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Differentiation of adipose-derived adult stem cells into epithelial-like stem cells
Annals of Anatomy 195 (2013) 212–218
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Annals of Anatomy journal homepage: www.elsevier.de/aanat
Research article
Differentiation of adipose-derived adult stem cells into epithelial-like stem cells Yingjun Yan a,b , Yuxiao Liu c , Daqing Liu b , Lijuan He b , Lidong Guan b , Yunfang Wang b , Xue Nan b , Xuetao Pei b,∗ a b c
Department of Plastic Surgery, China Meitan General Hospital, Beijing 100028, China Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China Department of Neurosurgery, The First Affiliated Hospital of PLA General Hospital, Beijing 100037, China
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Article history: Received 14 December 2011 Received in revised form 6 October 2012 Accepted 10 October 2012 Available online 14 February 2013 Keywords: Adipose-derived adult stem cells Differentiation Epithelial cells All-trans-retinoic acid Cytokeratin
s u m m a r y Adipose-derived adult stem (ADAS) cells can be easily obtained in large quantities. Previous studies have suggested that all-trans-retinoic acid (ATRA) plays an important role in the differentiation of mesenchymal stem cells toward an epithelial lineage. In order to verify that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells, ADAS-cells were isolated and cultured. They were induced to differentiate toward an epithelial lineage in vitro. Differentiated epithelial cells were assayed as to whether they retain characteristics of stem cells by RT-PCR and cell cycle stage analysis, and were further induced to differentiate toward an osteogenic lineage. RT-PCR analysis revealed that no CK5, CK10 or CK19 mRNA was detected in ADAS-cells, CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10. After induction, the expression of CK19 was observed by immunofluorescent staining. Positive staining with alkaline phosphatase (ALP) and Von Kossa staining verified that differentiated epithelial cells still had potential to further differentiate toward an osteogenic lineage. These experiments provide proof that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells. © 2013 Published by Elsevier GmbH.
1. Introduction The loss of tissue due to trauma, tumor resection or vascular insult represents a significant clinical problem with few solutions. For patients with such problems, autologous tissue transfers have been applied to reconstruct the tissue defects, but donor site morbidity can be both cosmetically and functionally limiting. Regenerative medical therapy may well be useful in treating those suffering from tissue defects, and the use of stem cells may be promising for tissue regeneration and engineering (Mizuno and Hyakusoku, 2003). Although embryonic stem cells (ESCs) are highly beneficial, they are limited by cell regulation and ethical considerations. Bone marrow mesenchymal stem cells have been shown to possess multiple differentiation potential in vitro. However, bone marrow procurement is extremely painful for donors, and only few cells can be harvested (Mizuno and Hyakusoku, 2003). Stem cells in human adipose tissue have been studied in the past few years. The common terminology used for these cells is
∗ Corresponding author. E-mail address: [email protected] (X. Pei). 0940-9602/$ – see front matter © 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.aanat.2012.10.009
processed lipoaspirate (PLA) cells or adipose-derived adult stem (ADAS) cells (Gimble and Guilak, 2003; Schwarz et al., 2011; Zhang and Shao, 2011). Zuk et al. have shown that these cells have surface antigens and differentiation potential similar to those of mesenchymal stem cells (MSC) from human bone marrow stroma (Brzoska et al., 2005). ADAS-cells can be procured easily from the donor with little discomfort and donor site morbidity, which makes clinical application more feasible. Furthermore, these cells are derived from adults and therefore circumvent the ethical ambiguities of using embryonic stem cells. Once obtained and then modified in vitro, these cells can be reintroduced autologously, which has the advantage of reducing the probability of an immune reaction (Brzoska et al., 2005). ADAS-cells have been proven to differentiate into adipogenic, chondrogenic, myogenic, and osteogenic cells in vitro in the presence of lineage-specific induction factors (Zuk et al., 2001). All-trans retinoic acid (ATRA) can induce cytokeratin (CK) 18 expression in ADAS-cells and nearly abolish vimentin expression, indicating that ADAS-cells also have epithelial potential (Brzoska et al., 2005). In this study, we induced ADAS-cells to differentiate toward an epithelial lineage in vitro and further induced differentiated epithelial cells to differentiate toward an osteogenic lineage.
Y. Yan et al. / Annals of Anatomy 195 (2013) 212–218 Table 1 Primer pairs used for PCR. Primer pairs
Nucleotide sequences
Size of products (bp)
CK5
F: 5 -CCCAGTATGAGGAGATTGCCAACC-3 R: 5 -TATCCAGAGGAAACACTGCTTGTG-3
475
CK10
F: 5 -GCTGACCTGGAGATGCAAATTGAGAGCC-3 131 R: 5 -GGGCAGGATTCATTTCCACATTCACATCAC-3
CK19
F: 5 -GCTGGCCTACCTGAAGAAGA -3 R: 5 -CCGCTGGTACTCCTGATTCT-3
441
␣-Tubulin
F: 5 -CACCCGTCTTCAGGGCTTCTTGGTTT-3 R: 5 -CATTTCACCATCTGGTTGGCTGGCTC-3
528
2. Materials and methods 2.1. ADAS-cells isolation, culture and assay 2.1.1. ADAS-cells isolation and culture Lipoaspirates from patients undergoing cosmetic liposuction were processed according to methods first described by Zuk et al. (2001) and modified by Guan et al. (2006). Briefly, raw lipoaspirate (∼300 g) was digested with 0.2% collagenase type I plus 1% bovine serum albumin (BSA) for 45 min at 37 ◦ C. The stromal-vascular fraction was separated from remaining fibrous material and the floating adipocytes by centrifugation at 800 g for 4 min. The sedimented SVF-cells were filtered through an 80 m pore filter and then a 200 m pore filter, followed by an incubation step in an erythrocyte lysing buffer (160 mM NH4 Cl) for 10 min. For initial cell culture and expansion of the ADAS-cells, low-glucose DMEM medium (L-DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 g/mL streptomycin was used. Cultures were washed with phosphate-buffered saline (PBS) 24–48 h after plating to remove unattached cells and then fed with fresh medium. Cultures were maintained at 37 ◦ C with 5% CO2 and medium was changed 3 times per week. Cells were grown to confluence after the initial plating (P = 0), typically within 10–14 days. Once confluent, the adherent cells were released with 0.5% trypsin–EDTA and then either plated at 2 K/cm2 or used for experimental analysis. All cells used for analysis were early passage (passages 3–5). 2.1.2. ADAS-cell surface marker detection Flow cytometric characterization of ADAS-cells ranging from passage 3 to 5 was performed. Cells were suspended in PBS containing 1% BSA, and distributed to 1 × 106 cells per sample. All samples were incubated with antibodies conjugated with either FITC or PE, against the following antigens: CD29 (PE, Serotec), CD34 (PE, Miltenyi Biotec), CD44 (FITC, Serotec), CD45 (FITC, Serotec), CD49d (FITC, Serotec), CD71 (FITC, Serotec), and CD90 (FITC, Serotec), respectively. The cytometer is FACScalibur (Becton–Dickinson) with a 488 nm krypton–argon laser. Samples were compared with isotypematched controls. 2.1.3. Reverse transcription polymerase chain reaction (RT-PCR) analysis The total RNA from ADAS-cells was isolated using Trizol reagent (Gibco BRL), and reverse transcribed into complementary DNA (cDNA). PCR was performed on the cDNA using primers to detect CK5, CK10, and CK19 using a GeneAmp PCR System 2400 (PerkinElmer), with ␣-tubulin served as the housekeeping gene control. The primers used are listed in Table 1 (Abiko et al., 2004; CohenKerem et al., 2002; Kamiya et al., 2003). The protocol for PCR was 5 min at 94 ◦ C followed by 30 cycles of 30 s at 94 ◦ C, 30 s at 65 ◦ C, and 30 s at 72 ◦ C, with a final 7 min extension at 72 ◦ C. Amplified
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PCR products were analyzed by ethidium bromide staining after gel electrophoresis using a high-performance gel documentation and image analysis system (AlphaImagerTM 3300, Alpha). 2.1.4. Cell cycle analysis Duplicate samples were harvested. 2 × 105 cells were fixed for 1 h with 75% ethanol, then incubated with propidium iodide (PI) at a concentration of 20 g/mL for 30 min at 0 ◦ C. Cell cycle stage was assayed using a FACScalibur cytometer (Becton–Dickinson) with a 351 nm laser. 2.2. Epithelial differentiation of ADAS-cells 2.2.1. Epithelial differentiation protocol Isolated human ADAS-cells were plated in flasks and start differentiation process at passage 3. The epithelial differentiation culture medium was a mixture of L-DMEM and DF-12 (1:1) with 10% FBS and 1% ITS (Sigma), supplemented with 20 ng/mL epidermal growth factor (EGF, R&D), 5 ng/mL fibroblast growth factor (bFGF, R&D), 5 M all-trans-retinoic acid (ATRA, Gibco), and 0.1 M dexamethasone (Dex, Sigma). The epithelial differentiation duration is 7 days and culture medium was changed every 2 days. Differentiated cells were used for different analysis. The rest of differentiated cells were cultured in keratinocyte growth medium (KGM). The KGM was L-DMEM/DF12 (1:1) with 10% FBS, supplemented with 10 ng/mL epidermal growth factor (EGF, R&D), 5 ng/mL fibroblast growth factor (bFGF, R&D), 5 g/mL insulin (Sigma), 0.33 g/mL hydrocortione (Sigma), and 10 g/mL transferrin (Sigma). The KGM was changed every 2 days. 2.2.2. Epithelial differentiated cell surface marker detection Flow cytometric characterization of differentiated cells at passage 10 was performed using FITC or PE labeled antibodies against CD29 (PE, Serotec), CD34 (PE, Miltenyi Biotec), CD49d (FITC, Serotec), CD49f (FITC, Serotec), and CD71 (FITC, Serotec). 2.2.3. RT-PCR analysis The mRNA level of CK5, CK10 and CK19 of differentiated epithelial cells were checked by RT-PCT at passages 1 and 10. 2.2.4. Cell cycle analysis Cell cycle stage of differentiated cells was analyzed at passage 1 as described above. 2.2.4.1. Immunofluorescent staining. For immunofluorescence analysis, differentiated cells were first fixed in 4% paraformaldehyde for 15 min at room temperature and rinsed with PBS. Then cells were permeabilized with 0.1% Triton X-100 and 3% H2 O2 for 10 min. The primary antibodies (Mouse anti Human CK19, ZSGB-BIO) were diluted at a definite concentration in accordance with instructions. Incubation was performed at 4 ◦ C for 16 h. Then cells were incubated with a secondary antibody (FITC-Goat anti Mouse IgG, ZSGB-BIO) for 1 h at 37 ◦ C. The immunofluorescence images were captured using a fluorescence microscope. 2.3. Osteogenic differentiation of differentiated epithelial cells. 2.3.1. Osteogenic differentiation protocol Osteogenic differentiation was induced by culturing differentiated epithelial cells in an osteogenic medium (OM) for 3 weeks: L-DMEM supplemented with 10% FBS, 10 mM -glycerophosphate, 0.1 M dexamethasone, and 0.2 mM ascorbic acid (all from Sigma) (Cao et al., 2005). The osteogenic differentiation culture medium was changed every 3 days.
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Fig. 1. Morphological observation of differentiated epithelial cells by light microscopy. (Left) ADAS-cells exhibited fibroblast-like morphology (40×). (Middle) ADAS-cells after culture in epithelial differentiation medium for 3 days (40×). (Right) ADAS-cells after culture in epithelial differentiation medium for 7 days (40×).
2.3.2. Alkaline phosphatase (ALP) staining (Ca–Co technique) Three weeks post-differentiation, osteogenic differentiated cells were extensively washed with PBS, fixed in cool acetone for 10 min, washed with distilled water, incubated in 5 mL buffer [consisting of 20 g/L -phosphoric acid glycerin natrium (10 mL), 20 g/L barbital sodium (20 mL), 20 g/L addex-magnesium (1 mL), and 5 mL distilled water] (pH = 9.2, Zhongshan Biochemical, China) at 37 ◦ C for 6 h. Then they were washed with distilled water and incubated in a 20 g/L aqueous solution of cobaltous nitrate for 5 min. After another wash with distilled water, samples were incubated in aqueous solution of sulfon ammonium for 3 min. After a final wash, each sample was dehydrated, mounted, and examined by light microscopy (He et al., 2007). 2.3.3. Von Kossa staining Osteogenic differentiated cells were fixed with 4% paraformaldehyde for 60 min at room temperature. Cells were rinsed with distilled water and then overlaid with a 1% (wt/vol) silver nitrate solution in the absence of light for 30 min, then
developed under UV light for 60 min. After another wash with distilled water, the samples were dipped into 5% sodium thiosulfate to counteract silver nitrate. Each sample was dehydrated, mounted, and examined by light microscopy (Zuk et al., 2001). 3. Results 3.1. Morphology and surface markers of ADAS-cells Freshly isolated ADAS-cells were heterogenous, containing contaminating cells the numbers of which diminished over subsequent culture passages, leaving a homogenous population of cells exhibiting fibroblast-like morphology (Fig. 1 left). ADAS-cells were easily expanded in vitro and did not appear to require specific sera lots for expansion and multilineage differentiation (Zuk et al., 2001; Guan et al., 2006). Flow cytometric characterization of ADAS-cells showed that cultured cells were positive for CD29, CD44 and CD90, and negative for CD34 and CD45, with low expression of CD49d and CD71 (Fig. 2).
Fig. 2. Surface marker detection of ADAS-cell. ADAS-cells were positive for CD29, CD44, and CD90, and negative for CD34 and CD45, with low expression of CD49d and CD71.
Y. Yan et al. / Annals of Anatomy 195 (2013) 212–218
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Fig. 3. Surface marker detection of differentiated epithelial cell. Differentiated epithelial cells were positive for CD29 and negative for CD34 and CD49f, with low expression of CD49d and CD71.
3.2. Morphology and surface markers of differentiated epithelial cells To differentiate ADAS-cells toward an epithelial lineage, ADAScells were cultured in the epithelial differentiation culture medium for 7 days. The effect of induction could be observed by light microscopy. As documented in Fig. 1 middle and right, cells contracted gradually and assumed a uniform cobblestone-like appearance over time, presenting growth characteristics more like a cultured monolayer of epithelial cells. Flow cytometric characterization of differentiated epithelial cells showed that they were positive for CD29 and negative for CD34 and CD49f, with low expression of CD49d and CD71 (Fig. 3).
3.3. RT-PCR analysis No CK5, CK10 or CK19 mRNA was detected in ADAS-cells. CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1. In contrast, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10 (Fig. 4).
Fig. 4. RT-PCR analysis of ADAS-cells, differentiated epithelial cells at passage 1, and differentiated epithelial cells at passage 10. No CK5, CK10 or CK19 mRNA was detected in ADAS-cells. CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1. In contrast, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10.
Fig. 5. 87.42% of ADAS-cells in the G0–G1 stage, whereas 96.38% of differentiated epithelial cells in the G0–G1 stage.
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Fig. 6. After induction, the expression of CK19 was observed under a fluorescence microscope.
3.4. Cell cycle analysis Cell cycle analysis was performed in ADAS-cells and differentiated epithelial cells at passage 1. The results revealed 87.42% of ADAS-cells in the G0–G1 stage, whereas 96.38% of differentiated epithelial cells in the G0–G1 stage (Fig. 5). 3.5. Immunofluorescent staining After induction, the expression of CK19 was observed under a fluorescence microscope (Fig. 6). 3.6. Osteogenic differentiation of differentiated epithelial cells Osteoblasts differentiated from differentiated epithelial cells have typical osteoblasts cellular morphology as shown in Fig. 7 left. Cells developed a number of tiny, brown-black granules with positive stain for alkaline phosphatase (Fig. 7 middle), and cells formed irregularly-shaped, dense granules in the ECM with positive stain for Von Kossa suggesting indeed calcified ECM (Fig. 7 right). 4. Discussion Skin lesions resulting from burns or trauma are major healthcare problems. The prospective use of stem cells for the restoration of injured or diseased tissues has opened new fields of research. Plasticity is the first requirement for therapeutic potential (Zaragosi et al., 2006). Furthermore, an alternative source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous (Zuk et al.,
2001). ADAS-cells possess all of these advantages, and have been proven to differentiate into adipogenic, chondrogenic, myogenic, osteogenic, cardiomyogenic, neurogenic, and endotheliogenic cells in vitro in the presence of lineage-specific induction factors (Zuk et al., 2001; Guan et al., 2006; Cao et al., 2005; Kang et al., 2007; Huang et al., 2007; Planat-Bénard et al., 2004). Thus ADAS-cells may be an alternative stem cell source for regenerative medicine in the future. ADAS-cells are considered to be a heterogeneous cell population. Immunofluorescence and flow cytometry show that the majority of ADAS-cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells (Zuk et al., 2001). The adherent-cultured ADAS-cells assume fibroblast-like morphology as observed by light microscopy. The morphology can be maintained through repeated subculturing under non-stimulating conditions. ADAS-cells can be maintained in vitro for extended periods with stable proliferation and low levels of senescence (Zuk et al., 2001; Cao et al., 2005; Kang et al., 2007). Using flow cytometry to characterize the phenotype of adherent adipose-derived cells at the fifth passage, it had been shown that these cultured cells were positive for CD29 (1-integrin), CD44, CD90 (Thy-1), CD105 (endoglin), CD166, Flk1, and HLA-ABC. In addition, no expression of the hematopoietic and endothelial lineage markers (CD31, CD34, CD45, CD106, and CD184) was observed (Zuk et al., 2002; Brzoska et al., 2005; Cao et al., 2005; Kang et al., 2007). This phenotype is similar to that of cells isolated from the bone marrow, but ADAS-cells are positive for CD49d (VLA-4) and negative for CD106 (VCAM-1), while bone marrow-derived cells are positive for CD106 and negative for CD49d (Zaragosi et al. (2006)). In our study, ADAS-cells were positive for CD29, CD44, and CD90, and negative for CD34 and CD45, with low expression of CD49d and CD71. This profile is very similar to the phenotype having been reported in the aforementioned studies (Zuk et al., 2002; Brzoska et al., 2005; Cao et al., 2005; Kang et al., 2007). Previous studies have demonstrated that bone marrow-derived cells (BMDCs), embryonic stem cells, and umbilical cord blood stem cells can differentiate into an epithelial lineage in vitro and in vivo and not as the result of cell fusion (Borue et al., 2004; Nakagawa et al., 2005; P˘aunescu et al., 2007; Harris et al., 2004; Kamolz et al., 2006; Metallo et al., 2008). However, donor-derived cytokeratin 5-expressing cells were rare in Borue’s study (Borue et al., 2004), suggesting that BMDCs do not engraft as epithelial stem cells. The level of engraftment peaked and then decreased over time, which suggest that BMDCs may assist in early wound healing by engrafting as transit-amplifying cells (TACs), and then differentiate into keratinocytes. Recent studies have verified that ADAS-cells also have epithelial potential (Brzoska et al., 2005; Altman et al., 2009).
Fig. 7. In vitro osteogenic differentiation of differentiated epithelial cells. (Left) Differentiated epithelial cells after culture in osteogenic medium for 3 weeks (40×). (Middle) Some tiny, brown-black granules stained positively for alkaline phosphatase (200×). (Right) Irregularly-shaped, dense granules in the ECM stained positively with Von Kossa staining (100×).
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However, no study has investigated whether ADAS-cells can differentiate into epithelial stem cells or TACs. Epithelial stem cells have a high proliferative potential, are capable of self-renewal, and express 1 integrin but not CK1/CK10. The descendants of the stem cells are the TACs that express both 1 integrin and CK1/CK10 and belong to a partially differentiated pool of cells with a limited proliferative capability. Mitotically inactive keratinocytes (1 integrin− , CK1/CK10+ ) are the end-product of TACs differentiation (Gniadecki, 1997). Until now, identification of epithelial cells using molecular markers has been controversial. CD29+ , CD49fbri CD71dim , CD90+ , CK19+ and p63+ are commonly regarded as markers of epithelial stem cells in most studies (Gniadecki, 1997; Vorotelyak et al.,2006; Nakamura et al., 2006). The capacity of self-renewal makes stem cells particularly useful for transplantation medicine because they could provide an unlimited source of donor material for grafting or transplantation in theory. Moreover, most approaches to skin tissue engineering would require differentiated keratinocytes or keratinocyte progenitors, rather than undifferentiated stem cells, for seeding and attaching onto artificially synthesized matrices (Bannasch et al., 2003). In 2005, Broska et al. reported that adipose-derived adult stem (ADAS)-cells owned some characteristics of stem cells and could differentiate into epithelial cells in a suitable environment. In this study, we found that ADAS-cells could differentiate into an epithelial lineage and these ADSCs-derived epithelial cells owned some characteristics of stem cells, which could be induced to an osteogenic lineage. In detail, ADAS-cells were induced to an epithelial lineage under the influence of ATRA in vitro. As we observed, cells contracted gradually and assumed a uniform cobblestonelike appearance over time, presenting growth characteristics more like a cultured monolayer of epithelial cells. Cell cycle analysis also revealed more differentiated cells (96.38%) than ADAS-cells (87.42%) in the G0-G1 stage. RT-PCR analysis revealed no expression of CK5, CK10 and CK19 in ADAS-cells, only expression of CK19 in differentiated cells at passage 1, and expression of both CK10 and CK19 in differentiated cells at passage 10. Immunofluorescent staining for CK19 was chosen for the basal characterization of epithelial-like stem cells (Larouche et al., 2010). After induction, the expression of CK19 was observed. In order to evaluate whether differentiated epithelial cells have transdifferentiation potential, these cells were further induced to differentiate toward an osteogenic lineage. Positive staining with ALP and Von Kossa staining verified these cells were capable of osteogenesis. All these results indicated that ADAS-cells could differentiate into an epithelial lineage while retaining most of the characteristics of stem cells. However, differentiated epithelial cells were negative for CD49f (␣6-integrin) which should be present as an surface marker of epithelial stem cells (Vorotelyak et al.,2006; Nakamura et al., 2006). We thus deemed that these differentiated epithelial cells were not true epithelial stem cells and named them “ADSCs-derived epithelial-like stem cells”. Among the components of the epithelial differentiation culture medium used in our study, ATRA plays an important role in epithelial differentiation. Retinoic acid strongly upregulated expression of keratin 18 and the transcription factor p63 (p63 is involved in epidermal morphogenesis and ectodermal specification), while repressing early neural marker transcription (Metallo et al., 2008). In Brzoska’s study, ATRA had the expected inhibitory effect on cell proliferation with a tolerable impact on cell viability at a concentration of 5 M (Brzoska et al., 2005). p63, a homolog to the tumor suppressor protein p53, is chiefly expressed in epithelial tissues, including the epidermis. p63 affects cell death similar to p53, and also plays important roles in the development of epithelial tissues and the maintenance of epithelial stem cells. DeltaNp63alpha, a p63 isoform specifically expressed in basal keratinocytes, suppressed the expression of specific, late differentiation stage proteins, such
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as filaggrin and loricrin. A precise balance between p63 and keratinocyte growth factor (KGF) mediates the onset of epithelial cell differentiation, via c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) signaling (Ogawa et al., 2008). DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of embryonic stem cells to become K5/K14-positive stratified squamous epithelial cells (Medawar et al., 2008). The employment of bovine serum in the cell culture to induce epithelial differentiation is a major weakness as it represents a limitation for further implementation of these cells into a human organism. Thus, we intend to replace bovine serum in future study. Acknowledgments This work was supported by National High Technology Research and Development Program of China (No. 2006AA02A107), the Major State Basic Research Program of China (Nos. 2009CB941102 and 2010CB945504). Supplementary data Information about the transparent peer review is available as supplementary material in the database Science Direct. The reviewer comments and the authors reply can be viewed by clicking the link: http://dx.doi.org/10.1016/j.aanat.2012.10.009. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aanat.2012.10.009. References Abiko, Y., Hiratsuka, K., Kiyama-Kishikawa, M., et al., 2004. Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray. J. Oral Sci. 46, 19–24. Altman, A.M., Yan, Y., Matthias, N., et al., 2009. IFATS collection: human adiposederived stem cells seeded on a silk fibroin–chitosan scaffold enhance wound repair in a murine soft tissue injury model. Stem Cells 27, 250–258. Bannasch, H., Föhn, M., Unterberg, T., et al., 2003. Skin tissue engineering. Clin. Plast. Surg. 30, 573–579. Borue, X., Lee, S., Grove, J., et al., 2004. Bone marrow-derived cells contribute to epithelial engraftment during wound F healing. Am. J. Pathol. 165, 1767–1772. Brzoska, M., Geiger, H., Gauer, S., et al., 2005. Epithelial differentiation of human adipose tissue-derived adult stem cells. Biochem. Biophys. Res. Commun. 330, 142–150. Cao, Y., Sun, Z., Liao, L., et al., 2005. Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem. Biophys. Res. Commun. 332, 370–379. Cohen-Kerem, R., Lahat, N., Elmalah, I., et al., 2002. Detection of cytokeratins in normal and malignant laryngeal epithelia by means of reverse transcriptasepolymerase chain reaction. Ann. Otol. Rhinol. Laryngol. 111, 149–154. Gimble, J., Guilak, F., 2003. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 5, 362–369. Gniadecki, R., 1997. Effects of 1,25-dihydroxyvitamin D3 and its 20-epi analogues (MC 1288, MC 1301, KH 1060), on clonal keratinocyte growth: evidence for differentiation of keratinocyte stem cells and analysis of the modulatory effects of cytokines. Br. J. Pharmacol. 120, 1119–1127. Guan, L., Li, S., Wang, Y., et al., 2006. In vitro differentiation of human adiposederived mesenchymal stem cells into endothelial-like cells. Chin. Sci. Bull. 51, 1863–1868. Harris, R.G., Herzog, E.L., Bruscia, E.M., et al., 2004. Lack of a fusion requirement for development of bone marrow-derived epithelia. Science 305, 90–93. He, L., Nan, X., Wang, Y., et al., 2007. Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells. Sci. China Ser. C 50, 429–437. Huang, T., He, D., Kleiner, G., et al., 2007. Neuron-like differentiation of adiposederived stem cells from infant piglets in vitro. J. Spinal Cord Med. 30 (Suppl. 1), S35–S40. Kamiya, M., Ichiki, Y., Kamiya, H., et al., 2003. Detection of nonmelanoma skin cancer micrometastases in lymph nodes by using reverse transcriptase-polymerase chain reaction for keratin 19 mRNA. Br. J. Dermatol. 149, 998–1005.
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Annals of Anatomy journal homepage: www.elsevier.de/aanat
Research article
Differentiation of adipose-derived adult stem cells into epithelial-like stem cells Yingjun Yan a,b , Yuxiao Liu c , Daqing Liu b , Lijuan He b , Lidong Guan b , Yunfang Wang b , Xue Nan b , Xuetao Pei b,∗ a b c
Department of Plastic Surgery, China Meitan General Hospital, Beijing 100028, China Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China Department of Neurosurgery, The First Affiliated Hospital of PLA General Hospital, Beijing 100037, China
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Article history: Received 14 December 2011 Received in revised form 6 October 2012 Accepted 10 October 2012 Available online 14 February 2013 Keywords: Adipose-derived adult stem cells Differentiation Epithelial cells All-trans-retinoic acid Cytokeratin
s u m m a r y Adipose-derived adult stem (ADAS) cells can be easily obtained in large quantities. Previous studies have suggested that all-trans-retinoic acid (ATRA) plays an important role in the differentiation of mesenchymal stem cells toward an epithelial lineage. In order to verify that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells, ADAS-cells were isolated and cultured. They were induced to differentiate toward an epithelial lineage in vitro. Differentiated epithelial cells were assayed as to whether they retain characteristics of stem cells by RT-PCR and cell cycle stage analysis, and were further induced to differentiate toward an osteogenic lineage. RT-PCR analysis revealed that no CK5, CK10 or CK19 mRNA was detected in ADAS-cells, CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10. After induction, the expression of CK19 was observed by immunofluorescent staining. Positive staining with alkaline phosphatase (ALP) and Von Kossa staining verified that differentiated epithelial cells still had potential to further differentiate toward an osteogenic lineage. These experiments provide proof that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells. © 2013 Published by Elsevier GmbH.
1. Introduction The loss of tissue due to trauma, tumor resection or vascular insult represents a significant clinical problem with few solutions. For patients with such problems, autologous tissue transfers have been applied to reconstruct the tissue defects, but donor site morbidity can be both cosmetically and functionally limiting. Regenerative medical therapy may well be useful in treating those suffering from tissue defects, and the use of stem cells may be promising for tissue regeneration and engineering (Mizuno and Hyakusoku, 2003). Although embryonic stem cells (ESCs) are highly beneficial, they are limited by cell regulation and ethical considerations. Bone marrow mesenchymal stem cells have been shown to possess multiple differentiation potential in vitro. However, bone marrow procurement is extremely painful for donors, and only few cells can be harvested (Mizuno and Hyakusoku, 2003). Stem cells in human adipose tissue have been studied in the past few years. The common terminology used for these cells is
∗ Corresponding author. E-mail address: [email protected] (X. Pei). 0940-9602/$ – see front matter © 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.aanat.2012.10.009
processed lipoaspirate (PLA) cells or adipose-derived adult stem (ADAS) cells (Gimble and Guilak, 2003; Schwarz et al., 2011; Zhang and Shao, 2011). Zuk et al. have shown that these cells have surface antigens and differentiation potential similar to those of mesenchymal stem cells (MSC) from human bone marrow stroma (Brzoska et al., 2005). ADAS-cells can be procured easily from the donor with little discomfort and donor site morbidity, which makes clinical application more feasible. Furthermore, these cells are derived from adults and therefore circumvent the ethical ambiguities of using embryonic stem cells. Once obtained and then modified in vitro, these cells can be reintroduced autologously, which has the advantage of reducing the probability of an immune reaction (Brzoska et al., 2005). ADAS-cells have been proven to differentiate into adipogenic, chondrogenic, myogenic, and osteogenic cells in vitro in the presence of lineage-specific induction factors (Zuk et al., 2001). All-trans retinoic acid (ATRA) can induce cytokeratin (CK) 18 expression in ADAS-cells and nearly abolish vimentin expression, indicating that ADAS-cells also have epithelial potential (Brzoska et al., 2005). In this study, we induced ADAS-cells to differentiate toward an epithelial lineage in vitro and further induced differentiated epithelial cells to differentiate toward an osteogenic lineage.
Y. Yan et al. / Annals of Anatomy 195 (2013) 212–218 Table 1 Primer pairs used for PCR. Primer pairs
Nucleotide sequences
Size of products (bp)
CK5
F: 5 -CCCAGTATGAGGAGATTGCCAACC-3 R: 5 -TATCCAGAGGAAACACTGCTTGTG-3
475
CK10
F: 5 -GCTGACCTGGAGATGCAAATTGAGAGCC-3 131 R: 5 -GGGCAGGATTCATTTCCACATTCACATCAC-3
CK19
F: 5 -GCTGGCCTACCTGAAGAAGA -3 R: 5 -CCGCTGGTACTCCTGATTCT-3
441
␣-Tubulin
F: 5 -CACCCGTCTTCAGGGCTTCTTGGTTT-3 R: 5 -CATTTCACCATCTGGTTGGCTGGCTC-3
528
2. Materials and methods 2.1. ADAS-cells isolation, culture and assay 2.1.1. ADAS-cells isolation and culture Lipoaspirates from patients undergoing cosmetic liposuction were processed according to methods first described by Zuk et al. (2001) and modified by Guan et al. (2006). Briefly, raw lipoaspirate (∼300 g) was digested with 0.2% collagenase type I plus 1% bovine serum albumin (BSA) for 45 min at 37 ◦ C. The stromal-vascular fraction was separated from remaining fibrous material and the floating adipocytes by centrifugation at 800 g for 4 min. The sedimented SVF-cells were filtered through an 80 m pore filter and then a 200 m pore filter, followed by an incubation step in an erythrocyte lysing buffer (160 mM NH4 Cl) for 10 min. For initial cell culture and expansion of the ADAS-cells, low-glucose DMEM medium (L-DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 g/mL streptomycin was used. Cultures were washed with phosphate-buffered saline (PBS) 24–48 h after plating to remove unattached cells and then fed with fresh medium. Cultures were maintained at 37 ◦ C with 5% CO2 and medium was changed 3 times per week. Cells were grown to confluence after the initial plating (P = 0), typically within 10–14 days. Once confluent, the adherent cells were released with 0.5% trypsin–EDTA and then either plated at 2 K/cm2 or used for experimental analysis. All cells used for analysis were early passage (passages 3–5). 2.1.2. ADAS-cell surface marker detection Flow cytometric characterization of ADAS-cells ranging from passage 3 to 5 was performed. Cells were suspended in PBS containing 1% BSA, and distributed to 1 × 106 cells per sample. All samples were incubated with antibodies conjugated with either FITC or PE, against the following antigens: CD29 (PE, Serotec), CD34 (PE, Miltenyi Biotec), CD44 (FITC, Serotec), CD45 (FITC, Serotec), CD49d (FITC, Serotec), CD71 (FITC, Serotec), and CD90 (FITC, Serotec), respectively. The cytometer is FACScalibur (Becton–Dickinson) with a 488 nm krypton–argon laser. Samples were compared with isotypematched controls. 2.1.3. Reverse transcription polymerase chain reaction (RT-PCR) analysis The total RNA from ADAS-cells was isolated using Trizol reagent (Gibco BRL), and reverse transcribed into complementary DNA (cDNA). PCR was performed on the cDNA using primers to detect CK5, CK10, and CK19 using a GeneAmp PCR System 2400 (PerkinElmer), with ␣-tubulin served as the housekeeping gene control. The primers used are listed in Table 1 (Abiko et al., 2004; CohenKerem et al., 2002; Kamiya et al., 2003). The protocol for PCR was 5 min at 94 ◦ C followed by 30 cycles of 30 s at 94 ◦ C, 30 s at 65 ◦ C, and 30 s at 72 ◦ C, with a final 7 min extension at 72 ◦ C. Amplified
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PCR products were analyzed by ethidium bromide staining after gel electrophoresis using a high-performance gel documentation and image analysis system (AlphaImagerTM 3300, Alpha). 2.1.4. Cell cycle analysis Duplicate samples were harvested. 2 × 105 cells were fixed for 1 h with 75% ethanol, then incubated with propidium iodide (PI) at a concentration of 20 g/mL for 30 min at 0 ◦ C. Cell cycle stage was assayed using a FACScalibur cytometer (Becton–Dickinson) with a 351 nm laser. 2.2. Epithelial differentiation of ADAS-cells 2.2.1. Epithelial differentiation protocol Isolated human ADAS-cells were plated in flasks and start differentiation process at passage 3. The epithelial differentiation culture medium was a mixture of L-DMEM and DF-12 (1:1) with 10% FBS and 1% ITS (Sigma), supplemented with 20 ng/mL epidermal growth factor (EGF, R&D), 5 ng/mL fibroblast growth factor (bFGF, R&D), 5 M all-trans-retinoic acid (ATRA, Gibco), and 0.1 M dexamethasone (Dex, Sigma). The epithelial differentiation duration is 7 days and culture medium was changed every 2 days. Differentiated cells were used for different analysis. The rest of differentiated cells were cultured in keratinocyte growth medium (KGM). The KGM was L-DMEM/DF12 (1:1) with 10% FBS, supplemented with 10 ng/mL epidermal growth factor (EGF, R&D), 5 ng/mL fibroblast growth factor (bFGF, R&D), 5 g/mL insulin (Sigma), 0.33 g/mL hydrocortione (Sigma), and 10 g/mL transferrin (Sigma). The KGM was changed every 2 days. 2.2.2. Epithelial differentiated cell surface marker detection Flow cytometric characterization of differentiated cells at passage 10 was performed using FITC or PE labeled antibodies against CD29 (PE, Serotec), CD34 (PE, Miltenyi Biotec), CD49d (FITC, Serotec), CD49f (FITC, Serotec), and CD71 (FITC, Serotec). 2.2.3. RT-PCR analysis The mRNA level of CK5, CK10 and CK19 of differentiated epithelial cells were checked by RT-PCT at passages 1 and 10. 2.2.4. Cell cycle analysis Cell cycle stage of differentiated cells was analyzed at passage 1 as described above. 2.2.4.1. Immunofluorescent staining. For immunofluorescence analysis, differentiated cells were first fixed in 4% paraformaldehyde for 15 min at room temperature and rinsed with PBS. Then cells were permeabilized with 0.1% Triton X-100 and 3% H2 O2 for 10 min. The primary antibodies (Mouse anti Human CK19, ZSGB-BIO) were diluted at a definite concentration in accordance with instructions. Incubation was performed at 4 ◦ C for 16 h. Then cells were incubated with a secondary antibody (FITC-Goat anti Mouse IgG, ZSGB-BIO) for 1 h at 37 ◦ C. The immunofluorescence images were captured using a fluorescence microscope. 2.3. Osteogenic differentiation of differentiated epithelial cells. 2.3.1. Osteogenic differentiation protocol Osteogenic differentiation was induced by culturing differentiated epithelial cells in an osteogenic medium (OM) for 3 weeks: L-DMEM supplemented with 10% FBS, 10 mM -glycerophosphate, 0.1 M dexamethasone, and 0.2 mM ascorbic acid (all from Sigma) (Cao et al., 2005). The osteogenic differentiation culture medium was changed every 3 days.
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Fig. 1. Morphological observation of differentiated epithelial cells by light microscopy. (Left) ADAS-cells exhibited fibroblast-like morphology (40×). (Middle) ADAS-cells after culture in epithelial differentiation medium for 3 days (40×). (Right) ADAS-cells after culture in epithelial differentiation medium for 7 days (40×).
2.3.2. Alkaline phosphatase (ALP) staining (Ca–Co technique) Three weeks post-differentiation, osteogenic differentiated cells were extensively washed with PBS, fixed in cool acetone for 10 min, washed with distilled water, incubated in 5 mL buffer [consisting of 20 g/L -phosphoric acid glycerin natrium (10 mL), 20 g/L barbital sodium (20 mL), 20 g/L addex-magnesium (1 mL), and 5 mL distilled water] (pH = 9.2, Zhongshan Biochemical, China) at 37 ◦ C for 6 h. Then they were washed with distilled water and incubated in a 20 g/L aqueous solution of cobaltous nitrate for 5 min. After another wash with distilled water, samples were incubated in aqueous solution of sulfon ammonium for 3 min. After a final wash, each sample was dehydrated, mounted, and examined by light microscopy (He et al., 2007). 2.3.3. Von Kossa staining Osteogenic differentiated cells were fixed with 4% paraformaldehyde for 60 min at room temperature. Cells were rinsed with distilled water and then overlaid with a 1% (wt/vol) silver nitrate solution in the absence of light for 30 min, then
developed under UV light for 60 min. After another wash with distilled water, the samples were dipped into 5% sodium thiosulfate to counteract silver nitrate. Each sample was dehydrated, mounted, and examined by light microscopy (Zuk et al., 2001). 3. Results 3.1. Morphology and surface markers of ADAS-cells Freshly isolated ADAS-cells were heterogenous, containing contaminating cells the numbers of which diminished over subsequent culture passages, leaving a homogenous population of cells exhibiting fibroblast-like morphology (Fig. 1 left). ADAS-cells were easily expanded in vitro and did not appear to require specific sera lots for expansion and multilineage differentiation (Zuk et al., 2001; Guan et al., 2006). Flow cytometric characterization of ADAS-cells showed that cultured cells were positive for CD29, CD44 and CD90, and negative for CD34 and CD45, with low expression of CD49d and CD71 (Fig. 2).
Fig. 2. Surface marker detection of ADAS-cell. ADAS-cells were positive for CD29, CD44, and CD90, and negative for CD34 and CD45, with low expression of CD49d and CD71.
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Fig. 3. Surface marker detection of differentiated epithelial cell. Differentiated epithelial cells were positive for CD29 and negative for CD34 and CD49f, with low expression of CD49d and CD71.
3.2. Morphology and surface markers of differentiated epithelial cells To differentiate ADAS-cells toward an epithelial lineage, ADAScells were cultured in the epithelial differentiation culture medium for 7 days. The effect of induction could be observed by light microscopy. As documented in Fig. 1 middle and right, cells contracted gradually and assumed a uniform cobblestone-like appearance over time, presenting growth characteristics more like a cultured monolayer of epithelial cells. Flow cytometric characterization of differentiated epithelial cells showed that they were positive for CD29 and negative for CD34 and CD49f, with low expression of CD49d and CD71 (Fig. 3).
3.3. RT-PCR analysis No CK5, CK10 or CK19 mRNA was detected in ADAS-cells. CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1. In contrast, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10 (Fig. 4).
Fig. 4. RT-PCR analysis of ADAS-cells, differentiated epithelial cells at passage 1, and differentiated epithelial cells at passage 10. No CK5, CK10 or CK19 mRNA was detected in ADAS-cells. CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1. In contrast, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10.
Fig. 5. 87.42% of ADAS-cells in the G0–G1 stage, whereas 96.38% of differentiated epithelial cells in the G0–G1 stage.
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Fig. 6. After induction, the expression of CK19 was observed under a fluorescence microscope.
3.4. Cell cycle analysis Cell cycle analysis was performed in ADAS-cells and differentiated epithelial cells at passage 1. The results revealed 87.42% of ADAS-cells in the G0–G1 stage, whereas 96.38% of differentiated epithelial cells in the G0–G1 stage (Fig. 5). 3.5. Immunofluorescent staining After induction, the expression of CK19 was observed under a fluorescence microscope (Fig. 6). 3.6. Osteogenic differentiation of differentiated epithelial cells Osteoblasts differentiated from differentiated epithelial cells have typical osteoblasts cellular morphology as shown in Fig. 7 left. Cells developed a number of tiny, brown-black granules with positive stain for alkaline phosphatase (Fig. 7 middle), and cells formed irregularly-shaped, dense granules in the ECM with positive stain for Von Kossa suggesting indeed calcified ECM (Fig. 7 right). 4. Discussion Skin lesions resulting from burns or trauma are major healthcare problems. The prospective use of stem cells for the restoration of injured or diseased tissues has opened new fields of research. Plasticity is the first requirement for therapeutic potential (Zaragosi et al., 2006). Furthermore, an alternative source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous (Zuk et al.,
2001). ADAS-cells possess all of these advantages, and have been proven to differentiate into adipogenic, chondrogenic, myogenic, osteogenic, cardiomyogenic, neurogenic, and endotheliogenic cells in vitro in the presence of lineage-specific induction factors (Zuk et al., 2001; Guan et al., 2006; Cao et al., 2005; Kang et al., 2007; Huang et al., 2007; Planat-Bénard et al., 2004). Thus ADAS-cells may be an alternative stem cell source for regenerative medicine in the future. ADAS-cells are considered to be a heterogeneous cell population. Immunofluorescence and flow cytometry show that the majority of ADAS-cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells (Zuk et al., 2001). The adherent-cultured ADAS-cells assume fibroblast-like morphology as observed by light microscopy. The morphology can be maintained through repeated subculturing under non-stimulating conditions. ADAS-cells can be maintained in vitro for extended periods with stable proliferation and low levels of senescence (Zuk et al., 2001; Cao et al., 2005; Kang et al., 2007). Using flow cytometry to characterize the phenotype of adherent adipose-derived cells at the fifth passage, it had been shown that these cultured cells were positive for CD29 (1-integrin), CD44, CD90 (Thy-1), CD105 (endoglin), CD166, Flk1, and HLA-ABC. In addition, no expression of the hematopoietic and endothelial lineage markers (CD31, CD34, CD45, CD106, and CD184) was observed (Zuk et al., 2002; Brzoska et al., 2005; Cao et al., 2005; Kang et al., 2007). This phenotype is similar to that of cells isolated from the bone marrow, but ADAS-cells are positive for CD49d (VLA-4) and negative for CD106 (VCAM-1), while bone marrow-derived cells are positive for CD106 and negative for CD49d (Zaragosi et al. (2006)). In our study, ADAS-cells were positive for CD29, CD44, and CD90, and negative for CD34 and CD45, with low expression of CD49d and CD71. This profile is very similar to the phenotype having been reported in the aforementioned studies (Zuk et al., 2002; Brzoska et al., 2005; Cao et al., 2005; Kang et al., 2007). Previous studies have demonstrated that bone marrow-derived cells (BMDCs), embryonic stem cells, and umbilical cord blood stem cells can differentiate into an epithelial lineage in vitro and in vivo and not as the result of cell fusion (Borue et al., 2004; Nakagawa et al., 2005; P˘aunescu et al., 2007; Harris et al., 2004; Kamolz et al., 2006; Metallo et al., 2008). However, donor-derived cytokeratin 5-expressing cells were rare in Borue’s study (Borue et al., 2004), suggesting that BMDCs do not engraft as epithelial stem cells. The level of engraftment peaked and then decreased over time, which suggest that BMDCs may assist in early wound healing by engrafting as transit-amplifying cells (TACs), and then differentiate into keratinocytes. Recent studies have verified that ADAS-cells also have epithelial potential (Brzoska et al., 2005; Altman et al., 2009).
Fig. 7. In vitro osteogenic differentiation of differentiated epithelial cells. (Left) Differentiated epithelial cells after culture in osteogenic medium for 3 weeks (40×). (Middle) Some tiny, brown-black granules stained positively for alkaline phosphatase (200×). (Right) Irregularly-shaped, dense granules in the ECM stained positively with Von Kossa staining (100×).
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However, no study has investigated whether ADAS-cells can differentiate into epithelial stem cells or TACs. Epithelial stem cells have a high proliferative potential, are capable of self-renewal, and express 1 integrin but not CK1/CK10. The descendants of the stem cells are the TACs that express both 1 integrin and CK1/CK10 and belong to a partially differentiated pool of cells with a limited proliferative capability. Mitotically inactive keratinocytes (1 integrin− , CK1/CK10+ ) are the end-product of TACs differentiation (Gniadecki, 1997). Until now, identification of epithelial cells using molecular markers has been controversial. CD29+ , CD49fbri CD71dim , CD90+ , CK19+ and p63+ are commonly regarded as markers of epithelial stem cells in most studies (Gniadecki, 1997; Vorotelyak et al.,2006; Nakamura et al., 2006). The capacity of self-renewal makes stem cells particularly useful for transplantation medicine because they could provide an unlimited source of donor material for grafting or transplantation in theory. Moreover, most approaches to skin tissue engineering would require differentiated keratinocytes or keratinocyte progenitors, rather than undifferentiated stem cells, for seeding and attaching onto artificially synthesized matrices (Bannasch et al., 2003). In 2005, Broska et al. reported that adipose-derived adult stem (ADAS)-cells owned some characteristics of stem cells and could differentiate into epithelial cells in a suitable environment. In this study, we found that ADAS-cells could differentiate into an epithelial lineage and these ADSCs-derived epithelial cells owned some characteristics of stem cells, which could be induced to an osteogenic lineage. In detail, ADAS-cells were induced to an epithelial lineage under the influence of ATRA in vitro. As we observed, cells contracted gradually and assumed a uniform cobblestonelike appearance over time, presenting growth characteristics more like a cultured monolayer of epithelial cells. Cell cycle analysis also revealed more differentiated cells (96.38%) than ADAS-cells (87.42%) in the G0-G1 stage. RT-PCR analysis revealed no expression of CK5, CK10 and CK19 in ADAS-cells, only expression of CK19 in differentiated cells at passage 1, and expression of both CK10 and CK19 in differentiated cells at passage 10. Immunofluorescent staining for CK19 was chosen for the basal characterization of epithelial-like stem cells (Larouche et al., 2010). After induction, the expression of CK19 was observed. In order to evaluate whether differentiated epithelial cells have transdifferentiation potential, these cells were further induced to differentiate toward an osteogenic lineage. Positive staining with ALP and Von Kossa staining verified these cells were capable of osteogenesis. All these results indicated that ADAS-cells could differentiate into an epithelial lineage while retaining most of the characteristics of stem cells. However, differentiated epithelial cells were negative for CD49f (␣6-integrin) which should be present as an surface marker of epithelial stem cells (Vorotelyak et al.,2006; Nakamura et al., 2006). We thus deemed that these differentiated epithelial cells were not true epithelial stem cells and named them “ADSCs-derived epithelial-like stem cells”. Among the components of the epithelial differentiation culture medium used in our study, ATRA plays an important role in epithelial differentiation. Retinoic acid strongly upregulated expression of keratin 18 and the transcription factor p63 (p63 is involved in epidermal morphogenesis and ectodermal specification), while repressing early neural marker transcription (Metallo et al., 2008). In Brzoska’s study, ATRA had the expected inhibitory effect on cell proliferation with a tolerable impact on cell viability at a concentration of 5 M (Brzoska et al., 2005). p63, a homolog to the tumor suppressor protein p53, is chiefly expressed in epithelial tissues, including the epidermis. p63 affects cell death similar to p53, and also plays important roles in the development of epithelial tissues and the maintenance of epithelial stem cells. DeltaNp63alpha, a p63 isoform specifically expressed in basal keratinocytes, suppressed the expression of specific, late differentiation stage proteins, such
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as filaggrin and loricrin. A precise balance between p63 and keratinocyte growth factor (KGF) mediates the onset of epithelial cell differentiation, via c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) signaling (Ogawa et al., 2008). DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of embryonic stem cells to become K5/K14-positive stratified squamous epithelial cells (Medawar et al., 2008). The employment of bovine serum in the cell culture to induce epithelial differentiation is a major weakness as it represents a limitation for further implementation of these cells into a human organism. Thus, we intend to replace bovine serum in future study. Acknowledgments This work was supported by National High Technology Research and Development Program of China (No. 2006AA02A107), the Major State Basic Research Program of China (Nos. 2009CB941102 and 2010CB945504). Supplementary data Information about the transparent peer review is available as supplementary material in the database Science Direct. The reviewer comments and the authors reply can be viewed by clicking the link: http://dx.doi.org/10.1016/j.aanat.2012.10.009. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aanat.2012.10.009. References Abiko, Y., Hiratsuka, K., Kiyama-Kishikawa, M., et al., 2004. Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray. J. Oral Sci. 46, 19–24. Altman, A.M., Yan, Y., Matthias, N., et al., 2009. IFATS collection: human adiposederived stem cells seeded on a silk fibroin–chitosan scaffold enhance wound repair in a murine soft tissue injury model. Stem Cells 27, 250–258. Bannasch, H., Föhn, M., Unterberg, T., et al., 2003. Skin tissue engineering. Clin. Plast. Surg. 30, 573–579. Borue, X., Lee, S., Grove, J., et al., 2004. Bone marrow-derived cells contribute to epithelial engraftment during wound F healing. Am. J. Pathol. 165, 1767–1772. Brzoska, M., Geiger, H., Gauer, S., et al., 2005. Epithelial differentiation of human adipose tissue-derived adult stem cells. Biochem. Biophys. Res. Commun. 330, 142–150. Cao, Y., Sun, Z., Liao, L., et al., 2005. Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem. Biophys. Res. Commun. 332, 370–379. Cohen-Kerem, R., Lahat, N., Elmalah, I., et al., 2002. Detection of cytokeratins in normal and malignant laryngeal epithelia by means of reverse transcriptasepolymerase chain reaction. Ann. Otol. Rhinol. Laryngol. 111, 149–154. Gimble, J., Guilak, F., 2003. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 5, 362–369. Gniadecki, R., 1997. Effects of 1,25-dihydroxyvitamin D3 and its 20-epi analogues (MC 1288, MC 1301, KH 1060), on clonal keratinocyte growth: evidence for differentiation of keratinocyte stem cells and analysis of the modulatory effects of cytokines. Br. J. Pharmacol. 120, 1119–1127. Guan, L., Li, S., Wang, Y., et al., 2006. In vitro differentiation of human adiposederived mesenchymal stem cells into endothelial-like cells. Chin. Sci. Bull. 51, 1863–1868. Harris, R.G., Herzog, E.L., Bruscia, E.M., et al., 2004. Lack of a fusion requirement for development of bone marrow-derived epithelia. Science 305, 90–93. He, L., Nan, X., Wang, Y., et al., 2007. Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells. Sci. China Ser. C 50, 429–437. Huang, T., He, D., Kleiner, G., et al., 2007. Neuron-like differentiation of adiposederived stem cells from infant piglets in vitro. J. Spinal Cord Med. 30 (Suppl. 1), S35–S40. Kamiya, M., Ichiki, Y., Kamiya, H., et al., 2003. Detection of nonmelanoma skin cancer micrometastases in lymph nodes by using reverse transcriptase-polymerase chain reaction for keratin 19 mRNA. Br. J. Dermatol. 149, 998–1005.
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