Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint

S. Wakitani, K. Takaoka, T. Hattori1, N. Miyazawa2, T. Iwanaga2, S. Takeda2, T. K. Watanabe2 and A. Tanigami3

Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto,
1 Department of Orthopaedic Surgery, Osaka-Minami National Hospital, Kawachinagano and
2 Otsuka GEN Research Institute, Otsuka Pharmaceutical Co. Ltd, Tokushima and
3 Fujii Memorial Research Institute, Otsuka Pharmaceutical Co. Ltd, Otsu, Japan.


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Objective. To determine whether the joint space is a suitable environment for embryonic stem (ES) cells to grow and form cartilage.

Method. We transplanted ES cells into the knee joint and a subcutaneous space of mice with severe combined immunodeficiency.

Results. Teratomas formed in both areas. Those in the joints grew and destroyed the joints. The incidence of cartilage formation was the same in the knee joint and subcutaneous space, but the ratio of cartilage to teratoma was higher in the knee joint than in the subcutaneous space. The teratomas were proved to have been derived from the transplanted ES cells by detection of the neomycin-resistance gene that had been transfected into the ES cells.

Conclusions. It is currently not possible to use ES cells to repair joint tissues. Further optimization of donor ES cells to differentiate as well as inhibit tumour growth may help to meet these challenges.

KEY WORDS: ES cells, Joint space, Teratoma, Chondrocyte, Joint destruction.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The capacity of articular cartilage for repair is limited. Many attempts have been made to repair articular cartilage defects, including transplantation of various tissues or cells from joint tissues, bone marrow and periosteum, but none of these has been widely accepted for clinical use [1]. Recently, autologous cultured chondrocyte transplantation has been shown to improve symptoms and to result in some degree of repair [2]. However, this method involves the collection of autologous cartilage, which causes cartilage defects in the peripheral area. Thus, the search for new cell sources is continuing.

Embryonic stem (ES) cells are thought to be a possible source of tissue regeneration because they are self-renewing pluripotent cells that can differentiate into any tissue or cell type [3, 4]. Many attempts have been made to induce in vitro differentiation into many cell types, including haematopoietic precursors [5], heart and skeletal muscle [6], endothelium [7] and neural cells [8, 9]. In the orthopaedic field, ES cells are considered useful for regenerating articular cartilage, but thus far it has remained impossible to induce the formation of chondrocytes from ES cells in vitro [10]. It was reported, however, that ES cells could differentiate into neural cells in vivo when transplanted into the spinal cord [11]. This led us to conclude that the environment is one of the important factors influencing cell differentiation. We therefore transplanted ES cells into the knee joint and a subcutaneous space of mice with severe combined immunodeficiency (SCID mice) and observed their growth and chondrogenesis under different circumstances.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ES cell preparation
AB2.2 prime ES cell kits were purchased from Lexicon Genetics (Houston, TX, USA). The ES cells had been obtained from 129/Sv/Ev mice. They were cultured according to the instructions for the kit. Briefly, cells were cultured on the ESQ feeder cells that were supplied with the kit. Just before transplantation, the ES cells were embedded in collagen solution (type I collagen obtained from porcine Achilles tendon; Nitta Gelatin, Osaka, Japan) at 4°C and at a cell density of 107 cells/ml.

Surgery
Twenty-five female 5-week-old SCID mice (Fox Chase SCID C.B-17/Icr-scid Jcl; Nihon Clea, Osaka, Japan) were anaesthetized by intramuscular injection of ketamine (150 mg/kg; Sankyo, Tokyo, Japan) and xylazine (15 mg/kg; Bayer, Tokyo, Japan). Ten microlitres of the collagen solution containing the ES cells was injected into each right knee and into a subcutaneous space on the back of each mouse.

Evaluations
Five mice were killed at each of the time-points of 1, 2, 4, 6 and 8 weeks after surgery. The knee joints were collected, fixed with 10% buffered formalin, decalcified, embedded in paraffin and sectioned. The area of the back surrounding the injected subcutaneous space was examined, and the mass in the space itself was collected, fixed with 10% buffered formalin, embedded in paraffin and sectioned. Specimens were stained with haematoxylin–eosin and toluidine blue and analysed histologically. The widths of the tumours and of the cartilaginous area in the tumours were measured using a Scion image (Scion, Frederick, MD, USA).

Using Dexpad (Takara, Kyoto, Japan), DNA was extracted from two paraffin sections (5 µm thick) from each of the 15 selected preparations, i.e. all eight knees with tumours and seven subcutaneous tumours. The DNA was then amplified with the polymerase chain reaction to detect the neomycin (Neo)-resistance gene that had been transfected into the AB2.2 prime ES cells. Detection of the X11 gene served as a positive control as it was present in both the ES cells and the SCID mice. The primers for the Neo-resistance gene were set between neo p4 (5'-AGGATCTCGTCGTGACCCATG-3') and neo int2 (5'-TCAGAAGAACTCGTCAAGAAGGC-3'), and the size of the product was 250 base pairs. For the X11 gene, the primers were set between X11 KO-Hd-1 (5'-TGGGAGGGTGAACGCTATAC-3') and X11 KO-Hd-2 (5'-CTCACTGCGCGCTCATT-TTG-3'), and the size of the product was 260 base pairs.

For statistical analysis of data on the incidence of mass formation and the percentage of cartilaginous mass we used the Mann–Whitney U-test. Probability values less than 5% were considered significant. StatView (SAS Institute, Cary, NC, USA) was used for statistical analyses.

The procedure was approved by the Institutional Review Board.


    Results
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 Abstract
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 Materials and methods
 Results
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In the case of subcutaneous injection, small white fibrous tissues were found 1 week after transplantation, and with time these tissues became larger and the colour changed to black or red.

At short follow-up, it was difficult to detect tumours in the knees after injection with ES cells. When tumours had grown, they became identifiable. We found eight knees containing a tumour, and three of these tumours contained cartilage (Fig. 1Go, top). In two knee joints at 8 weeks, ES cells formed a large tumour that extruded from the knee joint and destroyed the knee structures (Fig. 1Go, middle).



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FIG. 1. Tumours formed in the knee joints and subcutaneous spaces. (Top) A tumour in a knee joint 4 weeks after transplantation. Toluidine blue staining; bar=1 mm. A tumour (arrow) containing three cartilaginous masses (arrowheads) was observed. The tumour measured 2.33 mm3 and the cartilaginous areas 0.18, 0.13 and 0.02 mm3. The proportion of cartilaginous areas to tumour was 14%. (Middle) A large tumour had destroyed the knee structure 8 weeks after transplantation. Haematoxylin–eosin staining; bar=1 mm. The tumour (arrows) had grown inside the knee joint and destroyed the knee structure. (Bottom) Tissues thought to have originated from ectoderm (skin), mesoderm (cartilage) or endoderm (mucous gland), 4 weeks after transplantation in a subcutaneous space. Toluidine blue staining; bar=100 µm. Dotted arrow indicates skin (zonal detachment of cells that have lost their nuclei); solid arrow indicates cartilage (round cells surrounded by matrix with metachromatic staining); arrowhead indicates a mucous gland (cells containing round, homogeneous materials arranged side by side facing the open space).

 
Some tumours were thought to be teratomas because they contained tissues that showed characteristics of tissues originating from the ectoderm, mesoderm and endoderm (Fig. 1Go, bottom).

The incidence of tumour formation in the knee (total of 8 tumours for 25 injections: 1/5, 1/5, 3/5, 1/5 and 2/5 at 1, 2, 4, 6 and 8 weeks after injection respectively) was much lower than that after subcutaneous transplantation (total of 22 for 25 injections: 3/5, 4/5, 5/5, 5/5 and 5/5 at 1, 2, 4, 6 and 8 weeks after injection respectively). The volume of the tumour became larger with time in both the knee joint and the subcutaneous space, but it was much larger in the subcutaneous space. The mean width of tumours in the knee joints was 0.24, 0.20, 1.31, 2.06 and 12.22 mm2 at 1, 2, 4, 6 and 8 weeks after injection respectively. Those in subcutaneous spaces were 3.65, 10.12, 18.23, 53.07 and 101.90 mm2 at 1, 2, 4, 6 and 8 weeks after injection respectively.

There was no significant difference between the incidence of cartilage formation in tumours in the knee joint (3/8) and in tumours in the subcutaneous space (6/22). The mean size of a single cartilage mass in a tumour in the knee joint (0.09 mm2) was almost the same as that in the subcutaneous space (0.08 mm2). Several cartilage masses were usually observed in a single tumour. Because the tumours in the subcutaneous transplantation sites were larger, the ratio of the width of the cartilaginous to the total width of the tumour in the knee joints (14.16, 10.50 and 2.67% in the three samples from joints) was significantly larger than that in the subcutaneous spaces (0.30, 1.06, 1.46, 0.49, 0.20 and 0.40% in the six samples from subcutaneous spaces) (P=0.0201).

The DNA analysis revealed that all samples contained the Neo-resistance gene, which had been transfected into the ES cells but was not present in the SCID mice, indicating that some cells in the histological sections had been derived from the transplanted cells (Fig. 2Go).



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FIG. 2. Gel electrophoresis of DNA amplified by the polymerase chain reaction. Lanes 1–7, DNA extracted from sections of subcutaneous tumours; lanes 8–15, DNA extracted from sections of knee joints with tumours; lane 16, DNA extracted from AB2.2 cells; lane 17, genomic DNA of an SCID mouse; lane 18, negative control.

 


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ES cells formed tumours both in the knee joints and in the subcutaneous spaces in SCID mice. This is the first report of ES cell transplantation into the knee joint. The incidence of tumour formation in the knee joint was low. The growth of tumour in the knee joint was slower than in the subcutaneous space. It thus appears that the joint environment is not optimal for the ES cells to grow and form a tumour. It is also conceivable that the confined space in the knee joint may have interfered with growth. However, some of these tumours subsequently grew larger and destroyed the knee joint.

We concluded that these tumours were teratomas because we identified tissues that showed characteristics of tissues originating from the ectoderm, mesoderm and endoderm. There was no significant difference between the incidence of cartilage formation in the tumours in the knee joint and that in the subcutaneous space. The ratio of cartilage to tumour was significantly greater for the joint space than the subcutaneous space. However, we consider that the joint environment can hardly be described as chondrogenic for ES cells. The volume of cartilage in the tumour was almost the same in the joint space as in the subcutaneous space. Even when the tumour became larger, the size of the cartilage mass within it remained constant. Thus, differences in the proportion of cartilage were due mainly to tumour size, not cartilage size.

We embedded the ES cells in type I collagen, which is not a constituent of articular cartilage. It is possible that type II collagen, which is a cartilage-derived collagen, may have a chondrogenic effect on ES cells. We did not use type II collagen, however, for two reasons. First, we wanted to make a clear distinction between the two environments. In the joint, but not in the subcutaneous space, the injected cells could have come into contact with type II collagen. If we had embedded the ES cells in type II collagen, the difference between the two environments would not have been clearly observed. Secondly, because type II collagen does not produce a hard gel, the embedded cells may not be arranged in three-dimensional position in a gel.

The Neo-resistance gene, which had been transfected into the ES cells, was not present in the cells of the SCID mice we used. However, it was detected in the histological sections, indicating that some cells in the histological sections had been derived from the ES cells. We examined all areas of all histological sections and found no abnormal tissues except for the tumours. This strongly suggests that the tumours were generated by the ES cells.

It is currently not possible to use ES cells to repair articular cartilage defects because tumours eventually grow and extrude from the joints and because cartilage formation is not satisfactory. Further optimization of donor ES cells to differentiate into cartilage [10] and inhibit tumour growth [12] may help to meet these challenges in the development of an ES-derived therapy for chondral defects.


    Notes
 
Correspondence to: S. Wakitani, Department of Orthopaedic Surgery, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Japan. E-mail: wakitani{at}hsp.md.shinshu-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 23 May 2002; Accepted 27 May 2002