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Published as doi: 10.1096/fj.06-5880com.
 (The FASEB Journal. 2007;21:2050-2063.)
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Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages

Lauren Crigler*, Amita Kazhanie*, Tae-Jin Yoon{dagger}, Julia Zakhari*, Joanna Anders*, Barbara Taylor* and Victoria M. Virador*,1

* Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA; and

{dagger} Department of Dermatology, College of Medicine, Gyeongsang National University, Jinju, Korea

1Correspondence: 9000 Rockville Pike, Bldg. 10, Rm. 2A33, Bethesda, MD 20892, USA. E-mail: vvirador{at}helix.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The skin contains two known subpopulations of stem cells/epidermal progenitors: a basal keratinocyte population found in the interfollicular epithelium and cells residing in the bulge region of the hair follicle. The major role of the interfollicular basal keratinocyte population may be epidermal renewal, whereas the bulge population may only be activated and recruited to form a cutaneous epithelium in case of trauma. Using 3-dimensional cultures of murine skin under stress conditions in which only reserve epithelial cells would be expected to survive and expand, we demonstrate that a mesenchymal population resident in neonatal murine dermis has the unique potential to develop an epidermis in vitro. In monolayer culture, this dermal subpopulation has long-term survival capabilities in restricted serum and an inducible capacity to evolve into multiple cell lineages, both epithelial and mesenchymal, depending on culture conditions. When grafted subcutaneously, this dermal subpopulation gave rise to fusiform structures, reminiscent of disorganized muscle, that stained positive for smooth muscle actin and desmin; on typical epidermal grafts, abundant melanocytes appeared throughout the dermis that were not associated with hair follicles. The multipotential cells can be repeatedly isolated from neonatal murine dermis by a sequence of differential centrifugation and selective culture conditions. These results suggest that progenitors capable of epidermal differentiation exist in the mesenchymal compartment of an abundant tissue source and may have a function in mesenchymal-epithelial transition upon insult. Moreover, these cells could be available in sufficient quantities for lineage determination or tissue engineering applications.—Crigler, L., Kazhanie, A., Yoon, T-J., Zakhari, J., Anders, J., Taylor, B., Virador, V. M. Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages.


Key Words: epidermal precursors • murine organotypic culture • keratinocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SKIN BIOENGINEERING HAS ADVANCED the production of in vitro human skin reconstructs for trauma patients with damaged areas too large to repair by the natural wound-healing process (1) . However, the growth of mouse skin reconstructs in vitro has not been as successful. Three-dimensional murine skin cultures would be useful to specifically study epidermal-mesenchymal cell signaling. Through the power of mouse genetics and using primary cells derived from genetically altered mouse skins, the underlying biology of a number of diseases would be studied. In vitro skin regeneration would be a useful model for the study of genetic determinants of wound healing. Human and mouse skin differ in epidermal thickness and appendage content, and thus it is possible that the niche responsible for epidermal renewal during wound healing may be different in the mouse and human (2 3 4) .

Many adult tissues contain populations that have renewal ability under circumstances such as trauma, disease, or aging (1 , 5) . There is also ample evidence that a population of skin-specific precursors capable of restoring skin integrity upon insult resides in a niche identified as the bulge region of the hair follicle (6 7 8) . Another such population, most likely responsible for normal epidermal homeostasis, is known to reside in the interfollicular basal layer of the epidermis (9 , 10) .

Epidermal-mesenchymal interactions are necessary to provide the context for tissue development (11) , differentiation (12) , and tissue renewal on injury (13 , 14) . In skin, the mesenchyme immediately adjacent to the hair follicle epithelium has an inductive effect on follicle development. Specialized mesenchymal cells, called dermal papilla cells, are embedded in the lower portion of the hair follicle and are surrounded by undifferentiated epithelial cells. These fibroblast-like dermal papilla cells produce and respond to the signals that regulate hair cycles (15 , 16) . Hair could be regrown when hair follicles were grafted onto nude mice with dermal papilla cells and not in their absence (17) . Furthermore, characteristics of the evolving hair were determined by the species of origin of the dermal papilla cell preparations. These findings could be expanded and explained with an in vitro model for murine epidermis.

In establishing such a culture system, we were unsuccessful in adapting methods reported for human epidermal cells in human skin reconstructs. However, selected epidermal subpopulations had the ability to survive while cultured in cell-to-cell contact with a dermal component at the air-liquid interface under reported culture conditions (18) . During the course of these studies, we identified a dermal subpopulation that could by itself expand in culture and express epidermal markers while cultured in the absence of contact with a dermal component at the air-liquid interface under epidermal growth conditions. This subpopulation had the ability to survive in minimal medium conditions over time, a property that might be expected of a progenitor population. For the purposes of these studies, a progenitor cell has the capacity to create progeny that are more differentiated than itself and the ability to survive and propagate under minimal culture conditions. Further analysis of the properties of our isolated subpopulation indicated characteristics consistent with a multipotent mesenchymal progenitor population.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue culture media and other chemicals
Minimum essential medium (S-MEM) without calcium (Life Technologies, Inc., Rockville, MD, USA) was used to prepare the two main types of culture media. Standard medium was S-MEM containing 8% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA, USA) treated with Chelex resin (Bio-Rad Laboratories, Hercules, CA, USA). High calcium medium contained 1.4 mM Ca2+. This medium was typically used to plate and attach keratinocytes or hair follicle buds and to culture some of the dermal-derived populations. Low calcium medium contained 0.05 mM Ca2+, typically used when cultures were grown under submerged conditions to allow for basal keratinocyte proliferation. In some experiments, cells were cultured in medium containing 2% serum. All other chemicals were from Sigma Chemical Company (St. Louis, MO, USA) unless stated otherwise. KGF (R&D Systems, Minneapolis, MN, USA) was used at 8.4 ng/ml in the 3-dimensional cultures.

Skin fractionation
Cellular subfractionation from both epidermis and dermis of neonatal BALB/c mice is summarized in Fig. 1 A and explained in detail in supplemental Methods.


Figure 1
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  		Figure 1. A) Simplified schematic of fraction preparation (for more detailed schematic, see Supplemental Fig. 1). B) Flow cytometry analysis of SP in epidermal subfraction B and dermal fractions D and E. C) Contour plots of CD34 and CD49f-positive populations contained within the CD117+ population of epidermal (subfraction A) and dermal fraction E. Percent CD117+ cells were 2.7 and 6.2, respectively. D) MRP8 staining of Epi, DHF, D, and E showing percent positive cells from 5 independent fields; DHF and D were negative. Top right panel: MRP8-stained neonatal skin; arrow points at area shown in inset. For description of epidermal subfractions A and B, see Supplemental Materials and Methods.

Epidermal fraction (Epi) was prepared as described (19) . Dermal fractions were made following a previously described method of preparing fibroblasts from the skin (20) . Briefly, dermises were separated from the epidermis by overnight trypsin incubation and chopped coarsely in 2 ml of 0.35% collagenase (Worthington, Lakewood, NJ, USA) per dermis. Collagenase suspension was incubated with agitation in a water bath at 37°C for 30 min, and DNase I (Worthington; 20,000 U/ml in PBS) was added at 125 µl/ml of collagenase suspension and incubated for 10 min. This suspension was filtered through a coarse 100 µ Nytex filter and diluted to 100 ml per five dermises. Tubes were centrifuged at 800 rpm for 5 min, and the supernatant was placed in new tubes to be centrifuged at 1400 rpm for 5 min. The pellets obtained at the end of this spin were pooled and designated fraction D.

The 800 rpm pellet was dissociated and centrifuged at 300 rpm for 5 min, yielding dermal-derived hair follicles (DHF). Supernatant from the 300 rpm pellet was centrifuged twice at 1000 rpm for 5 min, then both pellets were pooled and designated fraction E.

Organotypic (3-dimensional) cultures
Cells were allowed to adhere overnight to the upper side of an insert membrane while suspended in 1.4 mM Ca2+ medium. Insert membranes used were Millipore® (Multiwell), uncoated, 12 mm diameter, abbreviated Millipore uncoated membrane (MU) (0.4 or 3.0 µm pore size used), and collagen-coated Transwell®-COL Inserts (Corning Inc., Acton, MA, USA), 12 mm diameter, abbreviated Costar-coated membrane (CC) (0.4 or 3.0 µm pore size). These membranes were coated with an equimolar mixture of collagen (types I and III) derived from bovine placentas. The medium was changed to 0.05 mM Ca2+ and the cultures were submerged for 72 h. Then the upper side of the membrane was air exposed for 72 h to promote differentiation while a dermis in contact with the membrane was submerged in 0.25 mM Ca2+ medium containing 40 µg/ml ascorbic acid (18 , 21) . Cultures were fixed on day 6 in 3% formalin for 5 min and stored in 70% ethanol before histological processing. Dermatological punches and histological cassettes and sponges (all from Daigger, Vernon Hills, IL, USA) were used. Viability and morphology of the cultures were assessed by H&E staining of random sections. In some experiments, cells from fractions B, D, and E were attached on MU membranes and submerged in low Ca2+ medium containing KGF at 8.4 ng/ml. After 7 days, membranes were air exposed in high Ca2+ medium containing 40 µg/ml ascorbic acid. This exposure lasted 48 h to promote differentiation, followed by one or more rounds of submerged conditions to mimic the wound-healing environment. Cells were then fixed at different time points.

Cell sorting and analysis by flow cytometry
Cells were stained with antibodies to CD49f and CD90 (FITC, PharMingen, San Jose, CA, USA), CD34 and CD45 (PE, PharMingen), CD11b (PE) and CD117 (CyChrome, Caltag, Burlingame, CA, USA), and CD105 (Chemicon, Temecula, CA, USA), using standard procedures. Analysis was carried out on an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Cells were stained for the side population (SP) phenotype (22) by incubating cells (1x106 cells/ml in DMEM with 2% FBS) with 3 µg/ml Hoechst 33342 (Molecular Probes, Eugene, OR, USA) for 90 min (37°C, 5% CO2, 100% relative humidity). The cells were washed once in cold DMEM with 2% FBS and resuspended for sorting. Cell sorting was performed on an FACSVantage SE with DiVa option (BD Biosciences). Percents were compared from the unfractionated epidermis and dermis and individual fractions.

Cell survival and colony formation assays
Unsorted cells, SP, and CD49f-positive cells from fractions were placed in a monolayer culture using media containing different levels of calcium and serum. After ~4 wk, MTT analysis was performed to stain mitochondria to locate any living cells (Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer’s instructions. Prior to solubilization, bright-field pictures were taken of representative fields in all cultures. To study proliferative capacity, equal numbers of cells (enough to produce confluent monolayers) were plated in duplicate wells of 24-well plates. Epi were cultured in low calcium medium and dermal-derived fractions were cultured in high calcium medium. Cells were serially passaged until viable cells were no longer observed in the wells. To study whether some contaminating epidermal progenitors could affect the proliferative capacity of the dermal fractions, DNase was omitted from the dermal preparations, thus increasing keratinocye cross-contamination of the fractions.

Immunoblotting and immunostaining
Freshly isolated cell pellets were lysed with 0.25 M Tris HCl buffer, pH 6.8, containing 3% ß-mercapto ethanol and 5% SDS; cells that migrated through a membrane and adhered to tissue culture plates were lysed in situ. Electrophoresis was run on NuPage gels (Invitrogen, Carlsbad, CA, USA) and proteins were electrophoretically transferred onto PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA). Antibodies for immunoblotting included anti-keratins 1, 5, 10, 14 (Covance, Princeton, NJ, USA), all at 1:2000 dilution; and actin monoclonal (C4, Chemicon), rabbit polyclonal to ß-tubulin and to POU3F2 (Abcam, Canbridge, MA, USA) at 1:10000 and 1:1000 dilution, respectively. Secondary antibodies were anti-rabbit or anti-mouse IgG horseradish peroxidase linked (Amersham, Piscataway, NJ, USA). Chemiluminescent detection was done with West Pico (Pierce, Rockford IL, USA).

Immunostaining of paraffin-embedded sections was performed according to standard protocols using deparafination with Histochoice (Amresco, Solon, OH, USA) and antigen retrieval with 0.01% trypsin or postfixing frozen sections with 4% paraformaldehyde. Immunostaining of freshly isolated cells was performed according to standard protocols on flash-frozen cell spreads postfixed with 4% paraformaldehyde for 10 min. Primary antibodies were anti-keratins K14-FITC, K-10-FITC (Covance) at 1:200 dilution, anti-involucrin (a gift from Dr. Richard Eckert) at 1:500 dilution, anti-K15 (BD-PharMingen) at 1:100 dilution, anti-MRP8 (a gift from Dr. Joannes Roth) at 1:200 dilution, and {alpha}pep8/DCT (a gift from Dr. Vincent Hearing) at 1:500 dilution. Anti-smooth muscle actin (1:300) dilution was from Biocare Medical (Concord, CA, USA). Antidesmin was from DAKO (Carpenteria, CA, USA), used at 1:500 dilution. Secondary antibodies were either Alexa-488 or Alexa-594 conjugates (Molecular Probes). For confocal images, fluorescent cells were examined with a Zeiss LSM 510 Confocal Microscope (Carl Zeiss Inc., Thornwood, NY, USA) using a 40 x 1.3 NA Plan Neofluar objective. Images were collected sequentially where the FITC, TX Red, and DAPI signals were collected with a BP 505–550 filter, LP560 filter, and BP 385–470 filter after excitation with 488 nm, 543 nm, and 364 nm laser lines, respectively.

Multipotential differentiation assays
Differentiation assays for the osteogenic, adipogenic, myogenic, and chondrogenic lineages were performed according to refs. 23 , 24 . Before induction of differentiation, cultures containing epidermal subfraction B were grown to confluence in 0.05 mM Ca2+ and cultures containing fractions D or E were grown to confluence in 1.4 mM Ca2+-containing medium.

Osteogenic differentiation was induced with DMEM containing 10% FBS, 1% penicillin/streptomycin, nonessential amino acids, 50 µg/ml ascorbic acid, 10 mM ß-glycerol phosphate, and 10 nM dexamethasone for up to 3 wk (24) . Mineralization was visualized by staining with Alizarin Red S (2% w/v Alizarin Red S adjusted to pH 4.1 using ammonium hydroxide) for 5 min at room temperature, followed by a wash with water. Osteoblasts were shown by the formation of calcium-rich hydroxyapatite in the extracellular space, which stains orange-red with Alizarin red S. Adipogenic differentiation was induced by treating the cultures with DMEM containing 10% FBS, 1% penicillin/streptomycin, nonessential amino acids, 10% rabbit serum, 20 µM ETYA, 10 nM dexamethasone, and 25 µg/ml insulin (24) . For subsequent feedings, the medium lacked dexamethasone, and rabbit serum concentration was increased to 15%. Adipocytes were visualized by Oil Red O stain. Chondrogenic differentiation was induced by plating 5 million cells per well of a round-bottom 96-well plate. Cells were fed with 1.4 mM Ca2+ medium containing 50 µg/ml ascorbic acid, 100 nM dexamethasone, and 1 ng/ml TGF-ß (24) . After 3 wk in culture, cell clumps were harvested with a wide bore pipette tip, spread onto poly-L-lysine-coated slides, flash frozen, and postfixed with 4% paraformaldehyde, followed by staining with 0.1% Safranin O. Myogenic differentiation of freshly isolated fraction E cells plated at confluency was observed after infection with adenovirally expressed GFP (25) . Infection occurred in serum-free medium containing 2.5 µg/ml polybrene (Sigma) at 10–30 mulitiplicity of infection for 30 min at room temperature. Fresh complete medium was added thereafter. Cells were observed and photographed daily. Movie was acquired using an LSM 510 confocal microscope.

Array analysis
Focused arrays (GEArray S series Mouse Stem Cell Array, SuperArray, Bethesda, MD, USA) were used to establish the signature of epidermal and dermal subpopulations after 1 day in culture. The arrays contain 258 known genes that encode markers expressed by stem and differentiating cells as well as varied growth factors and other regulators of stem cell behavior. Cells from epidermal subfraction B, from dermal fractions D, E, and from a combination of D and E (termed Fb) were isolated and plated overnight in high Ca2+-containing medium. Total RNA was prepared with Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Arrays were hybridized according to manufacturer’s protocols for the nonradioactive format. For analysis, the manufacturer’s recommended method was used (www.superarray.com), which involved densitometric analysis of the 30 s exposures to X-ray film and superimposing figures with a grid pattern for quantification of raw pixels. From raw pixel data, most of row 17 (which contains no printed cDNAs) was used to subtract background. Once individual pixel values were obtained, the results were processed with Excel software. Rpl13a was selected as the housekeeping gene to normalize data, as Rpl13a was the least variable housekeeping gene among all four fractions. Then individual values were divided by the averaged value for Rpl13a in each fraction. A number of genes that were marginally up-regulated in only one or two fractions were removed from the analysis by setting up the threshold at 20% expression.

Statistics
Numerical values are expressed as mean ± SE. For a comparison among all groups in the study, multiple comparisons of more than two groups were done by 1-way analysis of variance (ANOVA). Unpaired Student’s t test was used for comparisons between two groups; P values are reported.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and characterization of epidermal and dermal subpopulations
In the search for murine skin subpopulations that would provide a good model for epidermal formation in vitro, we adapted sedimentation and differential adhesion protocols already used in our laboratory (26) to isolate cellular subpopulations from neonatal mouse skin. Flow charts of the protocols and characterization of all the fractions are presented in supplemental Fig. 1 and supplemental Table 1. By immunoblot, unplated, freshly prepared fractions contained varying amounts of basal and differentiated keratins (supplemental Table 1 and supplemental Fig. 1). Keratin 1 or 10 and K14 were detected mostly in epidermal and hair follicle-derived fractions. In the dermal-derived fractions, K1 and K10 were present in fibroblast (D and E) fractions, which could indicate a small keratinocyte contamination to these fractions; however, we noticed that keratinocyte-positive populations expanded from E upon culture in high or low calcium (supplemental Table 1 and Fig. 4 ) and in minimal medium conditions (Fig. 5 ). CD49f (integrin {alpha}6) and CD34 were used as markers of early keratinocyte precursors (27 28 29) . CD117 (c-kit) was used as a potential bulge marker (30) , as were K15 (31) and p63 (32 , 33) . All epidermal fractions had equivalent, nearly 100% expression of CD49f. Dermal fractions derived from hair follicles (26) had lower amounts of CD49F-positive cells (~30%). The dermal fibroblast fractions D and E contained the highest amount of CD34+ (~45%) cells and the lowest amount of CD49f+ (~10%). CD117+ cells had low abundance overall and were slightly higher in dermal fractions D and E, although not statistically significant. These CD117+ cells might be dermal melanocyte precursors (34) . Moreover, in epidermal fractions, CD117-positive cells were CD49 intermediate bright, whereas in dermal fraction E the CD117-positive cells were distinct from CD49f and CD34, thus arguing that the CD117+ population would not come from epidermal contamination of the dermal fractions (Fig. 1C ).


Figure 2
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  		Figure 2. Dermal fraction E has the potential for differentiation into epidermis in a 3-dimensional culture. A) Epidermal subfraction B was cultured under submerged conditions in 0.05 mM Ca2+, then air-exposed on a fresh dermis on an MU insert (0.4 µm) in 1.4 mM Ca2+ for 3 days. B) A mixture of fraction B and E was cultured as in panel A. For a description of epidermal subfraction B, refer to supplemental Materials and Methods.


Figure 3
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  		Figure 3. Survival and expansion of isolated fractions in monolayer culture. A) Isolated and/or SP-sorted fractions were plated in 0.05 mM Ca2+/2% serum containing SMEM medium for 1 month in triplicate wells, then an MTT assay was carried out. Cell survival was calculated from abs 490–650 (left panel) or abs 490–650/cell input (right panel). In a separate experiment, equal numbers of SP cells were plated and results followed the same pattern. *Significant difference in the survival of individual fractions compared with fraction E. Nonsorted fractions, P = 0.0018 B vs. E, P = 0.0244 C vs. E. Sorted fractions P = 0.0036 B vs. E, P < 0.0001 for both F and G vs. E. B) Fractions B, D, and E cells sorted based on CD49f were placed in 24-well plates at 10,000 cells/well and cultured with 2% serum and either 0.05 mM or 1.4 mM Ca2+ for 1 month, then an MTT assay was carried out. In CD49f-positive fractions, P = 0.00642 B vs. E. Right set of panels show MTT-stained cells. C) SP-sorted dermal subpopulations were plated in duplicate wells of a 24-well plate at 10,000 cells per well in minimal medium containing 1.4 mM Ca2+. In SP-sorted fractions, P < 0.0001 D vs. E. Right panel: MTT-stained cells. These experiments were repeated at least twice; one representative set of results is shown. For a description of epidermal and dermal subfractions, refer to supplemental Materials and Methods.


Figure 4
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  		Figure 4. Dermal fraction E does not require underlying dermis to survive in 3-dimensional cultures under epidermal conditions. Fraction E was plated in a 3 µm MU insert in high calcium-containing medium to favor attachment. The next day the medium was changed to low calcium-containing KGF and cultured under submerged conditions for 1 wk, followed by air exposure as detailed below. A) Air exposure for 2 days in medium containing 0.24 mM Ca2+. B) Air exposure for 6 days. C) Air exposure for 9 days. D) Higher magnification of the 2 days culture. E) K14-FITC stain of 2 days culture. F) Double stain K14-FITC and K10 with a Texas Red anti-rabbit secondary; some colocalization is observed as yellow signal (arrowhead). G) Fraction E was cultured, submerged, and air-exposed as in panel A, then submerged again for 4 days and air-exposed for an additional 7 days. H) K14-FITC stain of sample in panel G. I) Double stain of sample in panel G with K14-FITC and K10 with a Texas Red anti-rabbit secondary; yellow shows increased areas of colocalization of the Texas Red and FITC signals compared with panel F (arrowhead). J) Double stain of sample in panel G with K14-FITC and involucrin with a Texas Red anti-rabbit secondary. K) Fractions D and E were on MU inserts (3 µ pore size). Cells that migrated to the plastic well below were cultured in submerged conditions for 8 days with either 0.05 mM (Lo) or 1.4 mM (Hi) Ca2+ medium. Fraction B was cultured in 0.05 mM Ca2+ for 3 days as a control. Lysates for one set of wells of two replicates are shown. B-actin is shown as a loading control. Quantitation of averages for both replicates is shown.


Figure 5
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  		Figure 5. Differentiation assays. A) Osteogenesis differentiation, Alizarin Red stain. B) Adipogenic differentiation, Oil Red O stain. Fraction B did not survive these conditions. C) Chondrogenesis differentiation, Safranin O stain. D) Myogenic differentiation of adenovirally infected (GFP) confluent cultures grown in complete MEM medium for 3 wk; some cells within fraction E had myocyte morphology and were contractile (see supplemental video). E) Staining of cells cultured in MEM medium containing 0.05 mM Ca2+ and 2% FBS for a month. K5 denotes an active proliferating population expressing the basal keratinocyte marker K5. Tyrp2 denotes the melanoblast/early melanocyte marker dopachrometautomerase.

The side population (35) has been associated with mesenchymal stem cells, although skin stem cells may not share this marker (36) . SP-positive cells were much more abundant in epidermal fractions and in hair follicle buds from the dermis. Dermal fractions D and E presented the most reproducible data on SP; within them, fraction E had a higher percentage, albeit not significantly different from D (Fig. 1B) . These data suggested itwas possible to isolate early precursors but not keratinocyte stem cells by SP analysis of total skin (Fig. 1A ). In fractions D and E, CD34+ and CD49f+ represented distinct subpopulations within the SP. There were slightly more SP cells in E than in D, and 10% more CD34/CD49f double negative cells in fraction E; thus, we concluded that this fraction may contain a subset of precursors that is absent from dermal fraction D.

We used immunofluorescence of freshly isolated cell spreads to investigate K15, a marker of hair follicle bulge region, and p63 (supplemental Table 1 and Fig. 6 E). The percent of K15+ cells was higher in Epi and DHF and very low in fractions D and E (P<0.0001 Epi vs. E). Likewise, p63 distinguished Epi and DHF from fractons D and E (Epi vs. E, P=0.0051; D and E were not significantly different). MRP8, an inflammation-related molecule found in endothelial cells and keratinocytes (37) , was found in low amounts in Epi; within this fraction, it was present in fraction C. Most cells in dermal fraction E were positive (Fig. 1D ), and a small amount was detected in dermal subfraction G (see supplemental Table 1). The mesenchymal marker CD105 (38 , 39) was significantly higher in the hair follicle-containing populations Epi and DHF (Epi vs. E, P=0.0129; D vs. E, nonsignificant). CD90 (40) had a higher percent in the dermal fractions and slightly higher in E than D, albeit not significant (CD90 Epi vs. E, P=0.0032, D vs. E, nonsignificant). As MRP8 might be associated with immune infiltrates, we confirmed by CD11b that populations containing granulocytes, macrophages, and monocytes were not significantly different among the fractions. The sum of these findings suggested that our protocol selectively enriches for epidermal precursors associated with either the hair follicle bulge or dermal papillae, or with a yet uncharacterized mesenchymal progenitor cells present in the dermis.


Figure 6
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  		Figure 6. Additional characterization of the fractions. A) One min exposure of membrane arrays (for easier identification of genes). Some examples are highlighted: red indicates genes with a positive value in fraction E, blue indicates genes with positive values in more than one fraction and not in fraction E. Gene intensity values were calculated from a 30 s exposed film in which housekeeping genes (bottom row) were not saturating. B) Not all housekeeping genes in the array were evenly present in all fractions, thus Rpl13a was chosen for normalization. C) Bar graph showing percent contribution of each fraction to total expression levels; most genes that were negatively regulated belong to fraction E. D) Bar graph showing percent contribution of each fraction to the total expression levels in up-regulated genes; only genes in which fraction E had a positive value are shown. E) Immunostaining of freshly isolated flash-frozen fractions with markers p63 and K15. Quantitation of positive stained cells from five random fields is shown below; *significant differences between Epi and E (p63, P=0.0051; K15, P<0.0001). F) Immunoblots of proteins detected in fraction E in the array (K14, Fig. 6 B) or below detection in E (Pou3f2, Fig. 6 C). Left panel shows quantitation of their relative levels by NIH image.

Growth of the fractions in three dimensions
Epidermal populations containing keratinocytes and hair follicle buds grew occasionally and not reproducibly in 3-dimensional cultures. Reproducibility improved when we selected hair follicle buds (epidermal subfraction A) and when we used membranes in contact with fresh dermis (18) (Supplemental Fig. 2). We compared the growth of epidermal cells (subfraction B) with dermal fractions D and E. We consistently observed that fraction E but not D was able to form monolayers that stained H&E positive and appeared viable. Epidermal cells alone did not survive (Fig. 2 A), but a mixture of B and E did (Fig. 2B ). Taken together, these observations suggested that fraction E contains functional epidermal precursors.

To understand whether a small population of contaminating follicles could be responsible for the proliferative capacity of dermal fraction E, we looked at the proliferative capacity of Epi, DHF, D, and E in monolayer culture. When DNase was included in the dermal preparations, detectable levels of keratin 10 and 14 decreased, suggesting that the use of DNase substantially decreases cross contamination of fractions (supplemental Fig. 3). When we plated an equal number of cells from a preparation that did not include DNase, we were able to carry Epi in low calcium medium for three passages. Dermal-derived fractions (DHF, D, and E), cultured in high calcium medium, failed to survive past passage 7. When DNase was added to the preparation, Epi failed to survive past passage 2; DHF failed to survive past passage 9 and dermal-derived fractions D and E survived to passage 18. At this point, only fraction E had viable cells, which had largely senescent morphology and contained some adipocyte-looking cells in the culture. In the same experiment, we maintained cells that remained in the well after passage 1 for a month with only one medium change. In the epidermal fraction, no cells survived. In DHF, cells had lifted and died by 3 wk; however, a monolayer of cells from both fractions D and E was still present after a month, similar to what we report in Fig. 3 .

Survival and differentiation of monolayer cultures in reduced serum
We hypothesized that epidermal precursors in the dermis would mobilize to restore damaged epidermis, and we could mimic this process in vitro. We tested whether we could functionally identify which fraction would respond to wound-healing cues. Those cells, we hypothesized, would be able to survive harsh conditions through quiescence prior to responding to wound-healing signals. To test the former, we isolated and/or sorted these fractions and placed them in monolayer culture for 1 month in medium containing 2% serum and either 0.05 mM or 1.4 mM Ca2+. We then assayed cell survival by MTT. The unsorted epidermal subfractions B and C had increased survival; likewise, SP-sorted cells from hair follicle subfractions (B, F, and G) showed the greatest ability to survive in minimal media containing 0.05 mM Ca2+ (Fig. 3A ). Epidermal fractions sorted based on the integrin marker CD49f survived only under low calcium conditions (Fig. 3B ) The moderately increased survival of a CD49f population in high calcium suggested that expansion of an epidermal-like population under high calcium and air exposure (see Figs. 2 and 4 ) may originate in a population distinct from CD49f+. In SP-sorted fractions, a population of small, vigorously proliferating cells expanded from fraction E, and not from D or epidermal subfraction B, under high calcium conditions (Fig. 3C ). Selection by c-Kit (CD117) showed no differences between dermal fractions D and E (data not shown). Together, these observations suggested that a subpopulation of SP + dermal cells, distinct from the CD49+ population, contains epidermal precursors capable of expanding in higher calcium conditions. Therefore, the effects we had observed when fractions B and E were mixed and cultured in three dimensions (i.e., air-exposed in high calcium, Fig. 2 ) could be explained by the ability of selected dermal cells to form epidermis in vitro.

Survival and differentiation of dermal fraction E in 3-dimensional cultures
Based on the results in monolayer cultures, we reasoned that dermal subpopulations survived more readily than others because they contained early progenitor cells. Upon wounding, healing would arise from those populations and provide rapidly amplifying cells for epidermal formation. Accordingly, we simulated conditions that favor epidermal cell expansion. In 3-dimensional cultures, these conditions consisted of low Ca2+-containing medium with KGF when submerged and high Ca2+ and ascorbic acid-containing medium (21) when air exposed. Fraction E was plated in 1.4 mM Ca2+ to favor attachment to the membrane, and 24 h later the medium was changed to 0.05 mM Ca2+ medium containing KGF. Cultures were grown submerged for a week and air-exposed in medium containing 1.4 mM Ca2+ for 2 days, 6 days, and 9 days (Fig. 4A-C ). Fraction E was grown submerged and air-exposed as in panel A, then submerged again for 4 days and air-exposed for an additional 7 days (Fig. 4G ). The early cultures (Fig. 4D ) stained positive for CD34 (data not shown).

We compared the expansion of K10/K14 staining cells in the cultures. Figure 4E , H demonstrates K14 staining of the membranes shown in panels D, G. Figure 4F , I shows stronger staining for K10 of the panel G sample, suggesting that a second phase of submerge culture, followed by air exposure, directs differentiation of keratinocytes in vitro. Colocalization of a later keratinocyte marker involucrin with K14 further supported this differentiation (Fig. 4J ). This experiment was subsequently repeated with epidermal fraction B, but the cells were nonviable. Immunoblots of dermal subpopulations that migrated through 0.3 µ membranes adhered to the plastic underneath and were cultured in two individual wells with 0.05 mM or 1.4 mM Ca2+, suggested that fraction E more than fraction D contained cells able to migrate through membrane pores and expand keratin-positive cells under both calcium conditions; the differences, however, were not statistically significant (Fig. 4K ).

Differentiation assays
To test whether fraction E had multilineage potential, we conducted the following assays (24) . Fractions B, or total epidermal preparation, and D were used as comparison controls. Fraction E was positive for osteogenesis, chondrogenesis, and adipogenesis (Fig. 5A, B ). Under starvation and 0.05 mM Ca2+ conditions, fraction E and Epi but not D expanded a K5-positive population whereas fraction D more than E expanded a melanocyte-positive population (as marked by the early melanocyte marker Tyrp8/Dct) (Fig. 5E ). DCT, an early melanoblast marker, has been proposed to be a marker of the bulge (16) .

In addition to these findings, both fractions D and E, but most prominently fraction E, gave rise in culture to cells with contractile behavior (Fig. 5D and supplemental movie). We observed this phenotype on confluent monolayers and on air-exposed 3-dimensional cultures as well (data not shown).

Stem cell and differentiation markers were differentially expressed in skin subpopulations
Epidermal cells (subfraction B) and dermal fractions D and E were plated in complete high calcium medium. Total RNAs were prepared from the overnight-adhered cells and hybridization to mouse stem cell arrays was carried out. These arrays confirmed functional observations of differences between epidermal and dermal fractions and within dermal subfractions D and E. Differentiation and stem cell markers were differentially expressed in those populations (Fig. 6A ). As examples, genes circled in red were up-regulated in fraction E; blue circles mark genes that were below detection limits in fraction E while being up-regulated in the others. Figure 6C shows that many genes were exclusively below the limits of detection in E but up-regulated in other fractions, suggesting that negative selection might enrich for stem cells in fraction E (24 , 41) . Specifically, genes involved in cellular adhesion such as integrin ß4 and ICAM 5 were below detection in E and highest in D. Figure 6D represents the genes detected in E, as well as in other subfractions, and their percent of distribution. Changes in the expression of K14, 10, and of the transcription factor Pou3f2 were confirmed at the protein level by immunofluorescence and immunoblotting (Fig. 6F ).

In vivo engraftment and differentiation
Cells from Epi, DHF, D, and E fractions were engrafted into skin of immunodeficient mice in two ways: they were either subcutaneously injected or grafted under a silicon dome covering the graft bed as typical epidermal grafts.

Isolated subpopulations were injected at defined subcutaneous sites on the back of immunodeficient mice. Mice were sacrificed at 1 and 2 wk postinjection. In the 1 wk histology sections, clusters of cells in the injected sites of fractions Epi and DHF could not be found. Fraction D formed a fibrotic mass that had abundant vascularity, consistent with previous reports (42) . In the fraction E group, 1 wk after subcutaneous injection some pigmented aggregates were observed within host muscle (Fig. 7 A, Fontana Masson stain). In addition, both fraction D and E, but most prominently fraction E, displayed fusiform structures that stained positive for desmin and smooth muscle actin in the periphery and positive for smooth muscle actin toward the center (Fig. 7A , 1 wk). These structures were not as prominent in the 2 wk histology sections. These staining patterns suggested early differentiation of the mesenchymal cells into the myogenic lineage. In the 2 wk postinjection set, we were able to find the injected Epi and DHF cells, which formed abundant cysts that were larger in DHF (Fig. 7A , 2 wk), but fraction D cells could not be found. Fraction E showed a few small cysts, numerous melanocytes surrounding these and interspersed in the dermal tissue, as well as some nonmelanized, fusiform structures (Fig. 7A , arrowhead).


Figure 7
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  		Figure 7. Engraftment and differentiation of fraction E in vivo. Isolated subpopulations were injected at defined subcutaneous sites on the back of immunodeficient mice. Mice were sacrificed 1 and 2 wk postinjection. A) In the fraction E group, pigmented aggregates are seen within host muscle (arrow and upper right inset). Arrowhead and lower left inset show positive Fontana Masson fusiform structure, which stains positive for desmin and smooth muscle actin in the periphery and positive for smooth muscle actin toward the center (stained sections are from the area in the lower left inset). Clusters of cells in the injected sites of fractions Epi and DHF could not be found in the animals. At 2 wk, injected Epi and DHF cells formed abundant cysts, which were larger in DHF. Fraction E had some small cysts, numerous melanocytes surrounding these and interspersed in the dermal tissue, as well as some nonmelanized fusiform structures (arrowhead). Fraction D cells could not be found in the animals. B) Week 3 postgrafting with silicon chambers. Arrows point in graft area in all four panels. A dark healed wound was seen in DHF, which contained numerous hair follicles and cysts, as observed in the Fontana Masson panel below. Animals grafted with fraction E displayed a dark ring around the edges of the wound site; pigmented nests were deep within the host dermis, as seen in the Fontana Masson panel below. In the graft central areas, away from wound edges, very few melanocytes are observed in Epi and many more appear in fraction E.

For the epidermal grafts, 5 days postgrafting the silicon domes were removed to facilitate wound healing. At that time animals grafted with fraction E had a significantly bloodier wound, which nonetheless healed faster, as shown in Fig. 7B . All four subpopulations had graft take as evidenced by the presence of dermal melanocytes not associated with hair follicles, consistent with previous reports (43) . A very dark healed wound was appreciated in DHF, which contained numerous hair follicles and cysts as observed in the histology (Fig. 7B ). Animals grafted with fraction E displayed a dark ring around the edges of the wound site (Fig. 7B , arrow). On histology, it could be observed that there were pigmented nests deep within the host dermis, where a few contaminating hair follicles accumulated. The environment of the wound edge has been reported to be more favorable to hair follicle development (43 , 44) . In the graft central areas, away from wound edges, very few melanocytes were observed in Epi and D fractions; in DHF they were concentrated in areas surrounding preexisting follicles and cysts, whereas in fraction E melanocytes could be found in the center of the graft. Melanocytes appeared vacuolated in Epi and were lighter in color than those in the E fraction (Fig. 7B ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Murine skin bioengineering lags behind progress made in the field of human skin bioengineering (45 , 46) . A complete epidermis may be reformed after the growth of mouse keratinocytes on nonviable, de-epidermized dermis (10 , 47) , and some studies have shown that mixing human and murine skin components may reconstruct an epithelial layer (48) . Collectively, these studies suggest there may be different requirements for the in vitro formation of mouse and human skin, such as growth factors and cellular selection. In vivo, an epidermis can be formed by grafting epidermal cells with a dermal component onto nude mice (49) . This dermal component was made up of a combination of fractions D and E, described in this paper. Weinberg et al. were able to demonstrate hair growth when hair follicles were grafted with dermal papilla and not in their absence (17) . Our work aimed at expanding these findings in vitro. We could not consistently replicate the in vitro formation of an epidermal layer when we used neonatal mouse epidermal cells cultured on artificial inserts with gamma-irradiated dermal cells cultured on the underside (50) . Therefore, we focused on isolating different cellular subpopulations from neonatal mouse skin, taking advantage of properties such as differences in buoyant density or differential attachment to extracellular matrix components (51) , which can selectively enrich for hair follicle-derived cells (17 , 52) . Our isolation scheme selected seven populations [three derived from the epidermis (A, B, C) and four from the dermis (D, E, F, G)], characterized as shown in supplemental Table 1. Again, we had limited success with subpopulations obtained from the epidermal compartment on organotypic cultures. Among the dermal fractions, F grew well under submerged conditions in low calcium but failed to grow in 3-dimensional cultures. Cocultures of epidermal cells and dermal fractions, designed to enrich mesenchymal cells, grew successfully in 3-dimensional cultures. Moreover, we observed that de-epidermized dermis controls often reformed an epithelium even after gamma irradiation, and thus the dermis may contain radio-resistant progenitors. We reasoned that our isolation procedure could shed cells from the epithelial fractions into the mesenchymal component of the dermis. Those precursors would be derived from the bulge or the dermal papilla sections of the hair follicle and would, in conjunction with fibroblasts, favor epidermal formation in vitro. We tried to assess the effects of contamination of epidermal cells in the dermal preparations. Increasing keratinocyte contamination was found when DNase was left out of our isolation protocol (the use of DNase is a current practice in our laboratory, as described in ref. 15 , which does not specify why it was added). To our knowledge, other papers published that have examined dermal cells, such as ref. 53 , which describes DEEP-1 cells, fail to use DNase; we demonstrate that the use of DNase in preparations of dermal cells significantly reduces hair follicle contamination.

When plated on membranes, mesenchymal cell preparations formed multiple layers and migrated through pores to the underside. Furthermore, in submerged conditions, keratinocyte monolayers formed that stratified at the air-medium interphase and expressed keratin 14 and other markers, as seen in Fig. 4 .

The mesenchymal cells expressed cell surface markers that distinguished them from keratinocytes. They expressed low levels of CD49f and were rich in CD34; they had detectable CD117 and a higher level of CD90 expression. These cells distinctly expressed MRP8 (S1000A8), and this was not due to differential content in cells that express the type 3 complement receptor, CD11b. Changes in the expression of S100 A8 have been reported in wound fibroblasts (54) . A distinguishing characteristic of the mesenchymal population was the ability to survive and propagate under minimal culture conditions. This may have parallels in vivo, as reserve or hematopoietic stem cells are reported to survive for long periods in a poorly oxygenated niche (55) . Thus, the long-term survival of a mesenchymal precursor population may require that the cells have enhanced survival capabilities under stressful conditions.

The mesenchymal cells (from fraction E) could differentiate into different lineages, thus forming osteocytes, chondrocytes, myocytes, and adipocytes. We repeatedly observed that when dermal cells were placed in 3-dimensional cultures under submerged conditions, only isolated cells survived on the membranes, and those cells were evenly spaced. We hypothesized that those cells would differentiate into epidermis if we gave them an opportunity to expand with appropriate growth factors; we tested this by simulating the skin wound-healing environment. Our simple model of skin wound healing consisted of the growth of fraction E alone under alternating submerged/air exposed-submerged + KGF/air-exposed conditions. Early cultures were positive for the stem cell marker CD34 and for the basal keratinocyte marker K14, whereas cultures that had undergone more than one round of submerged/air-exposed conditions acquired the early differentiation keratinocyte markers K10 and involucrin.

We have not determined the origin of these mesenchymal cells. It is possible that cells from the hair follicle bulge area become dislodged and mixed in with the dermal populations in our isolation protocol. It has been reported that putative human keratinocyte stem cells have the lowest amounts of desmoglein (56) and therefore could become loose and remain in the dermal subpopulations separated from the dermal hair follicle cells. Thus, these precursors could come from either the bulge (10) or the dermal papilla, which may contain early transient amplifying cells (16) . However, the significant lower levels of p63 and K15 between E, D, and DHF argue against bulge contamination of our cultures.

Stem cells and early precursors express lineage-specific genes at low levels before lineage commitment (41) . Analysis of gene array data showed that among the few genes detected in fraction E were the intermediate filament keratins 14 and 17. K14 is a basal keratin that precedes K1 and the later stages of epidermal squamous differentiation; K14 was relatively more abundant in fraction E than in D, thus confirming our immunoblotting results. K17 is an early epidermal progenitor marker (57 , 58) and a marker for bulge (16 , 59) . Injury to the skin results in an induction of K17 concomitant with activation of keratinocytes for reepithelialization. K17 was not significantly different in fraction E. Only Acta2 was more prominent in fraction E than in the others. Acta2 encodes the alpha form of smooth muscle actin. The contractile isoform alpha-2 actin is also called smooth muscle and aorta isoform; it is present in musculoskeletal connective tissue cells and has been demonstrated in early progenitors (60 , 61) . The expression of smooth muscle actin suggests that the population might contain myofibroblast precursors (62) . The bone morphogenic proteins BMP3 and BMP6, signals required for stem cells, the stem cell marker CD34, GJB1, a member of the gap junction connexin family, and nerve growth factor were expressed about equally in all fractions analyzed and thus were not specific to fraction E. However, Cnp1, a marker for myelin, was expressed at much lower levels in E and B than in D and Fb, suggesting that oligodendrocytes or their precursors could be present in the D preparation and absent in fractions E and B. Cnp-positive cells were detected in bulge cells after differentiation in culture (63) . Some genes whose expression was lower in the E fraction were Fzd1 and 8, which were more prominent in D and B, consistent with recently published patterns of frizzled genes in skin and hair follicles (64) . Many stem cells and differentiation genes from the array were below the limits of detection in E but up-regulated in the other fractions.

When injected subcutaneously, fraction E gave rise to structures that stained positive for smooth muscle actin and desmin. In silicon chamber grafts, which are conducive to epidermal differentiation, melanocytes not associated with hair follicles were abundant in the dermis. Smooth muscle actin-positive cells and melanoblasts are both neural crest derivatives, and one or the other may predominate, depending on the microenvironment (65) . Collectively, our grafting and subcutaneous injection results suggest that dermal fraction E has the ability to differentiate into muscle-like structures when subcutaneously grafted or into melanocytes when grafted in an environment in which the canonical ß-catenin pathway predominates.

In summary, we isolated a mesenchymal population devoid of hematopoietic cell markers and capable of forming adipocytes, chondrocytes, osteoclasts, functional smooth muscle cells, and keratinocytes in vitro. Moreover, this population was able to generate an epidermal layer in a 3-dimensional model of murine skin. By comparison, this population lacks the expression of markers shared by epidermal and other dermal subpopulations such as ICAM 5 and ß-4 integrin; the expression of a selected set of genes is strikingly different from that of other epidermal and dermal subfractions. We were able to demonstrate the relative absence from dermal fractions D and E of epithelial stem cell markers (K15, p63); CD90, a mesenchymal progenitor marker, distinguished D and E from Epi and DHF whereas Cd105 did not. MRP8 (S100A8) is a distinguishing marker to this fraction but CD11b did not distinguish it from the other fractions studied.

This isolation procedure will be useful for the study of genetically altered mouse strains as models for various human skin conditions. Further analysis of these cells may provide new insight into how mesenchymal-to epithelial transitions play a role in tissue regeneration.


   ACKNOWLEDGMENTS
 
The authors thank Stuart Yuspa, Ulrike Lichti, Julio Valencia, and Luowei Li for critical discussions and helpful advice. We also thank Kevin Taylor, Joseph Zakhari, and Susan Garfield for expert technical assistance. This research was supported in part by the Intramural Research Program of the National Institutes of Health National Cancer Institute, Center for Cancer Research.

Received for publication February 1, 2006. Accepted for publication February 6, 2007.


   REFERENCES
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 DISCUSSION
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