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Recovery of Stem Cells from Cryopreserved Periodontal Ligament

¹ Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, Building 30, Room 131, 30 Convent Drive MSC-4320, and ² Comparative Molecular Cytogenetics Core, Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; and ³ Department of Oral and Maxillofacial Surgery, College of Dentistry, Seoul National University, Seoul, Korea;

^* corresponding author, sshi{at}dir.nidcr.nih.gov

	ABSTRACT

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Human post-natal stem cells possess a great potential to be utilized in stem-cell-mediated clinical therapies and tissue engineering. It is not known whether cryopreserved human tissues contain functional post-natal stem cells. In this study, we utilized human periodontal ligament to test the hypothesis that cryopreserved human periodontal ligament contains retrievable post-natal stem cells. These cryopreserved periodontal ligament stem cells maintained normal periodontal ligament stem cell characteristics, including expression of the mesenchymal stem cell surface molecule STRO-1, single-colony-strain generation, multipotential differentiation, cementum/periodontal-ligament-like tissue regeneration, and a normal diploid karyotype. Collectively, this study provides valuable evidence demonstrating a practical approach to the preservation of solid-frozen human tissues for subsequent post-natal stem cell isolation and tissue regeneration. The present study demonstrates that human post-natal stem cells can be recovered from cryopreserved human periodontal ligament, thereby providing a practical clinical approach for the utilization of frozen tissues for stem cell isolation.

KEY WORDS: periodontal ligament stem cells (PDLSCs) • osteoblast • cementum • cryopreservation

	INTRODUCTION

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Post-natal stem cells have been successfully isolated from a variety of human tissues, including, but not limited to, bone marrow, peripheral blood, neural tissue, skeletal muscle, epithelium, dental pulp, and periodontal ligament (Gronthos et al., 2000; Bianco and Robey, 2001; Evers et al., 2003; Korbling and Estrov, 2003; Seo et al., 2004). With recent advances in stem cell therapies and tissue engineering, the effective preservation of stem cells is becoming an important issue for stem-cell-mediated clinical treatment (Korbling and Estrov, 2003). For decades, cryopreserved hematopoietic stem cells have been utilized for disease treatment in clinics. Recently, it has been reported that hematopoietic stem cells can be successfully used for stem cell transplantation following 15 yrs’ cryopreservation (Broxmeyer et al., 2003), suggesting that long-term cryopreservation is a reliable approach for stem cell storage. Additionally, investigators’ ability successfully to cryopreserve reproductive cells—including spermatozoa and oocytes, reproductive tissues, embryos, and nuclear material—has been a significant contribution to reproductive technology and medicine (Hubel, 1997; He et al., 2003; Hoffman et al., 2003; Rowley et al., 2003; Woods et al., 2004). However, whether cryopreserved solid human tissue is a resource for retrieving functional stem cells is unknown.

Recently, human periodontal ligament stem cells were isolated and characterized as a population of multipotent stem cells capable of forming cementum and periodontal ligament tissues upon in vivo transplantation (Seo et al., 2004). Periodontal ligament tissue collected from extracted teeth is an easily accessible human tissue that may not only serve as a practical resource for potential stem-cell-mediated therapies, but may also provide a sufficient number of tissue samples for the analysis of stem cell characteristics. In this study, we took advantage of the availability of human periodontal ligament to test the hypothesis that cryopreserved human periodontal ligament contains retrievable post-natal stem cells.

	METHODS

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Subjects and Cryopreservation Cell Culture
Normal human impacted third molars and attached bone chips were collected immediately following extraction from a total of 10 adults (19–29 yrs of age) at the Dental Clinic of the National Institute of Dental & Craniofacial Research under approved guidelines, with written consent obtained from patients as set by the NIH Office of Human Subjects Research. Periodontal ligaments were gently separated from the surface of the root, and then they were minced into tiny pieces (0.5 mm³) (Seo et al., 2004). The periodontal ligament pieces derived from the different individuals were mixed together, and half of the tissue sample was utilized for the isolation of fresh stem cells, as control, while the remaining half was mixed with fetal calf serum (Equitech-Bio Inc., Kerrville, TX, USA) containing 10% dimethyl sulfoxide (DMSO) in a cryotube (NUNC Inc., Naperville, IL, USA) at 4°C and then directly stored in liquid nitrogen. After being frozen for 3 and 6 mos, the tissues were thawed rapidly at 37°C in a water bath and subsequently digested in a solution of 3 mg/mL collagenase type I (Worthington Biochem, Freehold, NJ, USA) and 4 mg/mL dispase (Roche, Mannheim, Germany) for 1 hr at 37°C. Single cell suspensions (5000 cells) were seeded into a T-25 flask (Costar, Cambridge, MA, USA) with alpha-modified Eagle’s Medium (GIBCO BRL, Grand Island, NY, USA), supplemented with 15% fetal calf serum (Equitech-Bio Inc., Kerrville, TX, USA), 100 µM L-ascorbic acid 2-phosphate (WAKO, Tokyo, Japan), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Biofluids Inc., Rockville, MD, USA), and then incubated at 37°C in 5% CO₂. To assess colony-forming efficiency, we fixed day 10 cultures with 4% formalin, and then stained them with 0.1% Toluidine blue. Aggregates of 50 cells were scored as colonies. The proliferation rate of sub-confluent cultures (first passage) of periodontal ligament stem cells was assessed by bromodeoxyuridine (BrdU) incorporation for 12 hrs, with the use of a Zymed Laboratories BrdU staining Kit (Zymed Laboratories, South San Francisco, CA, USA). Conditions for the induction of calcium accumulation and adipogenesis were as previously reported (Gronthos et al., 2000, 2002). For in vitro type I collagen generation, the periodontal ligament stem cell pellet (2 x 10⁶ cells) was cultured for 6 wks in 15-mL polypropylene tubes in 1 mL of high-glucose (4.5 g/L) DMEM medium supplemented with 1% ITS+, 100 µM L-ascorbic acid 2-phosphate (WAKO, Tokyo, Japan), 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Biofluids Inc., Rockville, MD, USA), 2 mM pyruvate, and freshly added 10 ng/mL TGF-ß1. The medium was changed twice a week.

Antibodies
Rabbit antibodies included anti-TGFßRI (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-human-specific mitochondria (Chemicon, Temecula, CA, USA), anti-type I collagen (LF-67), bone sialoprotein (BSP LF-120), and alkaline phosphate (ALP LF-47), courtesy of Dr. Larry Fisher of NIDCR/NIH (Miura et al., 2003). Mouse antibodies included STRO-1 (courtesy of Dr. Stan Gronthos). Rabbit and murine isotype-matched negative control antibodies were also used (Caltag Laboratories, Burlingame, CA, USA).

Transplantation
Approximately 2.0 x 10⁶ of in vitro expanded cryopreserved periodontal ligament stem cells were mixed with 40 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic particle (Zimmer Inc., Warsaw, IN, USA) and then transplanted subcutaneously into the dorsal surfaces of 10-week-old immunocompromised beige mice (NIH-bg-nu-xid, Harlan Sprague Dawley, Indianapolis, IN, USA) as previously described (Krebsbach et al., 1997; Seo et al., 2004). These procedures were performed in accordance with specifications of an approved animal protocol (NIDCR #02-222). The transplants were recovered at 6–8 wks post-transplantation, fixed with 4% paraformaldehyde, decalcified with buffered 10% EDTA (pH 8.0), and then embedded in paraffin. Sections were deparaffinized and stained with H&E.

Immunohistochemistry
Cryopreserved periodontal ligament stem cells were subcultured into 8-chamber slides (2 x 10⁴ cells/well) (NUNC Inc., Naperville, IL, USA). The cells were fixed in 4% paraformaldehyde for 15 min and then blocked and incubated with primary antibodies (1:200 to 1:500 dilutions) for 1 hr, respectively. The samples were subsequently incubated with goat secondary antibodies of either IgG-Rhodamine Red or IgG-Cy^TM2 (Jackson ImmunoResearch, West Grove, PA, USA), for 45 min. For nuclei staining, we applied VectorShield^® Mounting Medium with DAPI (4',6-diamino-2-phenylindol, Vector Laboratories, Inc., Burlingame, CA, USA) to mount the slides, and then examined them by fluorescence microscopy (Axioplane2, Zeiss, Thornwood, NY, USA). For enzymatic immunohistochemical staining, the Zymed broad-spectrum immunoperoxidase AEC kit (Zymed Laboratories Inc., South San Francisco, CA, USA) was used according to the manufacturer’s protocol.

Statistical Analysis
We used Student’s t test to analyze the significance between the 2 groups. P-values of less than 0.05 were considered statistically significant.

	RESULTS

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To examine whether cryopreserved tissue contained post-natal stem cells, we preserved small pieces of periodontal ligament frozen in liquid nitrogen for 3 and 6 mos, and then used the frozen periodontal ligament as a tissue resource to isolate post-natal stem cells. Compared with the number of periodontal ligament stem cell colonies recovered from fresh periodontal ligament tissue, 40% of single-colony-derived periodontal ligament stem cells can be recovered from frozen-thawed periodontal ligament tissue following enzyme digestion (5000 cells per T-25 flask) (Fig. 1A). Although the number of single colonies derived from cryopreserved periodontal ligament was significantly decreased in comparison with the fresh, isolated periodontal ligament stem cells, they maintained a high proliferative capacity in terms of BrdU labeling for 12 hrs (Fig. 1B). Following histological examination of cryopreserved periodontal ligament, the frozen periodontal ligament tissue exhibited normal histological structures in H&E staining in the majority of the areas examined (Figs. 1C, 1D). However, cellular damage—such as anisokaryosis, variable sizes of nuclei, and clumping of cells—was noted in some areas (Fig. 1E). These cells were also negative for TUNEL staining (data not shown), indicating a non-apoptotic cell death, probably caused by the nucleation of lethal intracellular ice.

View larger version (61K): [in this window] [in a new window]   Figure 1. Isolation of cryopreserved periodontal ligament stem cells. (A) Periodontal ligament stem cells recovered from 6-month-cryopreserved periodontal ligament were capable of forming single-cell clusters after being plated at low density and cultured with regular culture medium for 10 days as described in METHODS. The number of single colonies derived from cryopreserved periodontal ligament (CP) was significantly decreased (*p < 0.05) in comparison with that derived from the fresh non-frozen periodontal ligament (P) when the same number (5000) of cells was plated (CP = 4.7 ± 0.6, P = 24.8 ± 1.5, mean ± SD) (N = 3). (B) The proliferation rates were assessed by bromodeoxyuridine (BrdU) incorporation for 12 hrs. Cryopreserved periodontal ligament stem cells (CP) maintain a high level of proliferation rate, similar to that of the regular periodontal ligament stem cells (P), showing that there is no significant difference between the regular periodontal ligament stem cells and cryopreserved periodontal ligament stem cells (73.8 ± 3.7, 67.4 ± 10.3, respectively, mean ± SD) (N = 3). (C) H&E staining of non-frozen human periodontal ligament tissue. (D,E) H&E staining of periodontal ligament cryopreserved for 6 mos. Most areas of periodontal ligament tissue showed normal histological structures in H&E staining. However, some nuclear anisokaryosis was found in frozen periodontal ligament (E, arrow), indicating that the cryopreservation can cause some tissue damage (scale bar, 200 µm for C-E). (F-M) Cryopreserved periodontal ligament stem cells expressed STRO-1, one of the early progenitor markers for mesenchymal stem cells. The cryopreserved periodontal ligament stem cells may co-express STRO-1 with bone sialoprotein (BSP) and TGFß receptor type I (TGFßRI), as shown on the merged Figs. Some cryopreserved periodontal ligament stem cells may express STRO-1 and BSP separately (scale bar, 50 µm for F–M).

To evaluate the capacity for multipotential differentiation in vitro, we supplemented established secondary cryopreserved periodontal ligament stem cell cultures with L-ascorbate-2-phosphate, dexamethasone, and inorganic phosphate, to induce osteogenic/cementogenic differentiation, as previously described (Miura et al., 2003; Seo et al., 2004). The results demonstrated that Alizarin-red-positive nodules formed in the cryopreserved periodontal ligament stem cell cultures following 4 wks of induction, indicating calcium accumulation in vitro (Figs. 2A, 2B). Next, we examined the potentiality of cryopreserved periodontal ligament stem cells to develop into adipocytes. In parallel to what has been previously demonstrated for adult periodontal ligament stem cells (Seo et al., 2004), cryopreserved periodontal ligament stem cells were also found to possess the potential to develop into Oil-red O-positive lipid-laden fat cells following 5 wks of culture with an adipogenic-inductive cocktail (Figs. 2C, 2D).

View larger version (80K): [in this window] [in a new window]   Figure 2. In vitro characterization of cryopreserved periodontal ligament stem cells. (A,B) Alizarin red staining showed mineralized nodule formation (A). In the regular culture conditions, the cryopreserved periodontal ligament stem cells were unable to form mineralized nodules (B). (C,D) Cryopreserved periodontal ligament stem cells were able to form Oil red O-positive lipid clusters (C). Regular culture medium could not induce any Oil red O-positive lipid clusters in cryopreserved periodontal ligament stem cells (D) (scale bar, 100 µm for A-D). (E) When periodontal ligament stem cells were cultured with 10 ng/mL TGFß1 for 4 wks, they formed distinct collagen fibers in vitro (open arrows). (F) The in vitro-generated fibers were positive for anti-type I collagen antibody staining (open arrows). (G) Cryopreserved periodontal ligament stem cells were also able to generate collagen aggregates in vitro when cultured with 10 ng/mL of TGFß1 for 4 wks. (H) The newly generated aggregates were positive for anti-type I collagen antibody staining (scale bar, 50 µm for E-H).

It has been demonstrated that periodontal ligament stem cells were able to form cementum/periodontal-ligament-like tissues upon in vivo transplantation. To confirm the tissue- regenerative capacity, we transplanted cryopreserved periodontal ligament stem cells into immunocompromised mice subcutaneously, using hydroxyapatite/tricalcium phosphate (HA/TCP) as a carrier. A typical cementum/periodontal-ligament-like structure was generated, in which a thin layer of cementum was formed on the surface of the HA/TCP and periodontal-ligament-like structures associated with the newly regenerated cementum (Fig. 3A). The cryopreserved periodontal ligament stem cell transplants yielded human-specific mitochondria-positive cementoblasts/cementocytes, indicating in vivo differentiation of human cryopreserved periodontal ligament stem cells (Fig. 3B). Moreover, collagen fibers were inserted perpendicularly into cementum-like tissue (Figs. 3C, 3D), mimicking the natural Sharpey’s fibers in the periodontal ligament. To gain a better understanding of in vivo differentiation of cryopreserved periodontal ligament stem cells, we selected 6 single colony strains of cryopreserved periodontal ligament stem cells and transplanted them into immunocompromised mice as described above. Four out of 6 colonies could generate cementum and PDL structures with variable amounts of cementum/PDL fibers, while the remaining 2 colonies showed only fibrous tissue within the transplants (Figs. 3E, 3F), implying that cryopreserved periodontal ligament stem cells maintain characteristics of regular periodontal ligament stem cells. Additionally, the regenerated cementum and cementoblasts were found to be positive for anti-type I collagen and BSP antibody staining (Figs. 3G–3I). Analysis of these data confirmed that cryopreserved periodontal ligament stem cells were capable of differentiating into cementoblasts and forming cementum in vivo.

View larger version (155K): [in this window] [in a new window]   Figure 3. In vivo characterization of cryopreserved periodontal ligament stem cells. (A) After 8 wks of transplantation, cryopreserved periodontal ligament stem cells were capable of forming a cementum-like structure (C) on the surfaces of the hydroxyapatite/tricalcium phosphate (HA) carrier, which was connected to periodontal-ligament-like tissue (PDL). (B) The cells responsible for cementum (C) formation were positive for anti-human specific mitochondria antibody staining (black arrows). Analysis of the immunohistochemical staining data indicated that transplanted cryopreserved periodontal ligament stem cells differentiated into cementoblasts/cementocytes and generated cementum in vivo. (C,D) Transplanted cryopreserved periodontal ligament stem cells were able to form cementum (C) on the surfaces of HA/TCP particles (HA) and were able to generate Sharpey’s fibers (black arrows) inserted into cementum and which were continuous with periodontal-ligament-like tissue (PDL), shown by H&E (C) and Trichrome staining (D). (E,F) Of 6 selected single-colony-derived cryopreserved periodontal ligament stem cell strains, only 4 (67%) were capable of generating a cementum/periodontal-ligament-like structure (E). Newly formed cementum (C) was found to be adjacent to the surfaces of the HA/TCP carrier (HA) and was connected with periodontal-ligament-like tissue (PDL) that was the same as Sharpey’s fibers (back arrows). The remaining 33% (2 of 6) single-colony-derived cryopreserved periodontal ligament stem cell strains were unable to generate cementum in vivo (F). (G,H) Newly formed cementum (C) was positive for anti-type I collagen antibody staining (G), and cementogenic cells were positive for anti-bone sialoprotein (BSP) antibody staining (open arrows in H). (I) Pre-immunoserum control was negative for immunohistochemical staining of type I collagen and BSP antibodies (scale bar, 50 µm for A-I).

	DISCUSSION

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Previous experiments have shown that freshly isolated human periodontal ligament contains stem cells that are capable of differentiating into cementoblastic/osteoblastic cells in vitro and forming cementum/PDL-like tissues in vivo (Seo et al., 2004). The present study demonstrates that human post-natal stem cells can be recovered from cryopreserved human periodontal ligament, thereby providing a practical clinical approach for the utilization of frozen tissues for stem cell isolation. Importantly, human cryopreserved periodontal ligament stem cells maintain stem cell characteristics and in vivo tissue-regenerative capacity, suggesting a great potential for the use of cryopreserved periodontal ligament stem cells for periodontal tissue regeneration.

The rationale for the isolation of human post-natal stem cells from frozen tissues is the practical and effective preservation of clinical samples for subsequent stem cell recovery and potential stem-cell-mediated therapies. It is reasonable to speculate that cryopreservation of tissue in the clinic will be more practical than the isolation of stem cells, which may require additional equipment and professional personnel. In this study, we found that cryopreserved periodontal ligament stem cells are similar to the periodontal ligament stem cells with respect to their STRO-1-positive characteristics. Therefore, cryopreserved periodontal ligament stem cells may be derived from a population of perivascular cells (Gould et al., 1977; McCulloch, 1985). Moreover, cryopreserved periodontal ligament stem cells show a heterogeneous nature that may reflect differences in their developmental stages, or may even represent different periodontal ligament cell lineages analogous with non-frozen periodontal ligament stem cells (Seo et al., 2004). This is emphasized in experiments where each colony-derived cryopreserved periodontal ligament clonal stem cell line showed a variable capacity to generate cementum, ranging from a total absence of any cementogenesis to levels comparable with those in multi-colony-derived populations. It is notable that periodontal ligament stem cells and cryopreserved periodontal ligament stem cells were able to form collagen aggregates when cultured with TGFß1 in vitro, reflecting a specificity of these stem cells to form collagen fibers for maintaining periodontal ligament tissue homeostasis. Analysis of these data further supports the notion that cryopreserved periodontal ligament stem cells are functionally similar to periodontal ligament stem cells.

Interestingly, hematopoietic progenitors can be recovered following cryopreservation of whole bone marrow in which individual cells were suspended within a liquid phase (Lundell et al., 1999). Our study is the first to utilize frozen-thawed human tissue to isolate post-natal stem cells that were previously identified as stem cells at a functional level. Although the recovered number of single colonies from the six-month-frozen periodontal ligament was lower than periodontal ligament stem cells derived from fresh periodontal ligament, there was no difference in terms of stem cell characteristics, including marker expression, proliferation rate, G-band karyotype, and in vivo tissue-regenerative capacity. The reason for the lower stem cell colony recovery rate is not known. There are many factors that can influence the viability of successfully cryopreserved stem cells, including pre-freeze processing, variations in temperature and duration of storage, and post-freeze procedures (Hubel, 1997). The most common cause of cell death is the intracellular ice formation during the freeze-thawing process (Rowley et al., 2003; Woods et al., 2004). The method we used for tissue cryopreservation is derived from our regular mesenchymal stem cell cryopreservation technique, in which small pieces of tissue were mixed with 90% serum and 10% DMSO to maintain accessibility between frozen medium and cellular structures. There is great potential for our cell-freezing procedure to be improved to increase the post-thaw survival rate of cryopreserved stem cell, such as the use of trehalose, a non-reducing disaccharide of glucose (Eroglu et al., 2000; Guo et al., 2000). It is also important to note that there was no difference between the 3- and 6-month periods of frozen preservation with respect to the stem cell recovery rate, indicating that the duration of cryopreservation up to 6 mos may not be harmful to the survival of cryopreserved periodontal ligament stem cells.

Although Oil red O staining may have limitations for the identification of specific adipogenic differentiation, it has been widely used on a variety of cell types to indicate generation of adipocytes. We used Oil red O staining along with examining the mRNA expression level of peroxisome proliferator-activated receptor 2 (PPAR2), one of the master regulators of adipocyte differentiation, in our previous study, to indicate adipogenic differentiation of periodontal ligament stem cells (Seo et al., 2004). Therefore, Oil red O staining was used again in this study to confirm adipogenic differentiation of cryopreserved periodontal ligament stem cells.

This study demonstrated that postnatal stem cells could be successfully recovered from human tissues. The findings in this study, for the first time, to our knowledge, identify that postnatal stem cells can be retrieved from solid-frozen human periodontal ligament. It remains to be determined whether this technique is applicable for an extensive array of human tissues. It is also worthwhile to investigate whether post-natal stem cells can be recovered from long-term (greater than 6 mos) frozen tissues. The answers to these questions will be necessary for the use of cryopreserved tissues in post-natal stem-cell-mediated therapies and tissue engineering.

	ACKNOWLEDGMENTS

 
The Division of Intramural Research (US National Institute of Dental and Craniofacial Research, the National Institutes of Health, Department of Health and Human Service) provided research funding, space, and personnel for this study. The authors thank Drs. Larry Fisher and Stan Gronthos for providing BSP and STRO-1 antibodies, respectively.

Received November 15, 2004; Last revision February 9, 2005; Accepted May 24, 2005

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This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (14) Citing Articles via Google Scholar Google Scholar Articles by Seo, B.-M. Articles by Shi, S. Search for Related Content PubMed PubMed Citation Articles by Seo, B.-M. Articles by Shi, S.