Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2009 Nov 21;136(6):829–838. doi: 10.1007/s00432-009-0723-0

Transformation potential of bone marrow stromal cells into undifferentiated high-grade pleomorphic sarcoma

Qing Li 1,2, Hiroko Hisha 1,2, Takashi Takaki 1, Yasushi Adachi 1, Ming Li 1, Changye Song 1, Wei Feng 1, Satoshi Okazaki 1, Tomomi Mizokami 1, Junko Kato 1,3, Muneo Inaba 1, Naoki Hosaka 1, Masahiko Maki 1, Susumu Ikehara 1,2,4,✉
PMCID: PMC11828178  PMID: 19936790

Abstract

Purpose

Bone marrow adherent cells contain conventional bone marrow stromal cells and mesenchymal stem cells and these cells constitute the hematopoietic microenvironment. Mesenchymal stem cells have the capacity to give rise to multiple mesenchymal lineage cells and even ectodermal lineage cells. In the present study, we investigated what types of tumor cells are inducible from BM adherent cells by chemical carcinogens.

Methods

Bone marrow cells from neonatal C3H/HeN mice were collected within 24 h after birth and then cultured. Four days later, bone marrow adherent cells were obtained and the cells were treated with 3-methylcholanthrene.

Results

By this treatment, some transformed clones consisting of large spindle cells were obtained. The transformed cells were highly positive for CD44 and were positive for Sca-1, CD49d and CD106, whereas the cells were negative for hematolymphoid markers. The cell clones had the ability to support hematopoiesis in vitro. These results indicate that the transformed cell lines have the characteristics of BM stromal cells/mesenchymal stem cells. Moreover, during culture of the transformed cells, spontaneous bone nodule formation was observed. When the transformed cells were inoculated into immunodeficient mice subcutaneously, the neoplasms grew in the subcutaneous tissue of the mice. Microscopically and ultrastructurally, the neoplasms showed the typical morphology of undifferentiated high-grade pleomorphic sarcoma (UHGPS). Bone-related genes have been found to be expressed in both transformed cells and UHGPSs.

Conclusion

The present study suggests that UHGPSs are derived from BM stromal cells, probably mesenchymal stem cells.

Keywords: Undifferentiated high-grade pleomorphic sarcoma, Mesenchymal stem cell, Carcinogenesis, Chemical transformation in vitro, Mouse

Introduction

Mesenchymal stem cells (MSCs) present in bone marrow (BM) are thought to be capable of supporting hematopoiesis and of differentiating along multiple mesenchymal lineages (Prockop 1997; Pittenger et al. 1999) and various other types of cells, including skeletal muscle (Ferrari et al. 1998), neurons (Woodbury et al. 2000), and epithelial cells (Jiang et al. 2002), etc.

Recently, attempts have been made to examine the relationship between malignancies and MSCs. We have also proposed a concept of “stem cell disorders” including hematopoietic stem cells (HSCs) (Ikehara 1998) and MSCs (Ikehara 2003). Some researchers have also demonstrated that MSCs could be induced into malignant cells. After adult mouse MSCs were transduced with special genes, cell lines were established. The cell lines formed tumors of mesenchymal malignancy (Serakinci et al. 2004), Ewing’s sarcoma (Riggi et al. 2005) and myxoid liposarcoma (Riggi et al. 2006) in mice. Donor-derived gastric intraepithelial cancers were found in the recipient mice after lethal irradiation and BM transplantation with chronic infection of Helicobacter. The authors suggested the intraepithelial cancer cells probably originated from MSCs (Houghton et al. 2004).

The causes of most malignant soft tissue tumors in humans are still unclear, although genetic and environmental factors (irradiation, viral infections and immune deficiency, etc.) have been found to be associated with the development of usual malignant soft tissue tumors. Chemical carcinogens also have been suggested to play an important role in oncogenesis in humans (Fletcher et al. 2002a, b). The chemical assay inducing the in vitro transformation of mammalian cells was established in the 1960s, and revealed the tumorigenic potential of chemicals.

In this study, we developed a new chemical assay of in vitro transformation using a primary culture of BM cells (BMCs) from neonatal C3H/HeN (C3H) mice, and successfully established transformed clones. When the transformed clones were inoculated into immunodeficient mice, undifferentiated high-grade pleomorphic sarcomas (UHGPSs) were formed in the mice. The morphological, functional and phenotypical findings suggest that the malignant-transformed cells originated from BM stromal cells, probably MSCs.

Materials and methods

Animals

C3H mice and BALB/c nu/nu (nude) mice were purchased from Shizuoka Experimental Animal Laboratory (Hamamatsu, Japan), and C.B-17/Icr-scid/scidJc1 (scid) mice were purchased from Japan Clea Experimental Animal Laboratory (Tokyo, Japan). The C3H mice were mated before neonatal mice would be needed in experiments. All studies were approved by the Laboratory Animal Research Facility of the Kansai Medical University and adhered strictly to the university guidelines for the use and care of experimental animals.

Isolation of BMCs from neonatal C3H mice

The humeri, femora and tibiae were separated from the neonatal mice within 24 h after birth and BMCs were flushed from the BM cavities of the bones using a syringe with a 27-G needle with 2% fetal bovine serum (FBS)/PBS. Approximately 1 × 107 BMCs were cultured with 10% FBS/IMDM in a 25-cm2 flask in a 5% CO2 atmosphere at 37°C in moisture.

Chemical in vitro transformation method

On the fourth day of BMC culture, 3-methylcholanthrene (3-MCA), a chemical carcinogen having an aromatic polycyclic hydrocarbon structure (Sigma–Aldrich), was added to the cultured BMCs at the final concentration of 5 μg/ml and incubated for 20 h (a modified method of Catherine et al. 1973). The flask was then washed with 2% FCS/PBS, and the cells contained in the washing solution were recovered and returned back into the flask in order to serve as a kind of feeder cells. Fresh 10% FCS/IMDM was then added to the flask. One month later, when colonies of the transformed cell clones reached 0.5–2.8 cm in diameter in the flasks, each colony was collected separately using cloning cylinders (37847-0300, Scienceware) or cloning disks (F37847-003, Scienceware) and cultured in 12-well plates. When the cells in the wells reached 80–95% confluence, the transformed cells were detached with 0.02% EDTA solution alone and subcultured once every 4 days continually.

Examination of bone nodules (BNs) formed by the transformed cells

When the nodules appeared in the cultures, the culture media were treated with 9 μg/ml tetracycline (Sigma–Aldrich) in 10% FBS/IMDM for 24 h. After being fixed in Karnovsky fixative, the samples were examined on a confocal laser scanning microscope (Zeiss LSM 510 meta, Carl Zeiss). The nodules were also assessed by von Kossa staining to detect calcium mineral deposits.

Inoculation of cells into mice

The transformed cells were harvested from the culture and re-suspended in PBS. Nude and scid mice were subcutaneously inoculated with from 6 × 106–1 × 107 cells per animal.

Light microscopy and electron microscopy (EM)

Samples were routinely processed, and hematoxylin and eosin (H&E) sections and EM sections were prepared. The EM sections were observed using a Hitachi H-7000 electron microscope (Hitachi, Ibaragi, Japan).

Immunophenotyping by flow cytometry

The transformed cells were harvested from the culture, and reacted with PE-, FITC-, or biotin-conjugated monoclonal antibodies (mAbs). The cells labeled with biotin-conjugated mAbs were further reacted with avidin-conjugated FITC or PE. The stained cells were subjected to flow cytometric analysis using a FACScan (Becton–Dickinson, USA). The mAbs used were as follows: PE-CD11b, CD14, CD44, CD45, CD90, Gr-1, CD117, Sca-1, H-2Kk (BD Pharmingen) and CD31 (Invitrogen); FITC-conjugated CD11c, CD54 (BD Pharmingen) and CD34 (Invitrogen); biotin-conjugated CD49d (BD Pharmingen) and CD106 (Invitrogen).

Purification of HSCs

BMCs were collected from 8-week-old C3H mice. Low-density (LD)/lineage-negative (Lin−) HSCs were purified using discontinuous density-gradient centrifugation and magnetic beads separation as previously described (Wang et al. 2006).

Long-term culture (LTC) assay of HSCs on the transformed cells or fresh BM adherent cells and colony-forming unit in culture (CFU-C) assay

LTC assay

The transformed cells were cultured in flasks at around 80% confluence and then irradiated (20 Gy). The LD/Lin− HSCs (1 × 105) were inoculated on the adherent cell layers. As a control, the LD/Lin− HSCs were also cultured on fresh BM adherent cells that had been prepared by the culture of BMCs obtained from adult C3H mice. Every 7 days, the non-adherent cells were collected and used for CFU-C assay.

CFU-C assay

CFU-C assays were performed using “Methocult GF H3434” (Stemcell Technologies Inc., Vancouver, BC, Canada), following the manufacturer’s instructions.

RNA preparation and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from 3 UHGPS tumors, 5 transformed cell lines, 3-week-old mouse bones (positive control) and peripheral blood mononuclear cells (negative control) by RNA STAT-60 kit (Tel-Test, Inc., Friendswood, TX). RNA concentration was determined at an optical density of OD260. The RNA was transcribed into cDNA by ReverTraAce-α- (Toyobo, Osaka, Japan). Specific primers for the RT-PCR are shown in Table 1. The PCR was first denatured at 94°C for 2 min, and then at 94°C for 20 s, at 52–55°C for 20 s and at 72°C for 60 s for 30 cycles and with a final 8-min extension at 72°C. Ten microliters of PCR products was analyzed by 2.5% agarose gel with ethidium bromide staining.

Table 1.

Primer sequences used for polymerase chain reaction

Gene Primer sequence Product size Tm (°C)
Runx2 Forward: 5′-TACAAACCATACCCAGTCCCTGTTT-3′ 197 55
Reversed: 5′-AGTGCTCTAACCACAGTCCATGCA-3′
Osx Forward: 5′-ACTCATCCCTATGGCTCGTG-3′ 238 55
Reversed: 5′-GGTAGGGAGCTGGGTTAAGG-3′
Oc Forward: 5′-CTTGGTGCACACCTAGCAGA-3′ 208 54
Reversed: 5′-TTCTGTTTCCTCCCTGCTGT-3′
Bsp Forward: 5′-AAAGTGAAGGAAAGCGACGA-3′ 215 52
Reversed: 5′-GTTCCTTCTGCACCTGCTTC-3′
Opn Forward: 5′-TCTGATGAGACCGTCACTGC-3′ 170 53
Reversed: 5′-AGGTCCTCATCTGTGGCATC-3′
G3pdh Forward: 5′-ACCACAGTCCATGCCATCAC-3′ 450 56
Reversed: 5′-TCCACCACCCTGTTGCTGTA-3′
Open in a new tab

Osx Osterix, Oc osteocalcin, Bsp bone sialoprotein, Opn osteopontin, G3dph glyceraldehyde-3-phosphate dehydrogenase

Results

Transformation of BMCs by treatment with 3-MCA and cloning of the transformed cells

BMCs from neonatal C3H mice were cultured in flasks and a small number of BMCs adhered to the surface of the flasks and began to proliferate within 1–2 days. The adherent cells reached about 10–20% confluence in flasks on day 4 of culture (Fig. 1a). After treatment with 3-MCA, the adherent cells continued to proliferate and changed gradually into multilayered and randomly oriented spindle cells (Fig. 1b). The transformed BMCs were subcultured at a very low cell concentration but some clones soon appeared and continued to increase in size with the passage of time. Under the phase-contrast microscope, all colonies of the transformed cells were predominantly composed of randomly oriented, enlarged fibroblast-like spindle cells (Fig. 2). The colonies (0.5–2.8 cm in diameter) were formed sufficiently far apart that individual colonies (clones) could be easily isolated using cloning cylinders and disks. Before the subculture, the transformed cells were cultured at a high cell concentration, and we cannot therefore exclude the possibility that some of the colonies might have been derived from a common clone. The transformation experiments were performed three times using the neonatal mice. A total of 24 transformed clones were obtained (1, 5 and 18 clones in each experiment). Morphologically, it is speculated that the transformed clones were derived from adherent cells of the BM.

Fig. 1.

Fig. 1

Open in a new tab

Morphological characteristics of BM stromal cells before and after treatment with 3-MCA. a BM stromal cells on the fourth day in culture just before treatment with 3-MCA. A small number of clusters of spindle cells can be seen (×4). b BM stromal cells on the seventh day after treatment with 3-MCA. Multilayered, randomly oriented spindle cells can be seen (×10)

Fig. 2.

Fig. 2

Open in a new tab

Morphological characteristics of transformed cell colony. Margin of colony (×10)

Bone nodule (BN) formation of the transformed cells

Some nodules with a round or oval shape and surrounded by fibroblastic-like cells could be found in all the transformed 24 clones when they were cultured just in 10% FBS/IMDM (Fig. 3a). Hematoxylin and eosin (H&E) sections of the nodules showed a dense collagenous matrix containing ovoid osteocyte-like cells and flattened osteoblast-like cells at the periphery (data not shown), these cells being similar to the MSC-derived BNs reported in the literature (Turksen and Aubin 1991). After the cultures were treated with tetracycline, the fluorescence of tetracycline could be detected on the nodules using a confocal laser scanning microscope (Fig. 3b), and when the cultures were treated with von Kossa reagents, all the nodules were stained black or dark brown, indicating the existence of calcium deposits in the nodules (Fig. 3c). The formation of BNs is regarded to be the result of the capacity for osteogenesis of cells in culture, which is one of the characteristics of MSCs. This present finding showed that all the clones of the transformed cells had a similar capacity for osteogenic differentiation with MSCs.

Fig. 3.

Fig. 3

Open in a new tab

BN formation. BNs were found in the culture of the transformed cells. a Image of a BN in living state under inverted contrast microscope (×10). b Fluorescent image of the BN labeled with tetracycline being excited with the 405-nm laser emitted by laser diode in a confocal laser microscope (×10). c Calcium deposits in a BN detected by von Kossa reagents. The apparatus was set to focus on the lowest part of the BNs, and therefore the top part of the BNs and the multilayered transformed cells on the flask surface are not so clearly in focus in Fig. 3a, c

Phenotypic characterization of the transformed cells

All the 24 transformed clones were strongly labeled with CD44. The expression of Sca-1, CD49d, CD106 and H-2Kk was also observed. However, the cells were negative for CD45, Gr-1, CD31 and the macrophage markers (CD11b, CD11c and CD14) (Fig. 4). They were also negative for CD34, CD117, CD54 and CD90. These results indicate that the phenotype of the transformed cells resembles that of MSCs (Wang et al. 2006; Meirelles Lda and Nardi 2003).

Fig. 4.

Fig. 4

Open in a new tab

Phenotypic characteristics of transformed cell clone

Hematopoiesis-supporting capacity of the transformed cells in vitro

We attempted to examine, using an LTC assay, whether the transformed cells support hematopoiesis in vitro as do BM stromal cells/MSCs; LD/Lin− HSCs were cultured on the transformed clones. Floating cells in all the seven LTC flasks could be collected continually for 4 weeks and the cells indeed possessed the capacity to form CFU-C in methylcellulose assay, while the LD/Lin− cells cultured in the flasks without the transformed cells did not show any CFU-C (Fig. 5). Each CFU-C colony included granulocytes alone, macrophages alone, granulocytes–macrophages, or erythroblasts (data not shown). Both the irradiated and non-irradiated transformed cells had the capacity to support hematopoiesis in vitro (Fig. 5). The LTC assay was carried out seven times using seven different transformed clones, and all the transformed clones were able to support hematopoiesis in vitro, although the CFU-C number varied between experiments. This result confirmed that the transformed cells possessed the ability to support hematopoiesis in the same way as BM stromal cells/MSCs.

Fig. 5.

Fig. 5

Open in a new tab

Ability of transformed cell clone to support hematopoiesis in vitro. a Number of floating cells collected from the flasks where LD/Lin− cells were cultured on the irradiated or non-irradiated transformed cells or irradiated BM adherent cell of C3H mice (positive control). b Number of CFU-C formed by the floating cells. Representative data of seven independent experiments

Implantation of transformed cells into immune-deficient mice

When transformed cell clones were inoculated subcutaneously into BALB/c nu/nu and C.B-17/scid mice, neoplasms were found in the inoculated subcutaneous sites in the majority of the mice (17/24 clones) on and after days 21–30 of the inoculation. The neoplasms increased gradually in size and reached 2 cm in diameter or more largely with the passing of time. Ulcer formation on the tumors was seen in some mice. The cut sections of smaller tumors were firm and gray-white in color, whereas necrosis or hemorrhaging was observed in the sections of on some of the larger tumors. Microscopically, all of the neoplasms were predominantly composed of severely atypical spindle cells (fibroblast-like component) and, in some areas, were arranged in a storiform pattern in the H&E section (Fig. 6a, b). The atypical spindle cells were admixed with numerous large bizarre polyhedral cells (Fig. 6b, c), and multinucleated tumor giant cells (Fig. 6d). Tumors derived from nine transformed clones were examined with EM. Some neoplastic cells had ultrastructural features associated with fibroblasts and others contained lysosomes and phagosomes, the organelle characteristic of histiocytes. The dual ultrastructural characteristics of both fibroblasts and histiocytes were also seen (Fig. 7a, b). Desmosome-like structures were also seen between neoplastic cells (data not shown). We also found so-called primitive mesenchymal cells (Fu et al. 1975) (data not shown). To sum up, the malignant neoplasms found in the present study displayed an appearance typical of the UHGPS counterparts in humans, microscopically and ultrastructurally. In the subcutaneous inoculation of the transformed cells, no malignant tumors other than the UHGPSs were found.

Fig. 6.

Fig. 6

Open in a new tab

Histological analysis of neoplasms. Histological images of the neoplasms formed in immunodeficient mice after inoculating transformed cell clones (H&E sections). a Invasive growth into striated muscle (M) and fat tissue (F) at the margin of the neoplasm (×4). b Elongated spindle cells arranged in storiform pattern (solid arrow) (×10). c Pleomorphologic area with bizarre cells (arrow heads) (×40). d Multinucleated tumor giant cells (empty arrow) (×40)

Fig. 7.

Fig. 7

Open in a new tab

EM analysis of neoplasm. a, b The different types of tumor cells could be seen ultrastructurally. H Histiocyte-like cell, P phagosome, F fibroblast-like cell, DU dual ultrastructural cell (×1,500)

Expression of bone-related genes in transformed cells and UHGPS cells

To assess bone-related gene expression in the transformed cells and the UHGPS cells, we studied the expression of these genes using the RT-PCR assay. Both the transformed cells and UHGPS cells were found to synthesize messages of bone-specific proteins such as, Bsp, Oc, and Opn. These cells also expressed osteoblast-specific transcription factors, Runx2 and Osx (Fig. 8). Such mRNA expression was seen in all the samples of the transformed cells and UHGPS cells (Fig. 8). Thus, it is conceivable that BNs were formed during the culture of the transformed cells (Fig. 3). It was also shown that the UHGPS cells continued to retain their osteoblast genotype, although no bone differentiation was detected histologically.

Fig. 8.

Fig. 8

Open in a new tab

Expression of bone-related genes in the UHGPS and transformed cells. Total RNAs from the transformed cells and UHGPS cells were extracted and reversely transcribed. The cDNAs were amplified by PCR using the specific primers shown in Table 1. The PCR products were run on agarose gels and stained with ethidium bromide. A representative expression of bone-related genes in the UHGPS and transformed cells. M marker, Trans cells transformed cells, PBMNCs peripheral blood mononuclear cells, Blank blank control (water)

Conclusions

In this study, we successfully developed a new chemical in vitro transformation method using a primary BM culture of neonatal mice with 3-MCA (one of the most commonly used chemical carcinogens) and were able to readily establish some transformed cells. BNs were found in all the transformed cells, indicating that these cells have the potential for osteogenesis. The immunophenotype of the transformed clones resembles that of MSCs. Moreover, the transformed cells retained the ability to support normal hematopoiesis in vitro. This means that the transformed cells retained the capacity to provide a hematopoietic microenvironment for HSCs. It is well known that BM stromal cells/MSCs have such ability, and therefore it is speculated that the transformed cells were derived from BM stromal cells/MSCs. When the transformed clones were inoculated into immunodeficient mice, UHGPSs were formed at the subcutaneous sites of inoculation. Bone-related genes were found to be expressed in both the transformed cells and the UHGPSs. Thus, UHGPSs are considered to originate from BM stromal cells/MSCs.

UHGPS is the most common type of soft tissue sarcoma in older persons. The neoplastic cells forming these sarcomas were interpreted as being of histiocytic origin in the 1960s. Then, the term “malignant fibrous histiocytoma (MFH)” was formally incorporated into the classification (Rosenberg 2003 and Tos 2006). Later, this type of tumor was found to frequently express various antigens commonly expressed on many tumors (Lawson et al. 1987; Fisher 1990; Rosenberg et al. 1993; Montgomery and Fisher 2001). (This phenomenon might reflect the MSC-origin of UHGPSs, since MSCs have been reported to have the capacity for multi-lineage differentiation. However, such multi-immunotyping of human UHGPSs could be attributed in part to cross reactivity and aberrant expression, etc.) Importantly, no antigens specific to histiocytes were found in MFHs, essentially proving that MFH was not a true histiocytic neoplasm (Wood et al. 1986; Iwasaki et al. 1987). Recently, the term of UHGPS has been used for MFH and was considered to represent a small group of undifferentiated pleomorphic sarcomas with no definable line of differentiation (Fletcher et al. 2002a, b). The term “pleomorphic fibrosarcoma” has also been proposed for this tumor (Erlandson and Antonescu 2004). In our research we have found that UHGPSs have the typical microscopic and ultrastructural characteristics as seen in humans. The UHGPSs lack the features of any other type of poorly differentiated sarcoma seen in humans. Accordingly, we consider the UHGPS/storiform-pleomorphic MFHs to be a distinct clinicopathologic entity of malignancy.

In vitro BN formation of MSCs has been considered a good standard of their differentiation capacity to osteoblasts. We observed that the transformed cells could form BNs during culture (Fig. 3), whereas these cells formed UHGPSs (but not osteosarcomas) after being inoculated into the nude mice (Fig. 6). It can be speculated that the osteogenic activity of the transformed cells was lost when they were inoculated into the mice. The study of the gene expression (Fig. 8) showed that some bone-specific genes could be detected in both the transformed cells and the UHGPS cells. This phenomenon might be explained by de-differentiation of the transformed cells, induced by unknown in vivo environmental factors. Bone formation in vivo is a complex processes that is affected by some unknown factors. Recently, Yamate et al. have shown a rat MFH-derived cell line (MT-9) can be induced to differentiate into adipocytes, osteocytes and myofibrocytes. It was also shown that the MT-9 cells had a message of c-kit (a marker of tissue stem cells). In rat BM tissues, some cells were stained positively with a rat MFH cell-specific antibody (A3). From these findings, Yamate et al. suggested that progenitors of MFH might be involved in the lineage of BM stem cells capable of differentiating into mesenchymal cells (Yamate et al. 2007). Thus, both Yamate et al. and we have arrived at a similar conclusion, albeit by different experimental routes. However, the details how UHGPS cells loss the ability of in vivo bone formation in our experiment need further investigation.

We have not observed the development of malignant tumors other than UHGPSs in the present experiments. However, Liu et al. reported that BMCs could form various types of tumors, including epithelial tumors, neural tumors, muscular tumors, blood vessel endothelial tumors, tumors of fibroblasts, and also teratomas (Liu et al. 2006). Both Liu et al. and we cultured BM cells with 3-MCA. The only difference between their method and ours was that they used secondary cultures of adult mouse BMCs treated with 3-MCA, while we used the primary cultures (on day 4) of neonatal mouse BMCs treated with 3-MCA. It is therefore conceivable that in our experiments the MSCs were kept in an undifferentiated state so that UHGPSs/MFHs developed, while in Liu et al’s experiments the majority of the BMCs had become more differentiated cells towards multiple lineages, and therefore, a variety of differentiated malignant tumors developed in their experiments.

Very recently, Matushansky et al. have also shown that the stem cell-specific gene expression pattern of human MSCs (hMSCs) is significantly associated with that of a MFH cell line but not other sarcoma subtypes (liposarcoma, leiomyosarcoma, or fibrosarcoma, etc.) (Matushansky et al. 2007). Thus, MHF exhibits the closest association among the other sarcoma subtypes to hMSCs. This result is possibly an explanation for our observation that only UHGPS was formed in the immunodeficient mice that had been injected with the 3-MCA-transformed cells. They also reported that malignant tumors were formed in nude nice when SV40-large T antigen-immortalized hMSCs had been cultured for 2 weeks in the presence of DKK1 (a Wnt signaling inhibitor) and the transformed cells were then injected into nude mice subcutaneously. However, the authors also mention that the tumor did not display full degree of pleomorphism commonly observed in MFH. Pleomorphism is one of the fundamental characteristics in the UHGPS, as the term “pleomorphic” contained in UHGPS. The tumor that Matushansky et al. have reported could be another type of tumor but was not the true UHGPS/MFH, since MSCs have been reported to develop into several different types of malignancies (Serakinci et al. 2004; Riggi et al. 2005, 2006; Houghton et al. 2004; Matushansky et al. 2007) in different experimental systems. In our research, we developed the UHGPSs with full pleomorphism. Further studies are needed to clarify the mechanism involved in the transformation of MSCs.

Already, the early passage of Syrian hamster embryo cells (SHE), BALB/c 3T3 mouse fibroblasts (B/C 3T3) and C3H 10T1/2 mouse fibroblast lines (10T1/2) have been used in the in vitro cell transformation (Berwald and Sachs 1965; Aaronson and Todaro 1968; Reznikoff et al. 1973). As we know, SHE are the embryo cells and B/C 3T3 and 10T1/2 cells are eternal aneuploidy lines. Therefore, there must be some fundamental differences between these cells and normal postnatal cells. The BMCs used in the present study were postnatal and normal diploidy cells, but SHE, B/C 3T3 or 10T1/2 are not. Thus, our in vitro transformation method might provide a new strategy for screening chemical carcinogens toward normal postnatal cells.

Acknowledgments

Supported by a grant from the Haiteku Research Center of the Ministry of Education; a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology; a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology; a Grant-in-Aid for scientific research (B) 11470062; Grants-in-Aid for scientific research on priority areas (A) 10181225 and (A) 11162221, and Health and Labour Sciences research grants (Research on Human Genome, Tissue Engineering Food Biotechnology) and also a grant from the Department of Transplantation for Regeneration Therapy (Sponsored by Otsuka Pharmaceutical Company, Ltd.), a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd.; a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO). We thank Prof. Xiaohong Wang, MD., Department of Pathology, the 304th Hospital, PLA General Hospital, Beijing, China, and Hua Cao, M.D., Ph.D., Division of Medical Genetics, University of Washington, USA for kindly offering helpful information and advice. We also thank Ms. Y. Tokuyama, K. Hayasi and A. Kitajima for their expert technical assistance. We also thank Mr. Hilary Eastwick-Field and Ms. K. Ando for their help in the preparation of the manuscript. We declare that we have no conflict of interest.

References

  1. Aaronson SA, Todaro GJ (1968) Development of 3T3-like lines from Balb-c mouse embryo cultures: transformation susceptibility to SV40. J Cell Physiol 72(2):141–148 [DOI] [PubMed] [Google Scholar]
  2. Berwald Y, Sachs L (1965) In vitro transformation of normal cells to tumor cells by carcinogenic hydrocarbons. J Nat Cancer Inst 35(4):641–661 [PubMed] [Google Scholar]
  3. Catherine AR, John SB, David WB, Charles H (1973) Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res 33(12):3239–3249 [PubMed] [Google Scholar]
  4. Erlandson RA, Antonescu CR (2004) The rise and fall of malignant fibrous histiocytoma. Ultrastruct Pathol 28(5–6):283–289 [DOI] [PubMed] [Google Scholar]
  5. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F (1998) Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279(5356):1528–1530 [DOI] [PubMed] [Google Scholar]
  6. Fisher C (1990) The value of electronmicroscopy and immunohistochemistry in the diagnosis of soft tissue sarcomas: a study of 200 cases. Histopathology 16(5):441–454 [DOI] [PubMed] [Google Scholar]
  7. Fletcher CD, Sundaram M, Rydholm A, Coindre JM, Singer S (2002a) Soft tissue tumours: epidemiology, clinical features, histopathological typing and grading. In: Fletcher CD, Unni KK, Mertens F (eds) World Health Organization classification of tumours. Pathology and genetics of tumours of soft tissue and bone. IARC Press, Lyon, pp 12–18 [Google Scholar]
  8. Fletcher CD, Berg EV, Molenaar WM (2002b) Pleomorphic malignant fibrous histiocytoma/undifferentiated high grade pleomorphic sarcoma. In: Fletcher CD, Unni KK, Mertens F (eds) World Health Organization classification of tumours. Pathology and genetics tumours of soft tissue and bone. ARC Press, Lyon, pp 120–122 [Google Scholar]
  9. Fu YS, Gabbiani G, Kaye GI, Lattes R (1975) Malignant soft tissue tumors of probable histiocytic origin (malignant fibrous histiocytoma): general considerations and electron microscopic and tissue culture studies. Cancer 35(1):176–198 [DOI] [PubMed] [Google Scholar]
  10. Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, Cai X, Fox JG, Goldenring JR, Wang TC (2004) Gastric cancer originating from bone marrow-derived cells. Science 306(5701):1568–1571 [DOI] [PubMed] [Google Scholar]
  11. Ikehara S (1998) Bone marrow transplantation for autoimmune diseases. Acta Haematol 99(3):116–132 [DOI] [PubMed] [Google Scholar]
  12. Ikehara S (2003) A new concept of stem cell disorders and their new therapy. J Hematother Stem Cell Res 12(6):643–653 [DOI] [PubMed] [Google Scholar]
  13. Iwasaki H, Isayama T, Johzaki H, Kikuchi M (1987) Malignant fibrous histiocytoma. Evidence of perivascular mesenchymal cell origin immunocytochemical studies with monoclonal anti-MFH antibodies. Am J Pathol 128(3):528–537 [PMC free article] [PubMed] [Google Scholar]
  14. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418(6893):41–49 [DOI] [PubMed] [Google Scholar]
  15. Lawson CW, Fisher C, Gatter KC (1987) Malignant fibrous histiocytoma. Evidence of perivascular mesenchymal cell origin immunocytochemical studies with monoclonal anti-MFH antibodies. Histopathology 11(4):375–383 [PMC free article] [PubMed] [Google Scholar]
  16. Liu C, Chen Z, Chen Z, Zhang T, Lu Y (2006) Multiple tumor types may originate from bone marrow-derived cells. Neoplasia 8(9):716–724 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matushansky I, Hernando E, Socci ND, Mills JE, Matos TA, Edgar MA, Singer S, Maki RG, Cordon-Cardo C (2007) Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway. J Clin Invest 117(11):3248–3257 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meirelles Lda S, Nardi NB (2003) Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 123(4):702–711 [DOI] [PubMed] [Google Scholar]
  19. Montgomery E, Fisher C (2001) Myofibroblastic differentiation in malignant fibrous histiocytoma (pleomorphic myofibrosarcoma): a clinicopathological study. Histopathology 38(6):499–509 [DOI] [PubMed] [Google Scholar]
  20. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147 [DOI] [PubMed] [Google Scholar]
  21. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276(5309):71–74 [DOI] [PubMed] [Google Scholar]
  22. Reznikoff CA, Bertram JS, Brankow DW, Heidelberger C (1973) Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res 33(12):3239–3249 [PubMed] [Google Scholar]
  23. Riggi N, Cironi L, Provero P, Suvà ML, Kaloulis K, Garcia-Echeverria C, Hoffmann F, Trumpp A, Stamenkovic I (2005) Development of Ewing’s sarcoma from primary bone marrow-derived mesenchymal progenitor cells. Cancer Res 65(24):11459–11468 [DOI] [PubMed] [Google Scholar]
  24. Riggi N, Cironi L, Provero P, Suvà ML, Stehle JC, Baumer K, Guillou L, Stamenkovic I (2006) Expression of the FUS-CHOP fusion protein in primary mesenchymal progenitor cells gives rise to a model of myxoid liposarcoma. Cancer Res 66(14):7016–7023 [DOI] [PubMed] [Google Scholar]
  25. Rosenberg AE (2003) Malignant fibrous histiocytoma: past, present, and future. Skelet Radiol 32(11):613–618 [DOI] [PubMed] [Google Scholar]
  26. Rosenberg AE, O’Connell JX, Dickersin GR, Bhan AK (1993) Expression of epithelial markers in malignant fibrous histiocytoma of the musculoskeletal system: an immunohistochemical and electron microscopic study. Hum Pathol 24(3):284–293 [DOI] [PubMed] [Google Scholar]
  27. Serakinci N, Guldberg P, Burns JS, Abdallah B, Schrødder H, Jensen T, Kassem M (2004) Adult human mesenchymal stem cell as a target for neoplastic transformation. Oncogene 23(29):5095–5098 [DOI] [PubMed] [Google Scholar]
  28. Tos AP (2006) Classification of pleomorphic sarcomas: where are we now? Histopathology 48(1):51–62 [DOI] [PubMed] [Google Scholar]
  29. Turksen K, Aubin JE (1991) Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. J Cell Biol 114(2):373–384 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wang X, Hisha H, Taketani S, Adachi Y, Li Q, Cui W, Cui Y, Wang J, Song C, Mizokami T, Okazaki S, Li Q, Fan T, Fan H, Lian Z, Gershwin ME, Ikehara S (2006) Characterization of mesenchymal stem cells isolated from mouse fetal bone marrow. Stem Cells 24(3):482–493 [DOI] [PubMed] [Google Scholar]
  31. Wood GS, Beckstead JH, Turner RR, Hendrickson MR, Kempson RL, Warnke RA (1986) Malignant fibrous histiocytoma tumor cells resemble fibroblasts. Am J Surg Pathol 10(5):323–335 [DOI] [PubMed] [Google Scholar]
  32. Woodbury D, Schwarz EJ, Prockop DJ, Black IB (2000) Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61(4):364–370 [DOI] [PubMed] [Google Scholar]
  33. Yamate J, Ogata K, Yuasa T, Kuwamura M, Takenaka S, Kumagai D, Itoh K, LaMarre J (2007) Adipogenic, osteogenic and myofibrogenic differentiations of a rat malignant fibrous histiocytoma (MFH)-derived cell line, and a relationship of MFH cells with embryonal mesenchymal, perivascular and bone marrow stem cells. Eur J Cancer 43(18):2747–2756 [DOI] [PubMed] [Google Scholar]

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES