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Journal of Dental Sciences (2013) xx, 1e6
Available online at www.sciencedirect.com
journal homepage: www.e-jds.com
ORIGINAL ARTICLE
Surgical-derived oral adipose tissue provides early stage adult stem cells Juin-Hong Cherng a, Shu-Jen Chang a, Tong-Jing Fang b,c, Meng-Lun Liu d, Chung-Hsing Li e, Shih-Fang Yang a,f, Jiang-Chuan Liu g, Nien-Hsien Liou g, Ming-Lun Hsu a* a
Department of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC Department of Cardiology, SongShan Armed Forces General Hospital, Taipei, Taiwan, ROC c Department of Family Medicine, BeiTou Armed Forces Hospital, Taipei, Taiwan, ROC d General Surgery, Cheng-Hsin General Hospital, Taipei, Taiwan, ROC e Department of Dentistry for Children and Orthodontic Dental, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC f Eonway Health Maintenance Center, West Garden Hospital, Taipei, Taiwan, ROC g Department and Graduate Institute of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan, ROC b
Received 13 November 2011; Final revision received 10 January 2012 Available online - - -
KEYWORDS bone morphogenetic protein 4; human oral adipose tissue; nestin; octamer-binding transcription factor 4; telomere length assay
Abstract Background/purpose: Stem cells (SCs) are characterized by the ability to undergo self-renewal and differentiation into multi-lineage cell types. The low risk to donors, the ease of harvest, and the abundance in the human body of adipose-derived SCs make it an attractive source for adult SCs. Materials and methods: Oral adipose tissue was collected by clinical surgery as certified by the Institutional Review Board. SCs were isolated and purified with collagenase containing Dulbecco’s modified Eagle’s medium (DMEM) and confirmed by immunofluorescence staining with early stage SCs and germinal layer markers. Telomere length was assayed following the procedure provided with the TeloTAGGG kit, and the reverse transcription polymerase chain reaction (RT-PCR) of octamer-binding transcription factor 4 (Oct4) was detected with primers 50 GTA CTC CTC GGT CCC TTT CC-30 and 50 -CAA AAA CCC TGG CAC AAA CT-30 . Results: SCs derived from human oral adipose tissue (hOASCs) express Oct4 (an early stage stem cell marker) as well as bone morphogenetic protein 4 (BMP4) and nestin (both germ layer markers). Furthermore, the telomere length assay showed that hOASCs possess high-
* Corresponding author. Department of Dentistry, National Yang-Ming University, Number 155, Section 2, Linong Street, Beitou District, Taipei City 112, Taiwan, ROC. E-mail address:
[email protected] (M.-L. Hsu). 1991-7902/$36 Copyright ª 2013, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jds.2013.02.008
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J.-H. Cherng et al molecular-weight fragments of telomere, an indication of self-renewal capacity. Contrary to previous observations that the differentiation ability of adult SCs is affected by cell age and source of restructure, our results suggested that hOASCs possess pluripotential at the early stage and the ability to continually proliferate without curtailment of the telomere length. Conclusion: Human oral adipose tissues can be a beneficial source for isolating pluripotent adult SCs for clinical autologous applications. Copyright ª 2013, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction Stem cell (SC)-based therapies hold great promise for curing various human diseases. In recent years, progressive advancements have been made in SC therapy and restructuring techniques in various areas of study. The availability of SC resources has also drastically increased in conjunction with these advancements. SCs can be roughly categorized into three types: embryonic SCs, induced pluripotent SCs, and adult SCs. Among them, the adult SCs are ethically less controversial and bear no scruples on viral effect compared to other sources. Besides, those excess tissues left behind from various clinical operations, such as the umbilical cord, peripheral blood, fat tissue, and teeth, etc., can be great resources of adult SCs, benefiting potential recipients without causing additional damage to the donors. Recent studies showed that progenitor (stem) cells derived from oral tissues, such as dental pulp, periodontal ligaments, and dental papillae1e4 can differentiate into osteocytes, adipocytes, and chondrocytes.5e8 Yalvac et al proved that progenitor (stem) cells derived from the tooth germ can be reprogrammed to express early stage SC markers such as octamer-binding transcription factor 4 (Oct4), Krueppel-like factor 4 (Klf4), homeobox protein NANOG (Nanog), SRY (sex determining region Y)-box 2 (Sox2), and myelocytomatosis c oncogene (c-Myc).9 While searching for potential sources of adult SCs from other oral tissues, we found that human oral adipose tissue can develop into SCs (hOASCs). Other researchers have shown that the SCs from adipose tissue display similar morphological and phenotypical features to bone marrow mesenchymal SCs. Adipose tissue-derived SCs can differentiate into a variety of cells that include not only chondrocytes, adipocytes, osteoblasts, and myocytes10e12 but also neural lineages.13,14 Adipose tissues obtained from plastic surgery such as liposuction, wound reconstruction, and skin-tightening surgeries are readily accessible. We directed our attention at oral adipose tissues from dental surgery clinics as a potential source of adult SCs. In this study, the telomere length assay, a method that has been applied frequently for cell senescence studies, was used to characterize the stemness of OASCs.15,16
Materials and methods Isolation and purification of hOASCs Oral adipose tissue was collected by clinical surgery as certified by the Institutional Review Board of Tri-Service
General Hospital, Taipei, Taiwan. Approximately 1e3 mL samples were isolated from oral adipose tissue and incubated with transfer buffer (containing 0.1 M phosphate buffered saline (PBS; Ferak, Berlin, Germany), 1% penicillin/ streptomycin (Merck, Darmstadt, Germany), and 0.1% glucose (Ferak, Berlin, Germany), and then transferred immediately to our lab. The tissue was then cut into 1-mmdiameter sections and transferred into 10 mL Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 0.1% collagenase (Sigma, St. Louis, MO, USA) for 1 day in a 37 C incubator. Then, tissue sections were transferred to a DMEM/10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) solution for 24 hours. Cells were collected by centrifugation at 500 g for 5 minutes. The resulting pellet was suspended in keratinocyte serum-free medium (K-SFM; Gibco, Carlsbad, CA, USA) containing 5% FBS, antioxidants Nacetyl-cysteine and L-ascorbic acid-2-phosphate (Sigma, St. Louis, MO, USA).17 Cells were then placed in a 25-cm2 flask and incubated under 5% CO2 at 37 C. After 2e4 days of incubation, depending upon the growth rate of the cells, the primary cells generated were then collected after changing the medium. Under this growth condition, each generation took up to 2e3 days to reach confluency.
Growth rate of hOASCs The growth rate of the hOASCs was determined by planting 250,000 cells per 25-cm2 flask; each counting had three replicas and counted every 72 hours. Cells were counted before the next passage, and 250,000 cells were replanted for a total of the next passage. The growth rate was then calculated by dividing the total cell number after every 72 hours of culture.
Telomere length assay Purified genomic DNA was digested with an optimized mixture of frequent-cutter restriction enzymes. Following DNA digestion, the DNA fragments were separated by gel electrophoresis and transferred to a nylon membrane for Southern blotting. The blotted DNA fragments were hybridized to a digoxigenin (DIG)-labeled probe specific for telomeric repeats and incubated with a DIG-specific antibody covalently coupled to alkaline phosphate. Finally, the alkaline phosphatase can be detected with a commercial chemiluminescent substrate (CDP Star). The average length was determined by comparing with a molecular weight standard. In general, long telomeres are observed in SCs or cells that vigorously proliferate, whereas shorter lengths are typical of somatic cells.
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Early stage stem cells from oral adipose tissue
Cell preparation and immunostaining The cells were seeded with a density of 1 105 cells/well of a six-well culture plate for 1 day and then fixed with 4% paraformaldehyde. After washing with 1 PBS, the fixed cells were permeabilized with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA) for 15 minutes. Cells were then blocked for 20 minutes with PBS containing 10% normal goat serum (Gibco, Carlsbad, CA, USA) and incubated with primary antibodies (sc-8628, sc-6896, sc-23927, sc-29011, sc-6189; Santa Cruz Co., Santa Cruz, CA, USA) for 2 hours at room temperature. The primary antibody was removed, and cells were washed with 1 PBS and then incubated for 2 hours with rhodamine- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (61056-05-H488, 61056-05H555; Anaspec Co., Fremont, CA, USA ) for 2 hours. Fluorescent images were obtained with an epifluorescent microscope equipped with a digital camera (SPOT-RT; Diagnostic Instruments, Detroit, MI, USA).
RT-PCR Total RNA was extracted from cells with organic extraction reagent (TRIZOL; Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol and treated with 10 DNase buffer and 1 U/mL deoxyribonuclease (DNase; Invitrogen, Carlsbad, CA, USA) to remove any contaminating DNA. Oligo(dT) primers were used with reverse transcriptase (SuperScriptTM III RT; Invitrogen, Carlsbad, CA, USA) for
3 complementary DNA (cDNA) synthesis from 1 mg of total RNA following the manufacturer’s instructions. PCR was conducted with DNA Polymerase (Platinum Taq; Invitrogen, Carlsbad, CA, USA). We used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal standard for Oct4 RT-PCR. The primer sequences for human Oct4 were: 50 -GTA CTC CTC GGT CCC TTT CC-30 and 50 -CAA AAA CCC TGG CAC AAA CT-30 . The PCR products were separated on 1.5% agarose gels by electrophoresis. Digital images were captured on a digital gel image system (AlphaImager Mini System; Bucher Biotec, Basel, Switzerland).
Results Isolation of OASCs To isolate adult SCs from oral adipose tissue, we used a serum-free SC medium to select quality cells. After 3e4 days, fewer than half of the cells had adhered to the flask, and many had died (Fig. 1A). These adherent cells were then transferred to a new flask to begin the general growth process after initial culture (Fig. 1B). The medium-selected cells subsequently proliferated to confluence (Fig. 1C) and were used for immunofluorescence identification and molecular biological characterization of SCs. For continual maintenance, these cells need to be passaged every 2e3 days regardless of whether the flask is confluent or not. The growth curve of isolated cells showed that the cell number reached 2.5 107 after five generations (Fig. 1D).
Figure 1 (A) Cells derived from oral adipose tissue were selected by stem cell (SC) culture medium. (B) The secondary generation cells 2 days after first passage. (C) After proliferation, the third generation cells became confluent a further 2 days after secondary passage. (D) The growth curve of isolated cells in generations 1e5. Scale bar Z 200 mm. Please cite this article in press as: Cherng J-H, et al., Surgical-derived oral adipose tissue provides early stage adult stem cells, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/j.jds.2013.02.008
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J.-H. Cherng et al
OASCs characterized by telomere length assay To investigate the self-renewal characteristic of hOASCs, genomic DNA was purified from the 17th generation of cells, digested, and hybridized to a DIG-labeled probe specifically for telemetric repeats for the telomere length assay. Even though the telomere length for most cells tends to decrease as cells divide, the result of Southern blotting revealed that hOASCs still maintained long telomeres (w21 kbp, compared to the high-molecular-weight control DNA) after 16 passages (Fig. 2).
Bio-markers of SCs
Figure 2 The telomere length of oral adipose tissue-derived stem cells (OASCs) is maintainable (w21 kbp) after the 17th generation. HC Z high molecular weight control DNA; LC Z low molecular weight control DNA.
To identify hOASCs, we dissociated and plated cells on six-well culture plates without growth factors for 12e24 hours and immunostained them from a development point-of-view with Oct4 (a marker for SC-related transcription factors expressed during early development) and bone morphogenetic protein 4 (BMP4; a marker that acts as a morphogen in dorsal-ventral patterning of the mesoderm in human embryonic development). The results indicated that when compared to phase micrographs (Fig. 3A and D), almost all of the cells were immunepositive for Oct4 (Fig. 3B) and BMP4 (Fig. 3E). In order to make sure that adult SCs express early embryonic SC marker Oct4, we checked the messenger (m)RNA of Oct4 in these cells. The DNA agarose gel electrophoresis from the RT-PCR results also demonstrated that hOASCs produced Oct4 (Fig. 3C).
OASCs displayed the potential multi-lineages capability To better characterize and attempt to determine the origin and potential ability of hOASCs, we examined if they
Figure 3 (A,D) Stem cells derived from oral adipose tissue (OASCs) (B) expressed the embryonic stem cell marker Oct4 with FITC fluorescence. (C) The RT-PCR result showed that the mRNA of Oct4 expressed at 168 bp corresponded with the primer; the GAPDH expressed at 241 bp was the control. (E) BMP4, a early mesoderm marker, was also expressed in cells with rhodamine fluorescence. Scale bar Z 75 mm. Please cite this article in press as: Cherng J-H, et al., Surgical-derived oral adipose tissue provides early stage adult stem cells, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/j.jds.2013.02.008
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Early stage stem cells from oral adipose tissue
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Figure 4 (A,C,E) Stem cells derived from oral adipose tissue (B) were immunopositive for nestin, the neural stem cell marker. (D) OASCs were also positive for fibronectin, the mesoderm cell marker. (F) However, the OASCs did not express the neural crest cell marker p75NGFR. Scale bar Z 75 mm.
express markers associated with the mesoderm and mesoectoderm. Our results from immunolabeling revealed that most of the cells in the clusters were nestin-positive (Fig. 4A), but they did not produce the marker for neural crest SCs, p75NGFR, a low-affinity nerve growth factor receptor (Fig. 4C). By contrast, these cells also produced fibronectin, a protein produced by bone marrow mesenchymal SCs (Fig. 4B).
Discussion Oct4 is an important marker of SC-related transcription factors expressed during early stages of development. It was thoroughly studied in embryonic SCs and is confined to cells that display toti/pluripotent phenotypes. Oct4 regulates several genes expressed during early development, including Sox2 and Rex1,18 and it was suggested to be a master regulator of initiation, maintenance, and
differentiation of pluripotent cells.19,20 Reports suggested that Oct4 is not expressed in normal tissues because normal tissues mainly consist of terminally differentiated cells, as well as progenitor cells that have a decided fate.21 Our study serendipitously seems to suggest that normal tissues contain few adult SCs that are still in the very early stages. It is very possible that if we search for these early stage SCs, we may find a sufficient amount of Oct4-expressing cells in normal tissues. In human embryonic development, BMP4 is a critical signaling molecule required for early differentiation of embryos and establishment of the dorsal-ventral axis for differentiation of the later structures.22 BMP4 acts as a morphogen in dorsal-ventral patterning of the mesoderm including bone and cartilage development, and specifically in tooth and limb development. With the gradients, BMP4 also stimulates differentiation of overlying ectodermic tissues.23 The fact that hOASCs express BMP4 indicates that cells still preserve the receptor for stimulating mesoderm
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6 or ectoderm tissues. This process provides a potential source for applications in oral reconstruction and tissue engineering. The characteristics of germinal layer marker also demonstrated the potential preserves of hOASCs. Studies showed that adult neural SCs do not produce fibronectin; they are prone to generating neural cells instead.24,25 Mesenchymal SCs do not easily produce neural proteins26e29 because they are inclined to generate cells of mesodermal origin.30 p75 is the nerve growth factor receptor marker for neural crest cells.31 In our study, hOASCs express both nestin and fibronectin, but not p75. These results suggested that hOASCs are a special, pluripotent type of adult SC capable of generating cells of more than one embryonic germ layer lineage. hOASCs derived from a potentially autologous, accessible adult source, oral adipose tissue, and can readily generate both ectodermal and mesodermal cell lineages to provide strategies for clinical applications. The results led us to discover the multi-lineage potential of early stage adult hOASCs that possess the capability of continual cell proliferation without curtailment of the telomere length.
Acknowledgments The authors would like to extend their gratitude to Dr NianTzyy for assisting with the application of Institutional Review Board certification, and Dr Yuan-Wu Chen for providing the medical waste oral adipose tissue. This work was supported by Cheng-Hsin/Yang-Ming corporate program, grant number 98F117CY21.
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