Cotransplantation of Mouse Embryonic Stem Cells and Bone Marrow Stromal Cells following Spinal Cord Injury Suppresses Tumor Development

Abstract

Embryonic stem (ES) cells are a potential source for treatment of spinal cord injury (SCI). Although one of the main problems of ES cell-based cell therapy is tumor formation, there is no ideal method to suppress tumor development. In this study, we examined whether transplantation with bone marrow stromal cells (BMSCs) prevented tumor formation in SCI model mice that received ES cell-derived grafts containing both undifferentiated ES cells and neural stem cells. Embryoid bodies (EBs) formed in 4-day hanging drop cultures were treated with retinoic acid (RA) at a low concentration of 5 × 10^–9 M for 4 days, in order to allow some of the ES cells to remain in an undifferentiated state. RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived graft cells. Mice transplanted with ES cell-derived graft cells alone developed tumors at the grafted site and behavioral improvement ceased after day 21. In contrast, no tumor development was observed in mice cotransplanted with BMSCs, which also showed sustained behavioral improvement. In vitro results demonstrated the disappearance of SSEA-1 expression in cytochemical examinations, as well as attenuated mRNA expressions of the undifferentiated markers Oct3/4, Utf1, Nanog, Sox2, and ERas by RT-PCR in RA-treated EBs cocultured with BMSCs. In addition, MAP2-immunopositive cells appeared in the EBs cocultured with BMSCs. Furthermore, the synthesis of NGF, GDNF, and BDNF was confirmed in cultured BMSCs, while immunohistochemical examinations demonstrated the survival of BMSCs and their maintained ability of neurotrophic factor production at the grafted site for up to 5 weeks after transplantation. These results suggest that BMSCs induce undifferentiated ES cells to differentiate into a neuronal lineage by neurotrophic factor production, resulting in suppression of tumor formation. Cotransplantation of BMSCs with ES cell-derived graft cells may be useful for preventing the development of ES cell-derived tumors.

Keywords

Embryonic stem cells Bone marrow stromal cells Spinal cord injury Cotransplantation Tumor suppression

Introduction

A traumatic spinal cord injury (SCI) is considered to be an appropriate condition for treatment with cell-based therapy (15,20), for which several candidate cell sources have been proposed, such as fetal spinal cord tissue, neural stem cells (NSCs) from adult brains, mesenchymal stem cells from bone marrow and other organs, and embryonic stem (ES) cells. Because patients with an SCI have an absolute loss of neurons and glia, it would be ideal if the transplanted cells possessed an ability to accommodate the need for cell replacement. Although fetal spinal cord tissue may best meet this demand, there are ethical issues regarding the use of fetal tissues as well as technical difficulties in obtaining NSCs from adult brains.

Mouse ES cells have the potential to generate cells of all three types of embryonic germ layers (10,28) and a number of recent studies have shown that they can differentiate into a variety of different cell types in vitro, including muscle cells (17), adipocytes (46), osteocytes (22), hepatocytes (49), and insulin-secreting pancreatic beta-like cells (42), as well as those composing dopaminergic (19,31), GABAergic (47), and serotonergic (23) neurons. Differentiation of ES cells into NSCs or neural progenitor cells has also been reported (3,4,23, (36). In our previous study, we found that transplantation of mouse ES cell-derived grafts into parkinsonian and SCI model mice resulted in improvements in behavioral assessment results (21,31). In addition, bone marrow stromal cells (BMSCs) are another attractive cell source for cell-based therapy (18,27,37), though their ability and efficiency for differentiation toward neural lineage is limited compared to ES cells.

There are a number of problems to solve before ES cell-based clinical trials can be commenced, one of which is associated tumor formation. Several authors have reported uncontrolled growth of mouse ES cell-derived grafts at the grafted sites, such as the liver (44), knee joint (45), renal subcapsular space (11), subretinal space (1), and brain (33,38). Because contamination by undifferentiated ES cells in the grafts is the cause of such tumor development, the use of purified fractions of ES cell-derived NSCs or ES cell-derived neurons and glia, uncontaminated by even a trace of undifferentiated ES cells, is essential for clinical application of ES cells for SCI. Thus, it is considered that use of fluorescence-activated cell sorting (FACS) for a particular cell type may be useful for this purpose (8,12).

In the present study, we attempted a novel approach, in which we used BMSCs as differentiating agents for ES cells. In addition to the multipotency of BMSCs during differentiation, they are also known to produce many kinds of neurotrophic factors, such as nerve growth factor (NGF) (7,14), glial cell line-derived neurotrophic factor (GDNF) (7,14,50), and brain-derived neurotrophic factor (BDNF) (7), which led us speculate that cotransplantation of ES cells and BMSCs would provide some advantages over transplantation of ES cells alone.

We examined whether cotransplantation of BMSCs prevented tumor formation in SCI model mice that received ES cell-derived grafts containing undifferentiated ES cells. Embryoid bodies (EBs) formed in 4-day hanging drop cultures were treated with retinoic acid (RA) for 4 days at a low concentration of 5 × 10^–9 M, and RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived grafts. This concentration of RA is unsuitable for efficient neural induction, and ES cell-derived grafts prepared from such RA-treated EBs contain undifferentiated ES cells and neural stem cells. We found that cotransplantation of ES cell-derived grafts with BMSCs prevented the development of tumors in SCI model mice. Mice that received ES cell-derived grafts alone developed tumors at the grafted site and behavioral improvement ceased after 3 weeks. In contrast, no tumor development was observed in mice cotransplanted with BMSCs and they also showed sustained behavioral improvement. In addition, cytochemical findings demonstrated that the expression of SSEA-1 disappeared in RA-treated EBs cocultured with BMSCs, while mRNA expressions of the undifferentiated markers Oct3/4, Utf1, Nanog, Sox2, and ERas were also attenuated. Furthermore, we confirmed the production of NGF, GDNF, and BDNF in cultured BMSCs, as expected, and noted their survival in the spinal cord along with a maintained ability of neurotrophic factor production 5 weeks after transplantation, which suggested that the cotransplanted BMSCs played an important role in inducing neural differentiation of ES cells.

The present results suggest the usefulness of cotransplantation of ES cells and BMSCs to prevent the development of ES cell-derived tumors, as well as increase the safety of ES cell-based cell therapy for SCI.

Materials and Methods

Murine ES Cell Line

We utilized the mouse ES cell line G4–2 (129/SvJ mouse ES cells, a kind gift from Dr. Hitoshi Niwa, RIKEN Center for Developmental Biology, Kobe, Japan) (32) in the present study. G4–2 ES cells were derived from EB3 ES cells and carried the enhanced green fluorescent protein (EGFP) gene under the control of the CAG expression unit. The EB3 cells were a subline derived from E14tg2a ES cells (16) and carried the blasticidin S-resistant selection marker gene driven by the Oct-3/4 promoter (active under undifferentiated status) (32). Undifferentiated G4–2 ES cells were maintained on gelatin-coated dishes without feeder cells in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; GIBCO/BRL, Grand Island, NY), 0.1 mM 2-mercaptoethanol (Sigma), 10 mM nonessential amino acids (GIBCO/BRL), 1 mM sodium pyruvate (Sigma), and 1400 U/ml of leukemia inhibitory factor (LIF; GIBCO/BRL). For some of the experiments, G4–2 cells were cultured in medium containing 10 μg/ml of blasticidin S to eliminate differentiated cells.

Preparation of Graft Cells

For graft cells, ES cells and BMSCs were prepared as shown in Figure 1A. ES cell-derived graft cells were prepared by formation of EBs using RA. To ensure the development of ES cell-derived tumors, we avoided the use of RA at optimal concentrations for neural differentiation, which range from approximately 5 × 10^–7 to 5 × 10^–6 M. Briefly, undifferentiated ES cells were dissociated into single-cell suspensions and then cultured in hanging drops to induce EB formation at an initial cell density of 500 cells per drop (20 μl) of ES cell growth medium in the absence of LIF for 4 days. The resulting EBs were collected and cultivated in suspension for 4 days with RA at a final concentration of 5 × 10^–9 M, which was lower by approximately 100- to 1000-fold than the concentration generally utilized for efficient induction of ES cells toward a neuronal lineage (4,34,36). The RA-treated EBs were transformed into single cells by treatment with 0.25% trypsin and 1 mM EDTA (trypsin-EDTA solution) at 37°C for approximately 5 min, then used as grafts consisting of undifferentiated ES cells and neural stem cells.

Figure 1.

Preparation of graft cells and outline of the experiment. (A) Preparation of ES cell-derived graft cells and BMSC graft cells. ES cell-derived graft cells. Undifferentiated ES cells were cultured in hanging drops to induce EB formation at an initial cell density of 500 cells per drop (20 μl) of ES cell growth medium and the resulting EBs were cultivated in suspension for 4 days with RA at a final concentration of 5 × 10^–9 M. The RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived grafts. BMSC graft cells. BMSCs from the bone marrow of young adult C57BL/6 mice were seeded into plastic culture dishes and allowed to grow to near confluence by removing nonadherent cells until the first passage. In the third culture, following the second passage, adherent cells were cultured with BrdU at a final concentration of 3 μg/ml for 72 h. After labeling with BrdU, adherent cells were washed with PBS and prepared as single cells by enzymatic digestion, then used for transplantation. For coculturing with EBs, cells were used without BrdU pulsation. (B) Outline of the experiment. Thirty SCI mice were randomly divided into three groups of 10 animals each. Eight days after undergoing SCI, mice in the PBS group received 3.0 JLLl of PBS into the injured site of the spinal cord to assess the natural course after SCI, while the ES group received 3.0 μl of PBS containing 1.5 × 10⁴ ES cell-derived graft cells into the injured site of the spinal cord, and the ES/BMSC group received 3.0 μl of PBS containing 1.5 × 10⁴ ES cell-derived graft cells and 1.5 × 10⁴ BMSCs.

BMSCs were prepared from young adult female C57BL/6 mice bone marrow samples, which were displaced from mouse femurs using a 22-gauge needle connected to a 10-ml syringe and placed into dishes with modified Eagle's medium (MEM; Nacalai Tesque, Kyoto, Japan). Dissociated bone marrow cells were then seeded into plastic culture dishes for primary cultures in MEM supplemented with 10% FBS (GIBCO/BRL). After 48 h, nonadherent cells were removed and fresh medium added, which was replaced three times a week. After the adherent cells had grown to near confluence, they were detached by incubation with trypsin-EDTA solution for 10 min and then seeded into new dishes (first passage and second culture). After one more passage, the adherent cells in the third culture were used as BMSC grafts. Before transplantation, 5-bromo-2′-deoxyuridine (BrdU; Roche Applied Science, Indianapolis, IN) was added to the dishes at a final concentration of 3 μg/ml and cultured for the final 72 h in order to label the cells. After culturing with pulsation by BrdU, adherent cells were extensively washed with PBS and prepared as single cells by digestion with trypsin-EDTA solution, then used for transplantation. For coculturing with EBs, cells were used without BrdU pulsation.

Animal Preparations

All animal procedures were conducted in accordance with our institutional guidelines as well as those of the National Institutes of Health. Adult female C57BL/6 mice (10–12 weeks old, Japan SLC Ltd., Hamamatsu, Japan) were used. Each was anesthetized under pentobarbital anesthesia (60 mg/kg, IP) and given a laminectomy at T9–T10. An SCI was produced through the dura matter by a directed impact device (INP-150 pneumatic impactor; Scholar Tec, Osaka, Japan), which has been previously described in detail (41). The impactor consisted of a double-acting, stroke-constrained, pneumatic cylinder with a 5.0-cm stroke and 1.0-mm bore. The cylinder was rigidly mounted in a vertical position on a crossbar and could be precisely adjusted in the vertical axis over the rigidly stable head, while the lower rod end had an impactor tip attached. The upper rod end was attached to the transducer core of a linear variable differential transformer. The impact pressure was adjusted by controlled gas pressure applied to the impactor at 40 psi, which was adopted for producing an impact velocity of approximately 3000 mm/s, based on the results of our pilot study. In the present experiments, the depth and duration of the impact were kept constant at 0.5 mm and 50 ms, respectively. Most human spinal cord injuries involve tissue damage caused by rapid movements of the vertebral column and impact of bone against the spinal cord. A pneumatic impact device can produce a single, high-speed, and reproducible contusional impact to the spinal cord of laboratory mice.

Experimental Groups

Thirty mice were randomly divided into three groups of 10 animals each, the PBS group, ES group, and ES/BMSC group. Eight days after initiating SCI, mice in the PBS group received 3.0 μl of phosphate-buffered saline (PBS) into the injured site of the spinal cord to assess their natural course after injury, while the ES group received 3.0 μl of PBS containing 1.5 × 10⁴ ES-derived graft cells into the injured site of the spinal cord, and the ES/BMSC group received 3.0 μl of PBS containing 1.5 × 10⁴ ES cell-derived graft cells and 1.5 × 10⁴ BMSCs. Grouping and an outline of the experiment are shown in Figure 1B. The day of transplantation was designated as day 0.

Coculture of EBs with BMSCs

A total of 20 RA-treated EBs, prepared using a 4-day suspension culture with 5 × 10^–9 M of RA following a 4-day hanging drop culture, were cocultured in a 35-mm culture insert with 1 × 10⁶ BMSCs in 60-mm dishes across a 0.4-μm Millicell CM membrane (Millipore Corp., Bedford, MA) for 4 days.

RNA Extraction and Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis

Total cellular RNA was prepared using an acid guanidinium thiocyanate-phenol-chloroform method. Two micrograms of DNase-treated total RNA was used for the first-strand cDNA, and the reaction was performed with Super Script II and random hexa-nucleotide, following the protocol of the manufacturer (GIBCO/BRL). cDNA samples were subjected to PCR amplification with specific primers under linear conditions in order to reflect the original amount of the specific transcript. The cycling parameters were as follows: denaturation at 94°C for 1 min, annealing at 56–64°C (depending on the primer) for 45 s, and elongation at 72°C for 1 min (25 cycles). The PCR primers and lengths of the amplified products are shown in Table 1.

Table 1.

List of Primers

Genes	Primer Sequences	Product Size (bp)	GeneBank Accession No.	Temp. (°)
Gapdh	Forward: 5′-accacagtccatgccatcac Reverse: 5′-tccaccaccctgttgctgta	452	NM_008084	58
Oct-3/4	Forward: 5′-gccaggctcctgatcaacagcatca Reverse: 5′-atggctggacacctggcttcagact	1227	NM_013633	58
Utf1	Forward: 5′-cgtcgctacaagttcctcaa Reverse: 5′-aacgcggtattcaacgactg	485	NM_009482	56
Nanog	Forward: 5′-agggtctgctactgagatgctctg Reverse: 5′-caaccactggtttttctgccaccg	341	NM_028016	62
Sox2	Forward: 5′-gcctgggcgcggagtgga Reverse: 5′-gggcgagccgttcatgtaggtctg	443	NM_011443	64
ERas	Forward: 5′-actgcccctcatcagactgctact Reverse: 5′-cactgccttgtactcgggtagctg	210	NM_181548	60
Nestin	Forward: 5′-ggtctccctcgaatctctc Reverse: 5′-gatccaggcagctcccatt	1037	NM_016701	56
MAP2	Forward: 5′-tcagacttccaccgagcag Reverse: 5′-aggggaaagatcatggccc	776	MUSMAP2A	56
GFAP	Forward: 5′-tcgaatgactcctccactccct Reverse: 5′-tggccttctgacacggatttgg	1088	NM_010277	61
Oligo1	Forward: 5′-tttcccaggcgcgtgtgaac Reverse: 5′-agacaggtacttggtggggc	616	NM_016968	56
NGF	Forward: 5′-ctgtggaccccagactgttt Reverse: 5′-gcacccactctcaacaggat	354	NM_013609	58
GDNF	Forward: 5′-attttattcaagccaccatta Reverse: 5′-gatacatccacaccgtttagc	437	NM_010275	56
BDNF	Forward: 5′-tggctgacacttttgagcac Reverse: 5′-tcagttggcctttggatacc	357	NM_007540	56

To determine the existence of undifferentiated ES cells, the pluripotency-related transcription factors Oct4 (39,40), Utf1 (35), Nanog (6,30), and Sox2 (2), and the tumorigenicity-determining transcription factor ERas (43) were selected. Nestin, MAP2, GFAP, and oligo 1 were used to assess the differentiation of ES cells into NSC-like cells, neurons, astrocytes, and oligodendrocytes, respectively. Furthermore, the mRNA expressions of NGF, GDNF, and BDNF, neurotrophic factors in BMSCs, were examined.

Immunocytochemical Analysis of EB Cryosections In Vitro

EBs were cocultured with or without BMSCs and fixed in 4% paraformaldehyde. After collection using 0.5% agarose gel, they were embedded in OCT compound (Tissue-TEK, Miles, Elkhart, IN) and frozen in liquid nitrogen. Sections were then cut into 10-μm-thick slices using a cryostat and placed on 3-aminopropyltriethoxysilane-coated slides. After fixing in 100% ethanol and rinsing in PBS, they were incubated overnight at 4°C in anti-SSEA-1 (mouse monoclonal IgM antibody, 1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and anti-MAP2 (rabbit polyclonal antibody, 1:200, Santa Cruz Biotechnology Inc.) diluted in PBS with 0.1% Triton X-100. The sections were then rinsed in PBS and incubated in media containing fluorescent-labeled secondary antibodies (goat anti-mouse IgM antibody, 1:100, Santa Cruz Biotechnology Inc., and goat anti-rabbit antibody, 1:200, Molecular Probes, Eugene, OR) in PBS with 0.1% Triton X-100 for 60 min at room temperature. After rinsing again in PBS, the sections were mounted and cover-slipped in aqueous permanent mounting medium. Fluorescent staining was evaluated using a laser confocal microscope.

Immunocytochemical Analysis of BMSCs In Vitro

Cultured BMSCs were fixed in 4% paraformaldehyde. After rinsing in PBS, they were incubated overnight at 4°C in anti-NGF (rabbit polyclonal antibody NGF, 1:500, Santa Cruz Biotechnology Inc.) and diluted in PBS with 0.1% Triton X-100. Next, they were rinsed in PBS and incubated in medium containing a fluorescent-labeled secondary antibody (goat anti-rabbit antibody, 1:200, Molecular Probes) in PBS with 0.1% Triton X-100 for 60 min at room temperature. After rinsing again in PBS, the sections were mounted and cover-slipped in aqueous permanent mounting medium. Fluorescent staining was evaluated using a laser confocal microscope.

Behavioral Assessment of Mice

The effects of ES cell-derived NSC transplantation on behavioral improvement in the mice was assessed by four rating tests (motor scoring, platform hanging, mesh walking, and rope walking), which were conducted according to methods previously reported (41). Briefly, motor scoring was based on an open field test of coordinated motor function (0–6 points), platform hanging assessed the ability of the hind limbs to assist the mouse in climbing to the top of a platform when the forelimbs were placed on the platform edge and the animal was suspended 30 cm above soft bedding (0–4 points), mesh walking assessed the ability to navigate down a 38-cm hardware cloth held at a 35° angle into a box filled with soft bedding (0–4 points), and rope walking assessed the ability to navigate a 38-cm length of sisal rope 1.3 cm in diameter horizontally suspended 30 cm above soft bedding (0–4 points). Prior to these tests, the mice were trained and then randomly divided into groups for each examination. Behavioral functions were assessed weekly for 5 weeks after transplantation.

Immunohistochemical Analysis of Grafted Spinal Cord Cryosections

On day 35, the grafted mice were anesthetized terminally using an overdose of pentobarbital IP, after which the spinal cords were removed and postfixed for 24 h in 4% paraformaldehyde in PBS at 4°C, then sectioned. The sections were equilibrated in 10% sucrose in PBS for 4 h at 4°C, then 15% sucrose in PBS for 4 h at 4°C, and finally 20% sucrose in PBS overnight at 4°C. Next, they were embedded in Tissue-TEK (Miles) and frozen in liquid nitrogen. The sections were cut into 10-μm-thick slices using a cryostat and placed on 3-aminopropyltriethoxysilane-coated slides. After rinsing in PBS, they were incubated overnight at 4°C in anti-GFP (goat polyclonal antibody, 1:200, Santa Cruz Biotechnology Inc.), anti-GFAP (rabbit polyclonal antibody, 1:200, Santa Cruz Biotechnology Inc.), anti-MAP2, anti-nestin (rabbit polyclonal antibody, 1:200, Santa Cruz Biotechnology Inc.), anti-O4 (mouse monoclonal antibody, Chemicon International Inc., Temecula, CA), anti-SSEA-1, anti-Oct-3/4 (goat polyclonal antibody, 1:200, Santa Cruz Biotechnology Inc.), anti-NGF, and anti-BrdU (rat monoclonal antibody, 1:50, Oxford Biotechnology, Oxford, UK), then diluted in PBS with 0.1% Triton X-100. The sections were then rinsed in PBS and incubated in medium containing fluorescent-labeled secondary antibodies. The secondary antibodies included fluorescent-labeled goat anti-rat, goat anti-rabbit, and bovine anti-goat antibodies (1:200, Molecular Probes), and goat anti-mouse IgM antibody (1:100, Santa Cruz Biotechnology Inc.). After rinsing again in PBS, the sections were mounted and cover-slipped in aqueous permanent mounting medium. Fluorescent staining was evaluated using a laser confocal microscope.

Digital Image Acquisition

Digital images of microscopic fields of tumor tissue were acquired using a Zeiss microscope (Axiovert 40C, Carl Zeiss International, Oberkochen, Germany) and a color digital camera (AxioCamMRc, Carl Zeiss International). Maximum tumor areas in the spinal cord in sagittal sections were measured using the analysis software package AxioVision AC (V 4.5.0.0, Carl Zeiss International).

Statistical Analysis

Data are presented as the mean ± SD. Statistical analyses were performed using analysis of variance and Fisher's protected least significant difference (Stat View 4.0; SAS Institute Inc.; Cary, NC). Differences were considered statistically significant at p < 0.05.

Results

Presence of Undifferentiated ES Cells in ES Cell-Derived Graft Cells

Four-day EBs prepared by hanging drop cultures were subjected to a suspension culture for 4 days in the presence of RA at a concentration of 5 × 10^–9 M. According to previous reports that documented the differentiation of ES cells toward a neuronal lineage by RA (4,34,36), the concentration for efficient neural induction ranges from approximately 5 × 10^–7 to 5 × 10^–6 M. We chose a rather low concentration of RA in order to allow some of the ES cells to remain in an undifferentiated state. RT-PCR analysis of the ES cell-derived graft cells demonstrated the expressions of nestin, MAP2, GFAP, and oligo1, suggesting that they contained neural stem cells, neurons, astrocytes, and oligodendrocytes (Fig. 2A). However, several markers of an undifferentiated state, such as Oct3/4, Utf1, Nanog, Sox2, and ERas, were also observed. Furthermore, an immunocytochemical study showed that the cells contained SSEA-1-positive ES cells (Fig. 2B). SSEA-1 is known to be expressed on undifferentiated ES cells, and disappears as they begin differentiation and lose their pluripotency (9,13). These results indicated the presence of undifferentiated ES cells in the ES cell-derived graft cell population, because RA-treated EBs were used following enzymatic digestion into single cells.

Figure 2.

Presence of undifferentiated ES cells in RA-treated EBs. (A) RT-PCR analysis of undifferentiated ES cells (ES) and RA-treated EBs (RA-EBs). Four-day EBs prepared by hanging drop cultures were cultured for 4 days with RA at a concentration of 5 × 10^–9 M, which was a rather low concentration to allow efficient neural differentiation. The RA-EBs expressed not only markers of neural lineage such as nestin, MAP2, GFAP, and oligo1, but also several markers of an undifferentiated state, such as Oct3/4, Utf1, Nanog, Sox2, and ERas. (B) Immunocytochemistry of SSEA-1 expression by RA-EBs. The expression of SSEA-1, a marker of undifferentiated ES cells, by RA-EBs was examined. Immunocytochemical results showed that the RA-EBs contained SSEA-1-positive ES cells. Scale bar: 100 μm.

Behavioral Assessment

Total behavioral scores are shown in Figure 3. Mice in all groups showed gradual behavioral improvement during the first 3 weeks after transplantation. However, improvement ceased and behavior worsened after day 21 in the ES group. On day 35, mice in the ES group showed significant deterioration in motor scoring (Fig. 3A) compared to the ES/BMSC group, as well as in platform hanging (Fig. 3B) compared to the PBS group and ES/BMSC group. In addition, a significant worsening in total sum scores was observed on day 35 in the ES group (Fig. 3E) compared to the PBS group and ES/BMSC group. In contrast, mice in the PBS group and ES/BMSC group showed continuous improvement during the entire 5-week observation period following transplantation without deterioration. No significant differences were observed between the PBS group and ES/BMSC group at any of the observation points.

Figure 3.

Behavioral assessment. Behavioral improvement was assessed by four different rating tests (motor scoring, platform hanging, mesh walking, and rope walking) and then total behavioral scores were calculated. Mice in all groups showed gradual behavioral improvement during the first 3 weeks after transplantation. After day 21, improvement ceased and behavior worsened in the ES group, and those mice also showed significant deteriorations in motor scoring, platform hanging, and total sum scores on day 35. Mice in the PBS group and ES/BMSC group showed continuous improvement without deterioration during the entire 5-week observation period following transplantation. ^*p < 0.05.

Immunohistochemistry of Grafted Sites

Immunohistochemical examinations were performed on day 35 after macroscopic observation of the spinal cord. In all 10 mice in the ES group, macro- or microscopic examinations revealed formation of GFP-immunopositive tumors, indicating that they had originated from ES cells. Maximum tumor areas in the spinal cord in the sagittal sections were calculated using a digital method (Table 2). In this group, two of the mice had tumors with a maximum area between 1.0 and 2.0 mm², five had tumors with a maximum area between 2.0 and 3.0 mm², and three had tumors greater than 3.0 mm². Figure 4A and B shows a representative tumor with a maximum area of 2.07 mm², which formed at the grafted site and was GFP immunopositive. Various histological features were observed in this tumor, such as neural tube-like formation (Fig. 4C), densely proliferated cells (Fig. 4D), and intratumoral hemorrhage (Fig. 4E). Nestin-, MAP2-, and GFAP-immunopositive cells were scarcely found among the GFP-immunopositive cells (Fig. 4F), suggesting that the ES cell-derived tumor was composed largely of nonneural tissues. No evidence of directed differentiation along a neural lineage was observed in any of the tumors from the mice in the ES group, regardless of size. Next, we determined whether undifferentiated ES cells remained in those tumors. There were recognizable numbers of SSEA-1- and Oct3/4-immunopositive cells in the tumors (Fig. 4G), although in only a few of the sections. Of the five tumors examined, two had SSEA-1- or Oct3/4-immunopositive cells.

Figure 4.

Tumor development in ES group mice. (A, B) Histological and immunohistochemical examinations of the spinal cord at the grafted site on day 35. Tumors were recognizable in the spinal cords of all 10 ES group mice. Results from a representative tumor in a sagittal section, with a maximum area of 2.07 mm², are shown. (A) H&E staining, (B) immunostaining for GFP. Scale bars: 500 μm. (C, D, E) Disorganization of the spinal cord. The tumors contained various features, such as neural tube-like formation (C, arrowheads), densely proliferated cells (D), and intratumoral hemorrhage (E). Scale bar: 50 μm (H&E staining). (F) Failure of directed differentiation along a neural lineage. The differentiation of ES cell-derived graft cells into neurons, astrocytes, and neural stem cells was examined by double immunostaining with GFP and MAP2 (top), GFAP (middle), and nestin (bottom), respectively. MAP2-, GFAP-, and nestin-immunopositive cells were scarcely found among the GFP-immunopositive cells. Scale bar: 50 μm. (G, H) Existence of undifferentiated ES cells in the tumors. Recognizable numbers of SSEA-1-immunopositive cells (G) and Oct3/4-immunopositive cells (H) were found in the tumors.

Table 2.

Tumor Development in ES Group

	Area of Tumor at the Sagittal Section
	<1	1–2	2–3	>3
No. of mice	0	2	5	3

Tumor development was observed at the grafted site of the spinal cord in all of 10 ES group mice.

In contrast, there was no tumor formation seen in the grafted sites of the spinal cords in the ES/BMSC group, even with microscopic observation (Fig. 5A). However, GFP-immunopositive cells were found at the grafted sites in these mice (Fig. 5B) and there were mature neuron-like cells in the core area of transplantation (Fig. 5C). In the periphery of the grafted sites, accumulation of reactive astrocytes was prominent (Fig. 5D). Double-staining analysis also showed that most of the GFP-immunopositive cells were positive for either nestin or MAP2, while few were positive for GFAP (Fig. 5E) or O4, suggesting preferential differentiation of the grafted cells into NSC-like cells and mature neurons. There were no cells immunopositive for SSEA-1 or Oct3/4.

Figure 5.

No tumor formation in ES/BMSC group. (A, B) Histological and immunohistochemical examinations of the spinal cord at the grafted site on day 35. (A) H&E staining, (B) immunostaining for GFP. There were no tumors detected in the spinal cord of ES/BMSC group mice, although GFP immunopositivity was found. Scale bars: 500 μm. (C, D) Histological examination of grafted site. Mature neuron-like cells were seen in the core area of transplantation (C) and reactive astrocytes in the periphery of the grafted site (D). Scale bar: 50 μm (H&E staining). (E) Directed differentiation along a neural lineage. The differentiation of ES-derived graft cells into neurons, astrocytes, and neural stem cells was examined by double immunostaining with GFP and MAP2 (top), GFAP (middle), and nestin (bottom), respectively. Most of the GFP-immunopositive cells were immunopositive for either MAP2 or nestin, while few were positive for GFAP. Scale bar: 50 μm.

Immunocytochemical and RT-PCR Analyses of EBs Cocultured with BMSCs

To examine the effects of BMSCs on undifferentiated ES cells in ES cell-derived graft cells, the cells were cocultured with BMSCs across a membrane with 0.4mm sized pores for 4 days (Fig. 6A). The mRNA expressions of markers of an undifferentiated state (Oct3/4, Utf1, Nanog, Sox2, and ERas) were negative or faint after the coculture (Fig. 6B). Furthermore, an immunocytochemical study demonstrated the disappearance of SSEA-1 expression from the ES cell-derived graft cells after the 4-day coculture, while MAP2-immunopositive cells appeared at the periphery of growth after 4 days (Fig. 6C). These results suggest that coculturing with BMSCs promoted the neuronal differentiation of undifferentiated ES cells that had been retained in the ES cell-derived graft cells.

Figure 6.

BMSCs promote differentiation of undifferentiated ES cells in vitro. (A) Schema of coculture of RA-treated EBs and BMSCs. RA-treated EBs and BMSCs were cocultured across a 0.4-μm Millicell CM membrane for 4 days. (B) RT-PCR analysis of EBs cultured with or without BMSCs. Oct3/4, Utf1, Nanog, Sox2, and ERas were faint or not detected after the coculture with BMSCs. (C) Immunocytochemistry of EBs cultured with or without BMSCs. SSEA-1 expression disappeared after 4 days of coculture with BMSCs. MAP2-immunopositive cells appeared at the periphery of the EBs cocultured with BMSCs. Inset: Higher magnification of double-staining with MAP2 and DAPI. Scale bar: 100 μm.

Synthesis of Neurotrophic Factors of BMSCs In Vitro and In Vivo

To investigate the mechanism by which BMSCs promote the differentiation of undifferentiated ES cells retained among the ES cell-derived graft cells, we examined the synthesis of neurotrophic factors by BMSCs in vitro, because BMSCs have been reported to produce NGF, GDNF, and BDNF. As expected, mRNA expressions of NGF, GDNF, and BDNF were observed in BMSCs (Fig. 7A). Furthermore, NGF was also confirmed in cultured BMSCs in an immunocytochemical examination (Fig. 7B), although we did not perform such experiments for determining the expression of GDNF or BDNF.

Figure 7.

Production of neurotrophic factors by BMSCs in vitro and in vivo. (A) RT-PCR analysis of BMSCs. mRNA expressions of NGF, GDNF, and BDNF were observed in the BMSCs. M: molecular weight marker. (B) Immunostaining for NGF in BMSCs. Scale bar: 10 μm. (C) Double immunostaining for NGF and BrdU in the spinal cord at the grafted site. BMSCs were pulsed with BrdU in vitro before transplantation. The estimated area of transplantation is shown by the outline. Double immunopositive cells at the grafted site are shown in the insets. Scale bars: 20 μm.

Next, we examined whether the grafted BMSCs survived in the spinal cord for 5 weeks after transplantation and whether the synthesis of neurotrophic factors was maintained. Because the BMSCs were pulsed with BrdU before transplantation, determination of their presence in the spinal cord was performed immunohistochemically by detecting BrdU-immunopositive cells. BrdU immunopositivity was seen in the spinal cord (Fig. 7C, BrdU) and most of the BrdU-immunopositive cells were restricted to the grafted sites, although some existed in the periphery outside of the grafted sites. Double-staining for BrdU and NGF showed that NGF production was maintained in the grafted BMSCs (Fig. 7C).

Discussion

A traumatic SCI results in severe damage, leading to paraplegia, tetraplegia, or worse. Currently, no effective medical therapy is available for patients with an SCI, though one of the proposed treatment strategies involves resupplying the cells that were damaged or lost with progenitor or stem cells. ES cells are thought to represent a potent resource for such cell transplantation therapy, although there are a number of technical hurdles, including tumor formation, to be overcome before clinical trials can be commenced.

For clinical application, the safety of ES cell-based cell therapy needs to be improved. In the present study, we performed simultaneous cotransplantation of BMSCs and ES cell-derived cells into SCI model mice in an attempt to prevent the development of ES cell-derived tumors. Tumors developed in mice that received ES cell-derived graft cells alone, while no tumor development was observed in those that underwent cotransplantation with ES cell-derived graft cells and BMSCs. The tumors were composed of GFP-immunopositive cells, indicating that they had originated from the grafted ES cells. These results suggest that the cotransplanted BMSCs prevented tumor development by the ES cells.

The most critical factor in development of ES cell-derived tumors is the presence of undifferentiated ES cells among the graft cells. We prepared the present ES cell-derived graft cells by EB formation for 4 days and subsequent culturing for 4 days in the presence of RA at a concentration of 5 × 10^–9 M, which was rather low for optimal differentiation along a neuronal lineage (4,34,36). It was previously reported that nearly 90% of 8-day-old EBs treated with 5 × 10^–6 M of RA during the final 4 days showed nestin immunopositivity (4,36). Although we did not examine nestin immunopositivity in the ES cell-derived graft cells in the present study, we considered that the proportion of those cells would be much less than that of EBs treated with 5 × 10^–6 M of RA. Another report that documented the effects of RA concentrations on differentiation of mouse ES cells (34) noted that a low concentration of RA (10^–9–10^–8 M) induced not only ectodermal, but also mesodermal and endodermal, differentiation. Furthermore, we anticipated the existence of undifferentiated ES cells among the ES cell-derived graft cells and confirmed their existence by detection of mRNA expressions of Oct3/4, Utf1, Nanog, Sox2, and ERas, as well as the presence of SSEA-1-immunopositive cells. Considering the differentiation method adopted in the present study and the presence of undifferentiated ES cells among the graft cells, it is considered normal that tumor development or uncontrolled cell growth occurred in the mice that received ES cell-derived graft cells.

In contrast, no tumor development was observed in mice cotransplanted with ES graft cells and BMSCs, regardless of the presence of undifferentiated ES cells among the grafts at a sufficient amount to induce tumor formation when transplanted alone. Although the entire mechanism of suppressed tumor development following cotransplantation remains to be elucidated, we considered that the BMSCs played an important role to prevent the development of tumors. To clarify that, we investigated the effects of BMSCs in vitro on the differentiation of undifferentiated ES cells remaining in the grafts. After coculturing with BMSCs across a membrane that permitted communication of humoral factors, mRNA expressions of Oct3/4, Utf1, Nanog, Sox2, and ERas, markers of an undifferentiated state, were negative or faint, and SSEA-1-immunopositive cells disappeared, whereas MAP2-immunopositive cells appeared. These results strongly indicate a neuronal differentiation-promoting effect of BMSCs on undifferentiated ES cells.

Based on our results, we considered how BMSCs may act on undifferentiated ES cells and promote their differentiation. It has been reported that BMSCs secrete some neurotrophic factors, such as NGF (7,14), GDNF (7,14,50), and BDNF (7), and also that they promote neuronal differentiation of fetal spinal cord-derived neurosphere cells (48) and mesencephalic neural stem cells (25). Therefore, we speculated that one of the mechanisms by which BMSCs promote the differentiation of ES cells is related to secreted soluble factors, including neurotrophic factors. In the present study, we found mRNA expressions of NGF, GDNF, and BDNF, as well as NGF production in cultured BMSCs using an immunocytochemical method. Furthermore, we observed that transplanted BMSCs survived in the grafted site for at least 5 weeks after transplantation and maintained an ability to produce NGF. From these results, we consider it very likely that the cotransplanted BMSCs promoted neuronal differentiation of the undifferentiated ES cells that were retained among the ES cell-derived graft cells. Recently, it was reported that transplanted BMSCs attenuated the loss of neurons in experimental brain injury models by the production of neurotrophic factors (7). Thus, in addition to promoting differentiation along a neuronal lineage, BMSCs might also help ES cell-derived neurons survive following differentiation through the production of neurotrophic factors.

BMSCs have also been reported as potential candidates in other transplantation therapies for various CNS diseases, in addition to SCI. Many investigators have demonstrated that transplanted BMSCs are able to trans-differentiate into cells with neural characteristics (5,24,26,29). However, the proportion of BMSCs that undergo differentiation in the brain was reported to be very small (5,24,29). In addition to their limited ability to replace injured CNS tissue, expression of neural markers does not necessarily mean that these cells are capable of functioning as mature neurons. In contrast, it is widely accepted that ES cells have an ability to differentiate into neurons in vitro as well as in vivo after transplantation into the CNS. The beneficial effects of ES cell transplantation are considered to be due to their replacement of lost tissues or cells, regardless of defective reconstitution that is far from complete regeneration of the CNS. However, one of the obstacles to overcome in ES cell-based cell therapy is associated tumor development. As a possible new approach to prevent the development of ES cell-derived tumors, we propose adding BMSCs to ES cell-based cell transplantation. Cotransplantation of BMSCs and ES cells for treatment of experimental SCI in the present study led to the prevention of ES cell-derived tumor development by the cotransplanted BMSCs, possibly via their production of neurotrophic factors, which led to sustained behavioral improvement.

Previously, we demonstrated that transplantation of mouse ES cell-derived ES NSCs, selectively prepared by a step-by-step induction method (23,31), was effective and useful to treat SCI model mice (21). The experiments were performed using a 129/SvJ → 129/SvJ syngeneic combination and no tumor development was observed. In contrast, the SCI mice used in the present study were the C57BL/6 strain. Although ES cells derived from 129/SvJ mice could develop into tumors in nonimmunosuppressed C57BL/6 mice, an allogeneic immunological barrier might potentiate the inhibition of tumor development. Therefore, we think that more extensive experiments using a syngeneic donor–host combination is required before concluding the usefulness of cotransplantation therapy for the prevention of ES cell-derived tumor development. Although there is no current consensus regarding the ideal source for cellular grafts in cellular transplantation strategies, a practical method for treating SCI in the future may include the use of ES cell-derived cells, such as ES cell-derived NSCs, after selective induction and co-transplantation with BMSCs.

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