| Sign In to gain access to subscriptions and/or personal tools. |
Recovery of Stem Cells from Cryopreserved Periodontal Ligament1 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; Correspondence: * corresponding author, sshi{at}dir.nidcr.nih.gov
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
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.
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-β1. The medium was changed twice a week.
Antibodies
Transplantation
Immunohistochemistry
Statistical Analysis
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.
Ex vivo expanded cryopreserved periodontal ligament stem cells were found to express the cell-surface molecule STRO-1, an early mesenchymal stem cell marker, along with the co-expression of cementoblastic/osteoblastic markers TGFβR1 and BSP (Figs. 1F  –1M). In addition, some cryopreserved periodontal ligament stem cells expressed STRO-1 with BSP in a separate, non-overlapping manner (Figs. 1F–1I). This finding should be interpreted with caution, because it could be explained by the cells being in different stages of differentiation or maturity within the cell population.
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
Previously, it has been shown that transplanted human periodontal ligament stem cells were able to form type I collagen-positive periodontal-ligament-like tissue within the transplants (Seo et al., 2004), and also that TGFβ1 could induce collagen expression in bone marrow stromal stem cells (Bosnakovski et al., 2004). We analyzed whether TGFβ1 is able to up-regulate the expression of type 1 collagen, the main type of collagen present in periodontal ligament tissue. Under the induction of TGFβ1, periodontal ligament stem cells and cryopreserved periodontal ligament stem cells produced significant amounts of well-organized collagen fibers when cultured in vitro (Figs. 2E –2H).
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
To determine whether cryopreservation may influence the karyotype of cryopreserved periodontal ligament stem cells, we performed G-banded karyotype analysis to examine the chromosomal stability of cryopreserved periodontal ligament stem cells. The cryopreserved periodontal ligament stem cells exhibited a normal G-banded karyotype, compared with the regular periodontal ligament stem cells (data not show). Analysis of these data suggests that cryopreserved periodontal ligament stem cells may be useful for therapeutic purposes.
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 TGFβ1 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 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.
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 for publication November 15, 2004. Revision received February 9, 2005. Accepted for publication May 24, 2005.
Journal of Dental Research, Vol. 84, No. 10,
907-912 (2005) This article has been cited by other articles:
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
2 (PPAR
