing for BrdU staining. The dissociated cells were fixed initially in 1 ml of formal saline (Cancer Research UK) for 5 minutes and then in 1 ml of ice-cold 70% ethanol for 10 minutes. After centrifugation, cells were washed in PBS and resuspended in 1 ml of the 0.2 mg/ml pepsin solution (Sigma-Aldrich) containing 0.15 M hydrochloric acid and incubated at 37¡ãC in a water bath for 12 minutes. The reaction was neutralized by addition of 3 ml of PBS before centrifugation. After a wash in PBS, cells were incubated in anti-BrdU Ab (Becton Dickinson, Franklin Lakes, NJ, http://www.bd.com) at one in five in PBS/0.5% Tween 20 (Sigma-Aldrich)/1% FCS for 1 hour at room temperature and then in FITC-conjugated rabbit anti-mouse F(ab')2 fragments (DAKO) at 1 in 10 for 30 minutes in the dark. After the final wash, cells were resuspended in 50 µg/ml propidium iodide solution and left at room temperature in the dark for 15 minutes before analysis by FACScan.' T6 i i, ~- e! t/ L; z
7 v- X% r* Z( n$ H/ JFor SP and Dsg3 analysis, cells were harvested and resuspended at 1 x 106 cells per milliliter in DMEM supplemented with 10% FBS and 10 mM HEPES (Sigma-Aldrich). Cells were prewarmed to 37¡ãC before addition of 5¨C10 µg/ml Hoechst 33342 and incubated for 45 minutes, or for varied time points in a time-course analysis, at 37¡ãC, before being spun down at 4¡ãC and labeled for surface Dsg3 and RPE secondary on ice. Cells were washed and resuspended in ice-cold DMEM supplemented with 2% FBS and 10 mM HEPES and 5 µg/ml PI to exclude dead cells. The analysis was performed on a Moflo. Hoechst and PI were excited by an argon ion laser tuned to midrange UV light (350¨C356 nm) and collected on a linear scale through 424/44BP and 620LP filters (Dako Cytomation), respectively, using a 610DCL. The Dsg3-RPE was excited using the 488-nm argon ion laser and collected on a log scale through a 580/30BP filter. & O' J0 I* k4 S' L7 T3 g: x! x+ h6 t/ b, c, J5 K j
Colony-Forming Assay 9 } M- f; L" ~. z( k) u( x+ a2 g 7 F. [* P- x- x. G& eThe colony-forming assay and analysis were performed as described previously ., o; B: U- d3 v
1 i& _% C/ |: D( ~- pReverse Transcription and Quantitative Polymerase Chain Reaction8 L f- P$ Q5 V3 z3 A6 O8 @
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RNA was extracted from cells using TRIzol Reagent (Gibco BRL, Paisley, U.K., http://www.gibcobrl.com) according to the manufacturer's instructions. Quality and quantity of RNA were assessed by measuring the A260/A280 ratio on a NanoDrop machine (NanoDrop Technologies, Wilmington, DE, http://www.nanodrop.com). The integrity of the RNA was confirmed by gel electrophoresis. Reverse transcription was performed using 5 µg of total RNA, oligo(dT)12¨C18, and SuperScript II RT (Invitrogen, Paisley, U.K., http://www.invitrogen.com) in a total volume of 20 µl according to the procedure provided by the manufacturer. After the reaction, the enzyme was denatured at 70¡ãC for 15 minutes. The cDNA was diluted at 1:5; 2 µl of diluted cDNA was used for real-time polymerase chain reaction (PCR). Primers for TAp63, Np63, p63-tail, p63¦Â-tail, p63-tail, and GAPDH were as detailed elsewhere . PCRs were carried out in triplicate in a volume of 25 µl containing 2 µl of cDNA template, 1.5 µl (10 mM) of each primer, and 12.5 µl SYBR Green PCR master mix (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com). The products were detected with the ABI 7700 Sequence Detector (Applied Biosystems) and the cycle values (Ct) were determined as a measure of the cycle number at which a statistically significant increase in fluorescence intensity was first detected and normalized to the value for the control gene GAPDH to yield the relative abundance.' x$ {+ g1 N4 O- t
4 V, |6 u b& b. Q/ b7 e& ~( }Immunoblotting 8 i2 O# ?, r; J3 h' x' f . g" v* ^, v4 d* X# }9 SProtein extraction and Western blotting were carried out as described previously . For p63 analysis, protein was prepared by adding 1x sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.002% bromphenol blue, and 0.0625 M Tris HCl, pH 6.8). Cell lysate was treated by two cycles of boiling for 5 minutes at 95¡ãC and snap freezing for 5 minutes on dry ice. Protein concentration was measured on a NanoDrop machine (NanoDrop Technologies, Wilmington, DE, http://www.nanodrop.com reading at 280 nm. Equal amounts of protein samples were loaded on a 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (Hybond-P; Amersham Pharmacia, Buckinghamshire, U.K., http://www.amersham.com). The membrane was dried and then incubated for 1 hour at room temperature with the antibodies for p63 at dilution of 1:200, PCNA and actin both at 1:1,000. After the membranes were washed, they were incubated for 1 hour at room temperature with secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Amersham Pharmacia) diluted at 1:1,000 in TBS-0.01% Tween 20. Finally, the blots were detected with the ECL system (Amersham Pharmacia).4 @0 i8 j. p; y* ]* z
4 V1 Q( R' q& g+ tOrganotypic Culture on De-epidermalized Dermis8 w0 E& T) t% T+ I. | u5 ]
7 ~; x% R q& [- ?/ Y- n' tGlycerol-preserved skin (Euro Skin Bank, Beverwijk, the Netherlands, http://www.euroskinbank.org) was washed and stored in PBS (Ca2 /Mg2 -free), containing an antibiotic mix, at 37¡ãC for up to 10 days. The epidermis was removed mechanically (so-called de-epidermalized dermis), and the dermis was cut into 1.5 x 1.5-cm squares. A 1-cm-diameter stainless steel ring was placed on the reticular side of each dermal square, and suspensions of normal primary human fibroblasts were seeded at 5 x 105 cells per ring and grown for 1 day. Then, the sorted Dsg3dim and Dsg3bri keratinocytes of either primary or immortalized cell lines were plated at 2 x 105 cells per ring, or at the required density according to cell plating density experiments, on the papillary side and grown in normal keratinocyte culture medium for 1¨C3 days to reach a confluent monolayer. The grafts then were lifted on to the grids and cultured at the air-liquid interface for different time periods according to individual experiments before fixation and processing for paraffin embedding and frozen sectioning. For paraffin-embedded tissue, sections were cut at two different levels (approximately half-millimeter intervals) and stained with hematoxylin and eosin. A series of photomicrographs were taken from each section and subjected to image processing. Parameters such as the area and depth of epidermis formed by either the primary or immortalized cell lines were acquired using the software OPTILAB PRO 2.6.1 (Graftek France, Austin, TX, http://www.graftek.com) and subjected to statistical analysis. The sample size in each group was between 10 and 20, and data presented are the mean ¡À SEM. ( V. A$ r+ l4 B/ Q5 g5 ~ ! V5 ?+ o! |6 D; _0 HLaser Scanning Confocal Microscopy 5 J6 U3 r" c: F% v+ m 2 U: l4 I1 m$ J# J+ c* Q" aFrozen sections of the herein-reconstituted epidermis were dual labeled for Dsg3 and Dp using primary Abs mouse anti-Dsg3 5H10 and rabbit anti-Dp AHP320 and secondary goat anti-mouse Alexa Fluor 488- and anti-rabbit Alexa Fluor 546-conjugated IgG. Dual immunofluorescence images were acquired sequentially following the procedure described elsewhere . 4 c6 H* I9 \ g! O% l# x2 k! B8 r" r7 n9 f: F7 Z: M, p3 u
RESULTS : o. K8 J% h" N* E' }- T ' S" l: f0 ?/ H i4 s% v5 CPrimary Adult Dsg3dim Keratinocytes Show Comparable CFE to That of 6bri/CD71dim Cells * I! w9 G# P5 x7 z % J* }. f/ ^1 n$ Z. `9 JRecently, 6bri/CD71dim staining was identified as a marker for epidermal stem cells in neonatal foreskin epidermis . Cells were plated at 500, 1,000, 1,500, and 2,000 cells per well on feeder cells in 6-well plates, and grown for 12 days before fixation and staining for colonies. Figure 1B and 1C show the colonies (plated at 500 cells per well) and the statistical analysis of colonies from four wells in each group, respectively. Overall, there were no statistically significant differences between 6bri/CD71dim stem cells and Dsg3dim cells in terms of CFE, colony size, and density (p > .05). By contrast, the differentiated 6dim and Dsg3bri showed significantly lower CFE and colony density, and also a relatively higher percentage (approximately 50%) of abortive colonies (Fig. 1C). - A' C5 T5 a# n; B# E & X- R5 x+ k" D- EFigure 1. Colony characteristics of primary keratinocytes. (A): Comparative FACS sorting profiles of primary adult keratinocytes using (Aa) 6 integrin and CD71 transferrin receptor or (Ab) the cell surface marker Dsg3. (B): Representative images of colonies resulting from cells seeded on lethally treated feeder cells at 500 cells per well. (C): Quantitative analysis of colony-forming data (mean ¡À SEM; n = 4). The CFE, colony density, and colony size of 6bri/CD71dim cells, and Dsg3dim cells are comparable. Equally, 6dim differentiated cells and Dsg3bri cells are comparable (p > .05) and exhibit significantly lower CFE and colony density as well as smaller colony size compared with the other groups (p ( E; t& h1 \% N) x
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Cultured Dsg3dim Cells from Primary Keratinocytes and NTERT, but Not from HaCaT, Are Relatively More Proliferative Than Are Dsg3bri Cells' D! w- |1 [& g) m+ z7 M
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We labeled dissociated cells for DNA, with Hoechst 33342, and for Dsg3 on the cell surface with 5H10 and IgG:RPE conjugate and conducted FACS analysis. Dead cells were gated out with positive DAPI staining. Ten percent of either Dsg3dim or Dsg3bri cells were gated and displayed for cell cycle profile. As shown in Figure 2A, Dsg3dim primary keratinocytes had relatively more S¨CG2/M-phase cells compared with Dsg3bri cells, suggesting that Dsg3dim cells were more proliferative. NTERT cells exhibited a very similar profile (Fig. 2B). However, HaCaT cells exhibited more quiescent and slower-cycling kinetics (Fig. 2C). To confirm this more quiescent feature of Dsg3dim HaCaTs, we performed a BrdU incorporation assay on cells sorted for low or high Dsg3 expression. As presented in Figure 2Cc, the Dsg3dim HaCaT cells showed lower S¨CG2/M-phase cells (12.1% S-phase and 0.4% G2/M phase in Dsg3dim cells vs. 17.8% S-phase and 8.3% G2/M in Dsg3bri cells) and higher G0/G1 phase cells (87.3% G0/G1 phase in Dsg3dim cells vs. 73.1% G0/G1 phase in Dsg3bri cells) compared with the Dsg3bri cells. Thus, the acquisition of increased proliferative potential was not associated invariably with the Dsg3dim phenotype. 0 T7 U: w p/ J. M C! l+ G) a9 [% y% f
Figure 2. Cell cycle profiles of Dsg3dim and Dsg3bri cells of different keratinocytes. (A): Primary adult breast Kc were gated for 10% Dsg3dim populations Relatively more S¨CG2/M-phase cells exist in the Dsg3dim populations. (B) NTERT Kc (Ba) sorting profile (Bb) cell cycle profile showing more S¨CGz/M-phase cells in the Dsg3dim population. (C) HaCaT Kc (Ca) sorting profile (Cb) cell cycle profile showing more S¨CGz/M-phase cells in the Dsg3bri population; (Cc) confirmation of this by bromodeoxyuridine/fluorescence-activated flow cytometry analysis. Abbreviations: Dsg, desmoglein; Kc, keratinocytes; PE, phycoerythrin; SSC, side scatter.: a' o ~ U% e8 x
) h _9 [2 K5 i% I% PImmortalized Keratinocyte Cells with Low Levels of Dsg3 Expression Exhibit an Increased Colony-Forming Efficiency, Smaller Size, and a Relatively Larger Nuclear-to-Cytoplasmic Ratio ! J5 D! l/ @# g/ e, `* ?( I4 u- s" S* ~0 @# q
Because the immortalized cells had a more homogeneous appearance in culture than did the primary keratinocytes, we increased our sorting stringency. Thus, 5% of Dsg3 dim and 5% of Dsg3bri cells were sorted and plated at various densities in 6-well plates, with or without J2 feeder cells, and grown for 2 weeks. For HaCaT cells (n = 3) the Dsg3dim cells exhibited statistically significantly higher CFE and larger colony sizes compared to Dsg3bri cells (approximately a twofold increase; Fig. 3A). Similarly, Dsg3dim populations of the NTERT cells had a higher CFE (twofold increase) than did Dsg3bri populations (Fig. 3B), but there was no difference in colony size after 11 days of culture. These results are comparable to those obtained previously with primary keratinocytes . 0 B' \* p) t/ w. s6 e( U 8 L$ ]+ B% H6 V$ a7 T0 K& XFigure 3. Enhanced CFE of stringently sorted Dsg3dim cells from HaCaT and NTERT lines. (A): Data from three independent experiments (triplicate in each experiment) with stringently sorted HaCaT cells (mean ¡À SEM; n = 6). Significantly higher CFE and colony size were observed in Dsg3dim HaCaTs compared with Dsg3bri cells. (B): NTERT line (mean ¡À SEM; n = 6) with Dsg3dim cells showing significantly higher CFE compared with Dsg3bri cells (p .05). Abbreviations: CFE, colony-forming efficiency; Ct, cycle values; Dsg, desmoglein. - D7 y: u3 T4 I # Z9 S9 p- r) ]3 m$ y0 `/ HPrimary human keratinocytes (Fig. 4A) and NTERT Dsg3dim cells (Fig. 4B) showed lower scatter profiles compared with Dsg3bri cells (Fig. 4Ae¨C4Ag; Fig. 4Be¨C4Bg), even after 3¨C5 days of culture after cell sorting (supplemental online Fig. 1). Phase contrast microscopic images showed that Dsg3dim cells exhibited a small, round appearance with a relatively larger nuclear-to-cytoplasmic ratio in contrast to Dsg3bri cells, which were larger in size with a relatively small nuclear-to-cytoplasmic ratio (Fig. 4Aa¨C4Ad, 4Ba, 4Bb). This morphology was more evident after a short time period of trypsinization (Fig. 4Bc, 4Bd). By FACScan (Fig. 4Ah, 4Bh) both the Dsg3dim and Dsg3bri primary and NTERT cells retained their relatively low and high expression profile of Dsg3 after overnight culture. Interestingly, the primary keratinocytes exhibited a sustained Dsg3dim and Dsg3bri phenotype after FACS sorting, whereas the Dsg3dim and Dsg3bri phenotype in both of the immortalized cell lines was rapidly reversible (online supplemental Fig. 2).' t/ [" v: e! U: Z- ]( Q. P$ K) G
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Figure 4. Size of sorted Kc. (A): Immunofluorescence (: overnight culture) images of Dsg3dim and Dsg3bri primary Kc. Dsg3dim cells appeared smaller and more adhesive than Dsg3bri cells. (Ae¨CAg): FACS scatter profile of overnight-cultured Dsg3dim and Dsg3bri primary Kc. (Ah): FACS analysis of Dsg3 expression levels of sorted cells after overnight culture. (B): Phase contrast images of NTERT cells (Ba¨CBd) before and after trypsinization after overnight culture. The smaller size and relatively larger nuclear-to-cytoplasmic ratio of Dsg3dim cells is even more apparent after trypsinization (Bc, Bd). (Be¨CBg): Fluorescence-activated flow cytometry analysis (FACS) scatter profile of overnight cultured Dsg3dim and Dsg3bri NTERT cells. (Bh): FACS analysis of Dsg3 expression levels of the sorted Dsg3dim and Dsg3bri cells after overnight culture. Abbreviations: Dsg, desmoglein; exp, expression; FSC, forward scatter; Kc, keratinocytes; SSC, side scatter.# e6 s& Y' x ^% G7 m. e/ s
: u$ ^/ a/ ^5 G4 t" MTo examine the expression levels of other desmosomal cadherins in these sorted Dsg3dim and Dsg3bri cells, 1 x 106 Dsg3dim and Dsg3bri cells, as well as the live control cells, were sorted from the primary and both immortalized cell lines, plated into 6-well plates, and grown for 1 day in culture to allow intercellular junction formation. Then, proteins were extracted, and total cell lysates were subjected to Western blotting analysis. As shown in Figure 5, except for Dsg2 and Dsc2, which showed increased expression in Dsg3dim primary keratinocytes, all of the desmosomal cadherins, together with Dp, appeared to be downregulated in Dsg3dim cells, suggesting that desmosomes were either smaller or less frequent in the Dsg3dim populations from all three cell lines. As expected, the differentiation-associated isoform Dsg1 was not detectable in either HaCaT or NTERT cell lines, whereas Dsc1 was not detectable in HaCaTs. ' ]! {7 [- D0 p6 a: F' W ) z5 a8 x4 E8 X8 h& T9 C2 E1 tFigure 5. Western blot analysis of desmosomal cadherin expression. Except for Dsg2 and Dsc2, which showed increased expression in Dsg3dim primary cells, the majority of desmosomal cadherins and associated proteins were downregulated in this population in comparison with Dsg3bri cells. In immortalized cell lines, Dsg2 and Dsc2 also showed decreased expression in Dsg3dim cells. Dsg1 was not detectable in HaCaT or NTERT cells. Abbreviations: ¦Â-act, ¦Â-actinin; Ct, cycle values; Dsc, desmocollin; Dp, desmoplakin; Dsg, desmoglein; Kc, keratinocytes.% ]1 d! f, l+ \# h
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Overexpression of p63 Isoforms by Dsg3dim Keratinocytes0 X3 r; R$ Y. H! G# v/ T2 J
3 l5 W1 v) ^% H( N; X; L+ }# jBecause the p63 transcription factor, a p53 homolog, has been characterized as an epithelial stem cell marker and shown to play an important role in regenerative proliferation in epithelial development , we analyzed p63 expression in sorted Dsg3dim and Dsg3bri primary cells. Expression of the various p63 isoforms was assessed using real-time PCR and Western blot analysis (Fig. 6). Dsg3dim primary keratinocytes showed a highly significant increase in expression of the p63¦Â and p63 isoforms, particularly at the transcript level, and this was reflected by the large increase in total p63 levels seen in Western blot (Fig. 6) experiments. In agreement with the cell cycle analysis, there was increased expression of the nuclear proliferation marker PCNA in the Dsg3dim cell population. The expression of p63 isoforms in the two immortalized lines was less clear cut, and we were unable to show increased p63 protein in Dsg3dim cells from either line (data not shown). : Z4 w6 e, z! z1 c4 A " \$ L7 t+ R7 X1 h- vFigure 6. Expression of p63 isoforms. Real-time reverse transcriptase polymerase chain reaction and Western blotting analysis of p63 isoform expression in freshly sorted primary keratinocytes. Significantly enhanced expression of p63¦Â and p63 isoforms was seen in the Dsg3dim keratinocytes at the transcript level. This was also evident at the protein level using an antibody that recognized all p63 isoforms. Abbreviations: Ct, cycle values; Dsg, desmoglein; Kc, keratinocytes., w7 u# l' ^, i. Y' r
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Cells with Low Levels of Dsg3 Expression Formed Thicker, More Orderly Stratified Epithelia ' w7 |3 k. t8 ]2 G5 ~) I: L0 } / h* s8 R7 m0 U+ C- \Sorted Dsg3dim or Dsg3bri primary breast and foreskin keratinocytes were plated on DED substrate. As shown in Figure 7, the Dsg3dim primary keratinocytes generated a thicker, and more cellularly diverse, stratified skin equivalent relative to the Dsg3bri cells. After 3 weeks of air-liquid interface culture, the Dsg3dim breast keratinocytes formed a typical stratified epidermis with a well-defined stratum granulosum and stratum corneum (Fig. 7A). Increased thickness in Dsg3dim skin equivalents might be attributable to the relatively greater number of Ki67-positive cells in both the basal and immediate suprabasal layers (Fig. 7C). Furthermore, when the keratinocytes were harvested with trypsin from 2-week organotypic cultures and replated back onto lethally (mitomycin C)-treated feeder layers, focal colonies were observed from Dsg3dim primary keratinocytes but none from Dsg3bri cells after 1 week culture (data not shown). This finding suggested that clonogenic capacity was retained in Dsg3dim primary keratinocytes but was lost completely in Dsg3bri cells. Comparable organotypic experiments with HaCaT cells (Fig. 8) and NTERT cells (Fig. 9) gave similar results with these long-established lines. After 2-week organotypic culture in the air-liquid interface, statistically significantly thicker skin equivalents, as determined by the parameters of mean depth and mean area per section, were observed with Dsg3dim cells compared with Dsg3bri cells seeded at the same density (p .05; Figs. 8A, 8B, 9A, 9B). By 4 weeks, in addition to a dramatic increase in the overall thickness of skin equivalents, these differences were even more apparent (p .001). Interestingly, after 4-week culture in the air-liquid interface, clusters of cells were observed in the deep stratified layer of the skin equivalent from Dsg3dim HaCaT cells, which appeared to be undergoing sebocyte-like differentiation with a pale, enlarged cytoplasm and small nucleus (Fig. 8A). Lipid staining with Oil Red O was performed and confirmed that these cells are lipid positive (Fig. 8C). Further staining for the sebocyte marker keratin 7 showed that they were keratin 7-positive, indicating they had undergone sebocyte differentiation (data not shown). However, this phenomenon was rarely seen in the skin equivalent generated from Dsg3bri cells and was observed much less frequently in the sorted control cells (Fig. 8C). Ki67 staining of 2-week-established organotypic cultures revealed increased numbers of the positive cells in the Dsg3dim skin equivalent with a greater frequency of Ki67-positive cells in the suprabasal layer (Fig. 9A; representative from NTERT cells; arrows and data not shown), indicating that the Dsg3dim cells now were relatively more proliferative. After 3 weeks of air-liquid interface culture, the Dsg3dim cells formed a typical stratified epidermis with a well-defined stratum granulosum and stratum corneum (Fig. 9C and inset). However, these granular cells were not observed, and the stratum corneum was rarely seen, in organotypic culture established from Dsg3bri cells (Fig. 9C). Although only approximately one-quarter enhancement of skin thickness was achieved by doubling the seeding cell number of both Dsg3dim and Dsg3bri populations, significant increases of skin thickness consistently were seen in cultures from Dsg3dim cells as compared with Dsg3bri cells (Fig. 9D). The thickness of skin formed by Dsg3bri cells at the higher plating density was similar to that established by Dsg3dim cells at the lower plating density at the 2-week culture period (Fig. 9D)." B0 V! ]9 \, t3 b/ O- o
n% v8 p4 c3 N% E E: N% o3 F6 {
Figure 7. Organization of regenerated skin equivalents in vitro. (A): Histological sections of skin equivalents formed by primary breast keratinocytes. Reconstituted skin contained polarized basal layer, spinous layer, differentiated granular layer, and stratum corneum when formed by Dsg3dim primary cells. Reconstituted skin formed by Dsg3bri cells lacked the differentiated granular layer and stratum corneum. (B): Quantitative data derived from multiple sections from independent experiments. (C): Histological sections of skin equivalents formed by foreskin keratinocytes (2-week culture) stained with Ki67 showing positive nuclear stained cells in the basal layer (arrows) and immediate suprabasal layer (arrow head). Scale bars = 100 µm. Abbreviations: Ct, cycle values; Dsg, desmoglein; Kc, keratinocytes. # u2 A1 u4 M% X/ z: J+ W # t' h- k0 @+ ?. o: D; N4 BFigure 8. Organization and thickness of skin equivalents from the HaCaT cell line. (A): Histological sections, hematoxylin and eosin staining. Clusters of cells located at the basal compartment of Dsg3dim reconstituted skin showed sebocyte-like differentiation (arrow). (B): Quantitative data derived from multiple sections from independent experiments. (C): Oil red O staining (orange) shows positive staining in sebocyte-like differentiated cells. Scale bars = 50 (A) and 25 µm (C). Abbreviations: Ct, cycle values; Dsg, desmoglein; wk, week. : u- n; \4 G L8 N$ [$ i- a% X& z- {' k# z; z
Figure 9. Skin equivalents from the NTERT cell line. (A): Histological sections stained with hematoxylin and eosin and Ki67 (2-week cultures). Ki67 nuclear positive cells were present in the basal layer and the suprabasal layers of skin reconstituted by Dsg3dim cells, but suprabasal positivity was not seen in skin reconstituted by Dsg3bri cells. (B): Quantitative data derived from multiple sections from independent experiments. (C): Organotypic culture after 3 weeks with a well-developed granular layer in Dsg3dim reconstituted skin. (D): Quantitation of histological sections of skin formed by sorted cells at different plating densities. Cells were plated at 1.5 x 105 (low density) or 3 x 105 (high density) per ring and grown for 2 weeks. Scale bars = 50 µm. Abbreviations: Ct, cycle values; Dsg, desmoglein; wk, week.7 y" K$ m0 ?6 x8 Z. E+ F
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To rule out the possibility that enhanced thickness of the regenerated skin by Dsg3dim cells resulted from desmosomal dysfunction, frozen sections of organotypic culture from each group were dual labeled for Dsg3 and Dp and subjected to confocal analysis. As demonstrated in Figure 10, well-developed desmosomes and highly organized desmosome structure were seen in the skin formed by the Dsg3dim primary keratinocytes after 2¨C3 weeks of culture. Dsg3 and Dp largely were colocalized at the intercellular junctional area. Thus, desmosome assembly occurs even though the cells have low levels of Dsg3 at the time of seeding. Recent studies have suggested that p63 genes are responsible for these processes . Conversely, the regenerated epidermis formed by Dsg3bri primary keratinocytes showed relatively low levels of Dp expression and less colocalization of Dsg3 and Dp at the cell-cell interface. Taken together, results from this functional assay provided further evidence to support the notion that Dsg3dim cells had a stem/progenitor cell-like potential whether derived from the primary strains or from the immortalized nonmalignant cell lines. 9 [& n. w% A8 Z6 G q V6 H* V& n& R5 ]( l! i) P: [* E
Figure 10. Confocal microscopy of reconstituted skin. Frozen sections of the reconstituted skin were dual-labeled for Dsg3 (green) and desmoplakin (Dp; red) using mouse antibody (Ab) 5H10 for Dsg3 and rabbit Ab AHP320 for Dp. (A): The reconstituted skin formed by Dsg3dim primary cells showed typical punctate desmosome structure and enriched peripheral colocalization of Dsg3 and Dp (Aa, Ag). Interestingly Dsg3bri cells overall showed weaker Dsg3 and Dp staining, in particular Dp (Ae), and a lack of desmosomal protein staining was evident in the dermal-epidermal junctional area (Ab, Ah). Sorted control cells were more similar to Dsg3dim cells (Ac, Ai). (B): Reconstituted skin from HaCaT (Ba¨CBc) and NTERT (Bd¨CBf) showed strong staining and linear peripheral colocalization of Dsg3 and Dp whether derived from Dsg3dim or Dsg3bri cells. Scale bars = 20 (Aa¨CAc, B) and 10 µm (Ag¨CAi). Abbreviations: Ct, cycle values; Dsg, desmoglein; wk, week. $ ^7 S+ B; A L" W% r) X: R * G# s" ?/ S- t3 R& ?) D6 qSP Cells Showed No Correlation with Dsg3 Expression Levels & b% c1 g/ E" n+ E) F ! ^3 C9 l6 E/ ^7 T8 qIt is reported that SP cells possess stem cell properties when derived from certain tissues, such as bone marrow . * d; M" K# _" x, Q2 y: G7 z6 o+ u' k
Figure 11. Side population (SP) cell analysis of various cell populations. (A): Example of specific SP detection in HaCaTs showing that the pattern of these cells can be blocked by the specific inhibitor Res (10 µM). (B): The gated Dsg3dim and Dsg3bri cells were displayed separately for SP cell profiles and their percentages from both the primary and immortalized keratinocyte cell lines calculated (details in Materials and Methods). No apparent correlation was observed between SP and Dsg3 expression levels in any cell line. Abbreviations: Dsg, desmoglein; Kc, keratinocytes; Res, Reserpine. . V) i: p# [; v/ k, i, ^- o & R3 a9 i4 G2 r, n# G: J/ n% I/ ]DISCUSSION! k& x) J& C; C/ b, H' x" _( n! n6 x
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Traditionally, desmosomes have been considered simply anchoring junction structures, providing mechanical resilience by maintaining tissue integrity through direct coupling to the intermediate filament network. However, increasing evidence suggests that, like the adherens junctions, desmosomal functions extend beyond mere structural and mechanical properties .6 m. P; I! T0 C1 Q! u
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Keratinocyte stem cells are self-renewing, quiescent, and small in size; they occur at a low incidence (1%¨C10%); and they have the ability to regenerate the tissue of origin . Primary adult breast keratinocytes from the same source were used in this experiment, and the CFEs were comparable for the two populations (Fig. 1). We do not know whether there is overlap between the two populations, but the very similar clonogenic properties make it plausible that they represent a similar subgroup of basal cells. In addition, these Dsg3dim cell populations had their cell cycle (Fig. 2A), cell morphology (Fig. 4A), p63 expression (Fig. 6A), and SP incidence (Fig. 11) analyzed as well as, most importantly, their in vitro skin reconstitution capacity. We found that Dsg3dim cells were small and adhesive (online supplemental Fig. 2) and more actively cycling (Fig. 2), and showed increased p63 and PCNA expression (Fig. 6), features of putative stem/progenitor cells. Using in vitro organotypic culture on de-epidermalized dermis populated with consistent numbers of primary fibroblasts from human foreskin (p4¨C7), we demonstrated that the skin equivalents generated by Dsg3dim cells exhibited significantly thicker skin and a well-ordered epidermal structure containing the stratum granulosum and stratum corneum; a feature distinct from the structures formed by Dsg3bri and sorted control cells (Fig. 8A, 8B). This finding indicates that this population of cells not only is self-renewing but also is capable of giving rise to hierarchical keratinocyte progenies; these are traits of epidermal stem cells. The observed high proliferative potential and active cycling of Dsg3dim cells as indicated by positive nuclear Ki67 staining in skin (Fig. 7C) and elevated p63 and PCNA expression (Fig. 6A), as well as the functionally normal assembled desmosomes, may contribute to this enhanced skin thickness. P* y' m$ W! b. k% ~8 _9 {% a- U
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The concept of retention of stem cell hierarchies has been proposed . To explore the potential of using Dsg3 as a marker in immortalized keratinocytes, which have been established in tissue culture for some time, we used HaCaT and NTERT lines. By adopting an enhanced FACS stringency (5% Dsg3dim and Dsg3bri cells), we demonstrated enhanced CFE for Dsg3dim populations from both cell lines (Fig. 3). The differences in CFE relative to sort-control cells were small and did not achieve significance (Fig. 3), but both cell lines have been maintained for in vitro growth for extended periods. In general, Dsg3dim NTERT cells resembled primary keratinocytes more closely than did Dsg3dim HaCaT cells. For example, cell cycle analysis showed that Dsg3dim HaCaTs differed from Dsg3dim NTERT in that the former were more quiescent, whereas the latter exhibited more active cycling relative to their Dsg3bri compartments (Fig. 2). However, in our in vitro skin reconstitution analyses for these two keratinocyte lines, we found that, like the primary cells, Dsg3dim cells generated thicker skin than did Dsg3bri cells, and such reconstituted skins appeared more differentiated with, for example, clear evidence of sebocyte-like formation in HaCaT skins and a granular layer and stratum corneum in NTERT skins (Figs. 7¨C9). Again, as shown by Ki67 staining, the high proliferative potential and active cycling state of Dsg3dim cells likely is responsible for this enhanced skin thickness (Fig. 9A and data not shown).) H. g4 }+ f9 l1 y0 r' C
! k, F, Z: `( P7 S( \" O% aThe transcription factor p63 belongs to the p53 gene family, and is essential for the early stages of skin development. A critical role for p63 in stem cell maintenance, or in the commitment to keratinocyte stratification, has been established from the generation of transgenic mice in which the p63 gene has been knocked out . We showed in this study that expression of all isoforms of p63 was significantly higher, at both the transcript and protein levels, in the sorted Dsg3dim primary keratinocytes (Fig. 6A) and the NTERT line (data not shown). These reverse correlations between p63 and Dsg3 expression in primary and NTERT cell lines provides further support for the possibility that low expression of Dsg3 can enrich for epidermal stem cells, while also providing confirmatory evidence for the existence of stem/progenitor cell-like populations in the immortalized keratinocyte cell lines. # ^. i; B; i7 |" E. m2 D* o 3 v4 ?2 ^6 O' D" r, |- T. JWe have monitored the stability and progressive changes in Dsg3 expression after FACS sorting and incubation of cells in culture. We found in NTERT cells that the steady-state Dsg3 expression levels underwent progressive changes when cells were plated in culture. Thus, low or high Dsg3 expression levels, in the sorted Dsg3dim or Dsg3bri cell populations, respectively, initially were retained after the overnight culture. However, these differences gradually diminished during culture and Dsg3 expression levels in each population eventually shifted to overlap with the sorted control cells (supplemental online Fig. 1). Why this should occur it not clear. It is plausible that there is an intrinsic mechanism in the control of homeostasis of these cultured immortalized cell lines, but further studies are necessary to elucidate the basis of this phenomenon. j" M1 C" Z( Q% t8 z. y1 K* u" \/ n* R r
The characteristics of SP cells are a reflection of the ability of such cells to efflux DNA-binding dyes and often is claimed as a trait of stem cells . 5 O2 `/ R2 ?) v! n7 G* N; P O0 x! F; i3 ?6 KIn summary, we demonstrate in this study that Dsg3dim cells from both primary and immortalized keratinocyte lines exhibit stem/progenitor cell properties as defined by their small size, active and prolonged cycling state, high CFE, elevated p63 expression and the ability to reconstitute full functional skin in vitro. These features show no correlation with SP characteristics.7 a. Z& ]+ w( F5 u& h" r
6 w1 ?. M& J+ N# [7 mDISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST8 t5 K8 s3 v- a8 S. o
! y& K' S- w3 Y$ E3 T. E* K }* \The authors indicate no potential conflicts of interest. ( n6 Q! u2 Y9 q5 ?" I3 J+ L4 B& \+ J: m$ p- O8 O8 Y$ [
ACKNOWLEDGMENTS ! F* D; o( {9 T( R o* R" w! D9 F) f3 t& l
We thank Masayuki Amagai in particular for providing the 5H10 antibody; David Garrod for his support and for providing antibody reagents; Mike Philpott for his antibody reagents; Robin A.J. Eady, Kathleen J. Green, and Fiona Watt for their support during this Fellowship; Kathleen J. Green for her critical reading of the manuscript; members in FACS Laboratory, Cancer Research UK, and London Research Institute, in particular Derek Davies; and to Hart Lab members, in particular Vera Fantl, Kairbaan Hodivala-Dilke, Richard Grose, and Louise Reynolds. This work was funded by an MRC Career Development Fellowship to H.W. - ?3 p6 { u" u+ N% K) X 【参考文献】' O% d- ~% q3 |4 T* }
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