KR20090033643A - Novel dental stem cells from tooth follicles and the method for culturing them - Google Patents

Abstract

A tooth stem cell derived from tooth follicles having excellent colony-forming activity and reproductive capability is provided to recover damaged tooth tissue. A tooth stem cell is derived from crown follicle toward cusp(CCF), crown follicle toward root(CRF) or root follicle(RPF). The tooth stem cell expresses a marker of mesenchymal stem cells, STRO-1. The tooth stem cell forms colony twice equivalent to dental pulp stem cells when the same number of initial loading cells are cultivated. The tooth stem cell is cultivated in an alpha-MEM culture medium wherein 20% of FCS, 50-200 muM of ascorbic acid, 1-10 mM/L of glutamine, 50-200 U/mL of penicillin and 50-200 mug/mL of streptomycin are added.

Description

Novel dental stem cells from tooth follicles and the method for culturing them

The present invention relates to a stem cell derived from a new dental vesicle and a method of culturing the same, and more particularly, an endodontic vesicle stem cell toward a ridge separated from an immature tooth having only a crown and an endodontic vesicle stem cell toward an root and an open tooth root. (opened root apex) relates to a root cell follicle stem cells isolated from the teeth having a separate and a culture method for optimal growth thereof.

Stem cells are present in several vertebrate tissues, including the hematopoietic stem, nervous system, intestine, gonads, skin, olfactory epithelium and teeth. Stem cells are typically defined as cells that have self-renewal and result in differentiated progeny. In addition, the discovery of tooth stem cells isolated from tooth tissue consisting of teeth is a recent advance in dental biology. The teeth developed through the epithelial-mesenchymal interaction process consist of enamel, dentin, chalky and pulp. Teeth have unique characteristics of morphogenesis and differentiation that are controlled at each stage and space of development. It is important to clarify and compare the identification and isolation of stem cells from immature and mature teeth because the properties of dental tissues vary with each stage of development. Moreover, it is well known that progenitor / stem cells are involved in the continuous maintenance and repair of most tissue forms. However, despite well-known tooth development and several specialized tooth-related cell morphologies, little is known about the properties and properties of the precursors of the cell populations present in acquired organisms.

In general, teeth develop as ectoderm organs where sequential and interactive epithelial-mesenchymal interactions regulate initiation, morphogenesis, early dentin formation, and cell differentiation through signal transduction pathways. Morphological tooth development begins with the formation of dental lamina that grows into epithelial buds. After the budding stage, epithelial tissues form certain wrinkles at the cap and bell stages. In the late stages, tooth stem cells differentiate into mesenchymal cells on the crust surface toward the epithelial-mesenchymal interface, preodontoblasts and odontoblasts forming dentin. The crown is covered with epithelium at the late stage of the species. After the crown is formed, root development occurs and a complete tooth develops. Morphological stages of tooth development-budding, hat, species and terminal differentiation-have been known for decades, but no clear process for root development is known.

The inventors found new follicle tissues of teeth with only crown and immature teeth with open root apex while examining root development and root regeneration. The present invention was completed by revealing that colony formation and proliferation are excellent and have differentiation ability.

It is an object of the present invention to provide a new stem cell derived from dental plaque tissue having excellent colony forming ability, proliferative ability and differentiating ability and an optimal culture method thereof.

In order to achieve the above object, the present invention provides a tooth stem isolated from crown follicle toward cusp (CCF), crown follicle toward root (CRF) or root follicle (RPF). Provide the cells.

In addition, the present invention

1) separating the crown vesicle towards the ridge, the crown vesicle towards the root or the root vesicle tissue from the adult tooth;

2) degrading the vesicle tissue of step 1); And

3) To provide a method for isolating tooth stem cells derived from the crown vesicle toward the ridge, the crown vesicle towards the root or the root vesicle, comprising the step of filtering the digested product of step 2).

The present invention also provides a culture method for the optimal growth of the stem cells derived from the dental vesicles.

Hereinafter, the present invention will be described in more detail.

The present invention provides a tooth stem cell derived from a new dental vesicle tissue.

We found a new follicular tissue in the immature tooth with crown only and the open root apex in the third molars of an adult, and optionally divided the electronic vesicle tissue into the crown and root portion, and the vertebral vesicles for ridges ( crown follicle toward cusp (CCF) and crown follicle toward root (CRF), respectively. The latter vesicle tissue was named root follicle (root follicle, RPF). Tooth stem cells were isolated from them and their characteristics compared to those of dental pulp stem cells (DPSCs), which have been reported as cell sources of dental biotechnology.

Colony assay, immunohistochemistry, adipocyte generation and calcification were used to identify stem cell properties. Human crown follicle toward cusp stem cells (CCFSCs), crown follicle toward root stem cells (CRFSCs), root follicle stem cells (RPFSCs) to measure tissue regeneration , Root vesicle stem cells) and DPSCs (dental pulp stem cells) were implanted into immunocompromised mice (n = 12).

First, colony formation assays and immunohistochemistry for expression of STRO-1 molecules were performed to confirm that the dental vesicle-derived tooth stem cells of the present invention maintain their stemness. Maintaining pluripotency along successive passages in culture has important implications for the use of proliferated tooth stem cells for cell and gene therapy. When the passage was repeated, STRO-1 positive cells were found to be present in all of the plaque stem cells. In addition, dental vesicle-derived tooth stem cells (CCFSCs, CRFSCs, RPFSCs) were significantly superior to DPSCs in colony formation and proliferation at the next passage. Specifically, the dental vesicle-derived tooth stem cells, compared to DPSCs, formed two or more colonies for the same number of initial loading cells.

Moreover, calcification to assess whether the tooth stem cells of the present invention have the ability to differentiate into other cell lineages, such as osteoblasts, resulted in sparse CCFSCs deposits on the adhesion layer, while CRFSCs, RPFSCs and DPSCs were 3 weeks after induction. The formation of more mineralized nodules and calcified precipitates throughout the layer. CCFSCs formed scattered calcified nodules, but CCFSCs and CRFSCs of immature teeth formed mineralized matrices faster than RPFSCs. Therefore, although differentiation patterns are different according to tissue members, it can be seen that all the tooth stem cells of the present invention can differentiate into other cell lines such as osteoblasts.

In addition, to examine the ability of CCFSCs, CRFSCs, RPFSCs and DPSCs to differentiate into adipocytes, these cell groups were cultured for 21 days and exchanged with adipocyte induction medium. Cultured CCFSCs, CRFSCs, RPFSCs and DPSCs developed into oil red O positive lipid masses. After 14 days, lipid vacuoles were found, cell size and lipid vacuoles increased in CCFSCs, CRFSCs and fewer lipid vacuoles were present on day 21 of induction of adipocyte formation in RPFSCs and DPSCs. This is because CCFSCs and CRFSCs are organizations in development. Therefore, they will have more active potential effects on pulp and tooth shape and overall tooth formation.

In addition, to demonstrate the ability of CCFSCs, CRFSCs, RPFSCs, and DPSCs to differentiate into functional cementoblasts / osteoblasts, immunosuppression is achieved by combining a variety of exogenous expanded follicular stem cells and DPSCs with HA / TCP powders. Mice were transplanted. As a result, the follicular stem cells produced dentin / dimension-like complexes along the HA / TCP particle surface 6-12 weeks after implantation, as do DSPCs in vivo. Implanted tooth stem cells also produced a significant amount of collagen fibers with newly formed chalky-like structures. These fibroblast-like cells may belong to a more primitive reserve cell group that is involved in dentin formation in secondary transplantation. These progenitor cells can develop treatments based on the regenerative capacity of pulp tissue instead of irreversible endodontic treatment, such as the current pulp capping treatment. On the other hand, RPFSCs and CRFSCs produce large amounts of chalky / dimension-like tissues, so it is thought that the use of plasma stem cells will help regenerate chalky, dentin and upper pulp. Previous studies have demonstrated that bone marrow derived stromal stem cells (BMSSCs) can also differentiate into neuro-like cells after in vivo transplantation. Pulp cells are also known to produce neurotrophic factors and heal motor neurons after spinal cord injury. These evidence support the view that stem cells from non-neural tissues can differentiate into neurons. Thus, it can be seen that the plaque stem cells are related to the origin of neural crest cells. Neuronal cells play an important role in fetal development resulting in a variety of cell types, including neurons, pigment cells, smooth muscle, craniofacial cartilage and bone.

As a result, it can be seen that the tooth stem cells derived from the new cystoid tissue of the present invention are acquired stem cells that proliferate widely and differentiate pluripotently.

Stem cells are involved in the production and maintenance of their population, but many stem cells obtained from adult tissues are insufficient in vitro. In practical terms, these small amounts of stem cells have limited clinical applications. In addition, it is important to test the proliferation and differentiation ability for tooth regeneration and engineering to separate a large number of stem cells to secure these populations. In general, these follicular stem cells retain their stem cell properties until they lose their proliferative capacity. The accumulated total number of these tooth stem cells decreased during the culture propagation of the organs, but the expandability was maintained when the cells were passaged. Dental vesicle stem cells were significantly better than colony forming and proliferating at the next passage. In this situation, the new tooth stem cells recovered from the plaque are sufficiently qualified and valuable for clinical application, and the CCFSCs, CRFSCs and RPFSCs used in the present invention represent a group of stem cells that surpass DPSCs that can be used as a useful resource for regenerative therapy. can do.

In addition, the present invention

1) separating the crown vesicle towards the ridge, the crown vesicle towards the root or the root vesicle tissue from the adult tooth;

2) degrading the vesicle tissue of step 1); And

3) To provide a method for isolating tooth stem cells derived from the crown vesicle toward the ridge, the crown vesicle towards the root or the root vesicle, comprising the step of filtering the digested product of step 2).

A crown follicle toward the ridge (CCF) and a crown follicle toward the root (CRF) are isolated from the vesicle tissue of the immature tooth with only the crown. It also separates root follicles (RPFs) from teeth with open root ends. These tissues were prepared at 1-10 mg / ml collagenase type I and 1-10 mg / ml dispase solution at 37 ° C. for up to 2 hours, preferably 2-5 mg / ml collagenase type I and 2-5 Decompose at 37 ° C., 1 hour in mg / ml dispase solution. The cells are then passed through a 70 μm cell filter (Falcon, BD Labware, Franklin Lakes, NJ, U.S.A.) to obtain a single cell suspension of stem cells derived from vesicles of the present invention.

In addition, the present invention provides a culture method for optimal growth of the stem cells derived from the dental vesicles.

Preferably the culture method of the present invention is added 20% FCS (fetal bovine serum), 50-200 μM ascorbic acid, 1-10 mM / L glutamine, 50-200 U / ml penicillin and 50-200 μg / ml streptomycin Alpha-MEM medium, preferably 20% FCS, 150-200 μM ascorbic acid, 2-5 mM / L glutamine, 80-120 U / ml penicillin and 80-120 μg / ml streptomycin added Alveolar vesicle stem cells facing uplift in medium, most preferably in alpha-MEM medium with 20% FCS, 200 μM ascorbic acid, 2 mM / L glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin Culturing (CCFSCs) and crown vesicle stem cells (CRFSCs) towards the root.

Stem cells derived from dental vesicles of the present invention are new tooth mesenchymal stem cells having superior colony forming ability, proliferation ability and differentiation capacity as compared to dental pulp stem cells (DPSCs), which have been reported as cell sources of dental biotechnology. It can be usefully used as a stem cell that can repair damaged tooth structure, induce pulp or dentin regeneration, treat periodontal disease or improve clinical application.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Cell Culture

The third molar of a normal person withdrawn from 12 adults (18-35 years old) was recovered after informed consent from Seoul, Seoul, and the Seoul National University Dental Hospital. The experimental procedure was approved by the hospital's Institutional Review Board (IRB). Crown follicle toward cusp (CCF) and crown follicle toward root (CRF) were isolated from vesicular tissue of immature teeth with crowns only. Root root vesicles (RPFs) isolated from teeth with open root tips were removed with a surgical scalpel at the site of attachment to the root dentin. Sterile dental burs were cut around the cemento-enamel junction to separate the pulp from the exposed pulp chamber. These tissues were digested in 3 mg / ml collagenase type I and 4 mg / ml dispase solution at 37 ° C. for 1 hour. After passing the cells through a 70 μm cell filter (Falcon, BD Labware, Franklin Lakes, NJ, U.S.A.), a single cell suspension was obtained.

To identify stem cells, single cell suspensions (1 × 10 ⁴ cells) were added with 10% fetal bovine serum (FCS: Gibco BRL), 100 μM / L ascorbic acid 2-phosphate (Sigma, St. Louis, MO), 2 100-mm dish with alpha modified Eagle's medium (alpha-MEM, GIBCO BRL, Carlsbad, CA, USA) added with mM / L glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco BRL) (NUNC, Denmark) and then cultured in 37 ℃, 5% carbon dioxide.

To measure colony formation efficiency, the cells were fixed with 70% ethanol on day 10 and then stained with 0.1% crystal violet. 50 or more cell aggregates were made into colonies. Stem cell proliferation was measured by MTT assay at 4, 7 and 10 days of culture.

As a result, isolated vesicle stem cells showed the form of fibroblast-like cells. In addition, plaque stem cells (CCFSCs, CRFSCs, RPFSCs) clearly show superior ability to form and proliferate colonies at the next passage than previously reported DPSCs. The ability of vesicle derived cells to form adherent colonogenic cell masses is evidenced by the formation of about 100-170 single colonies produced from 1,000 single cells cultured at low density. Compared to DPSCs, vesicle-derived stem cells formed more than double colonies for the same number of initial loading cells. In addition, colonies of isolated stem cell groups were easily observed beyond 10 passages.

Example 2 Immunohistochemistry

Immunohistochemistry was performed using STRO-1, a well-known mesenchymal stem cell marker, to demonstrate the existence of stem cells even after extensive passage. STRO-1 cells were initially identified as precursors of colony forming osteogenic isolated from bone marrow.

Putative stem cells isolated from CCF, CRF, RPF and pulp were dispensed on two-chamber slides (2 × 10 ⁴ cells / well; NUNC, Denmark) and incubated for 24 hours. After washing with phosphate buffered saline (PBS, pH 7.4) and fixing for 15 minutes with 2% ρ-formaldehyde, the sample was reacted for 3 hours with STRO-1 antibody (1: 200, R & D Systems, Minneapolis, MN, USA). I was. Cells were then reacted with anti-mouse IgG-Cy3 goat secondary antibody (Zymed Laboratories, Carlsbad, Calif., USA) for 1 hour and the nuclei stained with DAPI (1 μg / ml) for 30 minutes.

As a result, when the passage was repeated, mesenchymal stem cell marker STRO-1 was expressed in all the plaque stem cells.

Example 3 Differentiation into Adipocytes

For adipocyte differentiation, 5 × 10 ⁴ cells were aliquoted into 60-mm dishes and incubated in optimal culture medium for 3 days. The medium was replaced with adipogenic culture, an optimal culture medium with 1 μM dexamethasone, 0.1 mM indomethacin, 0.5 mM isobutylmethylxanthine and 10 μM insulin added. Adipocyte medium was fixed in 70% ethanol for 10 minutes and stained with fresh Oil Red-O solution (Sigma) for 30 minutes.

As a result, cultured CCFSCs, CRFSCs, RPFSCs and DPSCs developed into oil red O positive lipid mass. After 14 days, lipid vacuoles were found, cell size and lipid vacuoles were increased in CCFSCs and CRFSCs, and fewer lipid vacuoles were present on day 21 of induction of adipocyte formation in RPFSCs and DPSCs.

< Example  4> travertine

For calcification, 1 × 10 ⁴ cells were dispensed in 60-mm dishes and incubated for 3 days in optimal culture medium. It was then incubated in an optimal culture medium with 5 mM β-glycerol phosphate and 10 nM dexamethasone (Sigma) added to induce mineral formation. Nodule formation was observed for 3 weeks of culture. Mineralized medium was fixed with 70% ethanol for 10 minutes and stained with 40 mM alizarin red (pH 4.2) for 30 minutes in a 50 rpm shake incubator.

While CCFSCs precipitates were sparse over the adhesive layer, CRFSCs, RPFSCs and DPSCs formed more mineralized nodules and calcified precipitates throughout the layer three weeks after induction. CCFSCs formed scattered calcified nodules, but CCFSCs and CRFSCs of immature teeth had the ability to form mineralized matrices faster than RPFSCs and DPSCs. Also, during the same period, the lipid vacuoles of CCFSCs and CRFSCs were much higher than those of RPFSCs and DPSCs. This is because CCFSCs and CRFSCs are organizations in development

< Example  5> organization Regeneration  Implant to Measure

In order to demonstrate the ability of CCFSCs, CRFSCs, RPFSCs and DPSCs to differentiate into functional cementoblast / osteoblasts, a variety of exogenous expanded follicular stem cells and DPSCs in vitro were combined with HA / TCP powder in immunocompromised mice. Transplanted. HA / TCP has been successfully used worldwide as a bone substitute because of their biocompatibility. Approximately 5 × 10 ⁶ cells of CCFSCs, CRFSCs, and RPFSCs (third passage) were mixed with 50 mg HA / TCP ceramic powder (Zimmer, Warsaw, IN, USA), followed by 6 week old immunocompromised beige mice ( Subcutaneously implanted on the dorsal surface of NIH-bg-nu-xid, Harlan Sprague Dawley, Indianapolis, IN, USA. The same number of in vitro propagated DPSCs were used as controls. These procedures were performed according to the specifications of the approved small animal protocol (SNU050512-11, Seoul National University). Six to twelve weeks after transplantation, the cells were harvested, fixed with 4% formalin, decalcified with 10% EDTA buffer (pH 8.0), and placed in paraffin. Paraffin blocks were fractionated at 5 μm, paraffin was removed and stained with hematoxylin and eosin.

As a result, the follicular stem cells can produce dentin / dimension-like complexes along the HA / TCP particle surface 6-12 weeks after implantation, as do DSPCs in vivo. The dentin / dimension-like structure covering the surface of the tissue was measured using an image analysis based on Photoshop applying color information contained in each pixel. The in vivo developmental capacity of PAFSCs implants 12 weeks after transplantation accounted for 20.9% of the total surface area and the ability to develop into separate tissues was remarkable. Other tooth stem cell implants-CCFSCs, CRFSCs and DPSCs accounted for 2.06%, 10.7% and 7.65% of the total space, respectively. All implanted tooth stem cells produced collagen fibers with condensed sparse cells similar to the pulp structure.

< Example  6> Optimum Culture Conditions

Single cell suspensions (1 × 10 ³ cells / well) of CCF, CRF, RPF and pulp were inoculated in 96-well culture plates (NUNC, Denmark) to which medium was added. To optimize the culture conditions, three forms of media were used: RPMI, DMEM and alpha-MEM with 2 mmol / L glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco BRL). Bovine or fetal bovine serum (FCS: Gibco BRL) was added at a 10% or 20% concentration and tested for different concentrations of ascorbic acid (0, 50, 100 and 200 μM). The medium was changed every 2-3 days.

CCFSCs and CRFSCs showed optimal growth in alpha-MEM medium added with 20% FCS, 200 μM ascorbic acid, 2 mM / L glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin. Comparing CCFSCs, CRFSCs, RPFSCs and DPSCs showed that plaque-derived stem cells showed higher proliferation rates and a greater number of population doublings.

On the other hand, RPFSCs and DPSCs were cultured according to the optimal culture conditions described by Jo et al. ¹⁸ (Jo YY, Lee HJ, Kook SY, Choung HW, Park JY, Chung JH, Choung YH, Kim ES, Yang HC, Choung PH. Isolation and characterization of postnatal stem cells from human dental tissues. Tissue Eng 2007 13 : 767-73.)

Claims (17)

Tooth stem cells derived from crown follicle toward cusp (CCF), crown follicle toward root (CRF) or root follicle (RPF). The tooth stem cell of claim 1, wherein the stem cell expresses STRO-1, which is a marker of mesenchymal stem cells. According to claim 1, Tooth stem cells, characterized in that when the same number of initial loading cells (initial loading cells) to form two or more colonies than the pulp stem cells (DPSCs). According to claim 1, Tooth stem cells, characterized in that to form more than 10 passages colonies. 2. The dental stem cell of claim 1, wherein the crown vesicle stem cells toward the ridges and the crown vesicle stem cells toward the roots form a mineralized matrix faster than the root vesicle stem cells in the mineral induction medium. 6. The stem cell of claim 5, wherein the mineral induction medium is added with 1-10 mM beta-glycerol phosphate and 1-50 nM dexamethasone to the tooth stem cell culture medium. According to claim 1, Tooth stem cells, characterized in that the lipid vesicles are formed more than the root vesicle stem cells in the crown vesicle stem cells toward the ridge and the root vesicle stem cells toward the root in the adipocyte formation induction medium. The method according to claim 1, characterized in that the culture is differentiated into various cell types of cementoblast, odontoblast, osteoblast, adipocyte, neuron, pigment cell, smooth muscle, craniofacial cartilage or bone. Tooth stem cells. The tooth stem cell of claim 1, wherein the stem cells form dentin / dimension-like structures when transplanted into immunocompromised mice. The tooth stem cell of claim 1, wherein said stem cell is used for repairing damaged tooth structures, pulp or dentin regeneration, or periodontal disease. 1) separating the crown vesicle towards the ridge, the crown vesicle towards the root or the root vesicle tissue from the adult tooth; 2) degrading the vesicle tissue of step 1); And 3) A method for separating tooth stem cells from a crown vesicle toward a ridge, a crown vesicle toward a root or a root vesicle, comprising filtering the digested product of step 2). 12. The method for separating tooth stem cells according to claim 11, wherein the crown vesicle facing the ridge and the crown vesicle facing the root are separated from immature teeth having only the crown. 12. The method of claim 11, wherein the root vesicle is separated from the tooth having an open root apex separately. 12. The method of claim 11, wherein the vesicles are decomposed at 37 ° C. for 2 hours or less in 1-10 mg / ml collagenase type I and 1-10 mg / ml dispase solution. 12. The method of claim 11, wherein after passing the cells through the cell filter medium, a single cell suspension is obtained. Ridges in alpha-MEM medium with 20% FCS (fetal bovine serum), 50-200 μM ascorbic acid, 1-10 mM / L glutamine, 50-200 U / ml penicillin and 50-200 μg / ml streptomycin Method for culturing the crown vesicle stem cells toward the crown vesicle stem cells toward the root. The method of claim 1, wherein 1 to 10 mM beta-glycerol phosphate and 1 to 50 nM dexamethasone are added to the tooth stem cells.