Cytotherapy, 2010; 12: 455–465

ISSN 1465-3249 print/ISSN 1477-2566 online © 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)DOI: 10.3109/14653240903555827

∗These two authors contributed equally to this work. Correspondence : Dr Shiaw-Min Hwang , Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu, 30062 Taiwan. E-mail hsm@fi rdi.org.tw

(Received 7 July 2009; accepted 14 December 2009)

Establishment of immortalized mesenchymal stromal cells with red fl uorescence protein expression for in vivo transplantation and tracing in the rat model with traumatic brain injury

CHI-JEN HUNG 1 ∗, CHAO-LING YAO 1,2 ∗, FU-CHOU CHENG 3 , MEI-LING WU 1 , TZU-HAO WANG 4 & SHIAW-MIN HWANG 1

1 Bioresource Collection and Research Center, Food Industry Research and Development Institute, Taiwan, 2 Department of Chemical Engineering and Materials Science, Yuan Ze University, Chung-Li, Taiwan, 3 Department of Medical Research, Veterans General Hospital, Taichung, Taiwan, and 4 Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan

AbstractBackground aims . Human mesenchymal stromal cells (hMSC) play a crucial role in tissue engineering and regenerative medicine, and have important clinical potential for cell therapy. However, many hMSC studies have been restricted by lim-ited cell numbers and diffi cult detection in vivo . To expand the lifespan, hMSC are usually immortalized by virus-mediated gene transfer. However, these genetically modifi ed cells easily lose critical phenotypes and stable genotypes because of insertional mutagenesis . Methods . We used a non-viral transfection method to establish human telomerase reverse tran-scriptase-immortalized cord blood hMSC (hTERT-cbMSC). We also established red fl uorescent protein (RFP)-expressing hTERT-cbMSC (hTERT/RFP-cbMSC) by the same non-viral transfection method, and these cells were injected into a rat model with traumatic brain injury for in vivo detection analysis. Results. The hTERT-cbMSC could grow more than 200 population doublings with a stable doubling time and maintained differentiation capacities. hTERT/RFP-cbMSC could proliferate effi ciently within 2 weeks at the injury location and could be detected easily under a fl uorescent microscope. Importantly, both hTERT-cbMSC and hTERT/RFP-cbMSC showed no chromosomal abnormalities by karyotype analysis and no tumor formation in severe combined immunodefi cient (SCID) mice by transplantation assay. Conclusions . We have developed immortalized cbMSC with hTERT expression and RFP expression, which will be useful tools for stem cell research and translational study.

Key Words: mesenchymal stromal cells , non-viral transfection , red fl uorescent protein , telomerase reverse transcriptase , traumatic brain injury

Introduction

Human mesenchymal stromal cells (hMSC) hold attraction for tissue engineering, regenerative medicine and cell therapy because of their multiple differentia-tion abilities, no ethical issues and fewer immunologic problems (1,2). In addition, hMSC can be isolated from various tissues such as bone marrow (BM), cord blood (CB), cord tissue, adipose tissue, skeletal muscle and amniotic fl uid (3–7). However, long-term culture of hMSC in current culture conditions results in replicative senescence, limited proliferation ability (less than 40 population doublings; PD) and loss of differentiation capacity (8–10). Thus it is diffi cult to obtain enough numbers of functional hMSC for stem cell study and translational research.

Previous studies have shown that the telomere is relative to the lifespan of the normal somatic cell. Telomerase, a ribonucleoprotein, can maintain telomere length in cells. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase and controls the activity of telomerase (11,12). It has been showed that the lifespan of normal human fi broblasts can be extended by forc-ing hTERT expression, and immortalization with a stable karyotype has been accompanied by increasing or keeping the telomere (13,14).

hTERT-immortalized MSC are usually estab-lished by using retroviral or lentiviral vectors to deliver DNA into cells because of their high gene-transferring effi ciency (15–19). However, some disadvantages,

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such as viral vector-induced potential mutations and insertional mutagenesis, need to be overcome (20–22). Non-viral transfection methods, such as cationic polymer transfection, calcium phosphate precipita-tion, electroporation and lipofection can establish transient and stable exogenous cells without muta-genesis, but with low transfection effi ciency and high cell mortality (23,24). Therefore, the establishment of hTERT-immortalized hMSC by non-viral trans-fection methods is an important issue for stem cell study and translational research.

In this study, we have established hTERT- immortalized hMSC derived from cord blood (hTERT-cbMSC), and hTERT-cbMSC co- expressing red fl uorescent protein (hTERT/RFP-cbMSC), by non-viral polymer transfection. In comparison with primary cbMSC, hTERT-cbMSC have high hTERT expression and can extend their lifespan over 200 PD. In addition, hTERT-cbMSC maintain similar characteristics to primary cbMSC, includ-ing surface marker expression and differentiation abilities. Regarding in vivo tracing and detection, we are the fi rst to report the injection of hTERT/RFP-cbMSC into rat models with traumatic brain injury (TBI). After scarifi cation at set points, hTERT/RFP-cbMSC were found to proliferate effi ciently within 2 weeks at the injury location and could be detected easily under a fl uorescence microscope. Both hTERT-cbMSC and hTERT/RFP-cbMSC showed no chromosomal abnormali-ties by karyotype analysis and no tumor formation in severe combined immunodefi cient (SCID) mice by transplantation assay. We have established immor-talized cbMSC with hTERT expression and RFP expression, which would be useful tools for stem cell study and translational research.

Methods

Cell culture

cbMSC were collected and developed as described previously (25). cbMSC culture medium was composed of minimum essential medium alpha-modifi cation (α-MEM; Hyclone, Logan, UT, USA), 20% fetal bovine serum (FBS; Hyclone) and 4 ng/mL basic-fi broblast growth factor (bFGF; R&D Systems, Minneapolis, MN, USA).

Non-viral transfection of cbMSC

When cbMSC were cultured to reach 60–70% confl uence, they were treated with the hTERT expression vector pGRN145 (American Type Cul-ture Collection, Manassas, VA, USA), and jetPEI™ reagent (Polyplus Transfection, Illrich, France) for immortalization following the manufacturer’s

instructions. After a 5-h treatment, the cells were washed and cultured in fresh culture medium for 48 h. The treated cells were then subcultured in culture medium containing 30 μg/mL Hygromy-cin B (Invitrogen, Carlsbad, CA, USA). The drug-resistant and stably growing transfected cell line, hTERT-cbMSC (polyclonal), was designed as PD1 for subsequent characterization. The hTERT-cbMSC cell lines were also co-transfected with RFP expression vector, pDsRed-N1 (Clontech, Palo Alto, CA, USA), for the generation of hTERT/RFP-cbMSC. To isolate single-cell hTERT/RFP-cbMSC, the transfected cells were also cultured in growth medium with 30 μg/mL Hygromycin B and 200 μg/mL Geneticin (Invitrogen) by limiting dilu-tion and selected under fl uorescent microscopy to make sure of RFP expression.

RNA preparation, cDNA synthesis and reverse transcription–polymerase chain reaction analysis

Total cellular RNA was extracted by using Trizol reagent (Invitrogen) according to the manufactur-er’s instructions. Briefl y, reverse transcription (RT) of RNA was performed with oligo (dT) primers and RevertAid H Minus M-MuLV reverse tran-scriptase (MBI Fermentas, Vilnius, Lithuania). In order to display hTERT expression, the following primers were used: hTERT (product size 146 bp, forward 5′-CGGAAGAGTGTCTGGAGCAA-3′ and reverse 5′-GGATGAAGCGGAGTCTGGA′3) and β-actin (as internal control, product size 396 bp, forward 5′-TGGCACCACACCTTCTACAATGAGC-3′ and reverse 5′-GCACAGCTTCTCCTTAATGTCACGC-3′). The polymerase chain reac-tion (PCR) was programmed with pre-heating at 94°C for 2 min, 35 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, and 72°C for 5 min, then kept at 4°C. The PCR products were analyzed by 1.5% agarose gel electrophoresis.

Telomere repeat amplifi cation assay

Telomerase activity was determined by using a Chemicon TRAPeze® XL detection kit (Chemicon, Temecula, CA, USA) following with the manufac-turer’s instruction. Briefl y, cells were trypsinized, lysed in CHAPS buffer with RNase inhibitor (MBI Fermentas) on ice for 30 min, and then centrifuged at 12 000 g for 20 min at 4°C; 800 ng protein of each sample were used for the telomere repeat amplifi cation (TRAP) assay. The external positive control was TSR8, expressed as telomerase activity by total product-generated (TPG) unit. The logarith-mic plot of the TSR8 standard curve was plotted by net fl uorescence increase against the correspond-

Establishment of immortalized MSC with RFP expression 457

ing TPG unit of TSR8 ( r 2 �0.99). The TPG unit of each sample was obtained from the standard curve by using the relative net fl uorescence increase as kit procedures.

Long-term cell culture

In vitro long-term cell growth ability was determined by calculating cumulative PD level. When the cells reached 80–90% confl uence, they were trypsinized and counted by hemocytometer for PD calculation. Cumulative PD indicated the sum of PD, and the PD time (PDT) was also calculated.

Flow cytometry analysis

For surface marker analysis, the primary cbMSC and hTERT-cbMSC at different passages were trypsinized and suspended in phosphate-buffered saline (PBS; Gibco BRL, Rockville, MD, USA). Cells were immunolabeled with the following primary antibod-ies: CD14 and CD19 (Chemicon); CD34 and CD45 (Miltenyi Biotec, Bergisch Gladbach, Germany); CD90 and CD105 (R&D Systems); CD29, CD31, CD44, CD73, CD117, HLA-A, -B, -C and HLA-DR (Becton-Dickinson, San Jose, CA, USA); and mouse IgG (Becton-Dickinson) as isotype control. The anti-mouse IgG–fl uorescein isothiocyanate (FITC)

or IgG–phycoerythrin (PE) (Beckman Coulter, Hialeah, FL, USA) was used as the secondary antibody. Data were analyzed using FACSCalibur fl ow cytom-etey with CellQuest software (Becton-Dickinson).

In vitro differentiation towards osteogenic and adipogenic cells

The differentiation potential of cells was examined using a differentiation–induction protocol and dif-ferentiation assay described previously (25).

Karyotype analysis

For karyotype analysis, 30 chromosomal spreads per sample were captured and analyzed by standard G-banding techniques. Briefl y, cells at the exponen-tial phase in metaphase were arrested with 0.05 mg/mL colcemid (Invitrogen) for 1 h and then harvested by standard cytogenetic methods of trypsin dispersal, hypotonic shock with 0.075 M KCl and fi xa tion with 3:1 methanol/acetic acid fi xative. Slides for chromosome painting were dehydrated sequentially with 70%, 90% and 100% ethanol for 10 min each at room temperature and were then stored at –20°C until use. Cytogenetic analysis was performed with Wright’s stain (Sigma, St Louis, MO, USA). The descriptions of karyotypes

Figure 1. Analyzes of growth kinetics, telomerase gene expression and telomerase activity of primary cbMSC and hTERT-cbMSC. (A) The lifespan. The primary cbMSC stopped proliferating at PD25 and the hTERT-cbMSC grew stably until PD201. (B) The PDT. Primary cbMSC kept a constant PDT before PD14 but increased signifi cantly thereafter. The hTERT-cbMSC could grow with stable PDT until PD201. (C) hTERT gene expression was detected by RT-PCR. P, pGRN145 vectors as positive control; N, negative control. (D) Telomerase RT activity was detected by TRAP assay and shown as TPG unit. HeLa cells were set as a positive control.

458 C.-J. Hung et al.

Tumorigenicity assay

Exponentially growing cells were harvested at a den-sity of 2 � 10 6 cells/100 μL PBS, and then 2 � 10 6 cells injected subcutaneously per SCID mouse (6–8 weeks old, female, BALB/c strain) to evaluate their tumorigenic potential.

Transplantation of hTERT/RFP cbMSC in rats with TBI

Adult male SD rats (250–350 g) were housed indi-vidually under controlled environmental conditions (12/12 h light/dark cycle), with food and water avail-able before being used in the experiments. The rats were gas anesthetized with isofl uroane, and the body temperature maintained at 37°C with a heating pad (CMAz/150). TBI was induced with a dragonfl y fl uid percussion device (model HPD-1700; Drag-onfl y R&D, Silver Spring, MD, USA). Three days after the brain contusion, the animals were trans-planted with 1 � 10 5 hTERT/RFP cbMSC into the subventricle zone (SVZ). Some rats were killed at 7 days and some rats were killed at 14 days after trans-plantation, and brain slices were examined under a fl uorescence microscope. All animal experiments were performed in accordance with institutional guidelines approved by the animal ethical committee of the Veterans General Hospital (Taichung, Taiwan).

followed the International System for Human Cyto-genetic Nomenclature (ISCN:2009, Karger Ag, Basel, http://atlasgeneticsoncology.org/ISCN09/ISCN09.html).

Microarray profi ling of array-based comparative genomic hybridization analysis

Genomic DNA (250 ng) was used for array compara-tive genomic hybridization (CGH) using Affymetrix 10K mapping chips. CGcgh, a software program for CGH analysis using microarray data, was used for data analysis. Briefl y, on each chromosome, the CGcgh program scanned the genomic regions with a user-defi ned window size (the default value is 50 probes) and performed sequential Student’s t -tests for determining whether each scanning genomic region and the whole genome (except chromosome X) had the same mean values. The CGcgh program slid the window of genomic region along all the chromosomes and plotted the base 10 logarithm of the P -value. For the mean value of a window to be statistically signifi -cantly less than the mean values of the whole genome, CGcgh designated it with a negative log P -value. On the other hand, a positive P -value was given to the mean values signifi cantly greater than the mean values of the whole genome. The CGcgh program can also display the G-banding ideogram of each chromosome at an 850-band resolution.

Figure 2. The morphologies of primary cbMSC and hTERT-cbMSC. (A) primary cbMSC and (B) hTERT-cbMSC are shown at different PD. The primary cbMSC appeared as normal fi broblast-like cells before PD14, and then soon showed senescence morphology. The immortalized hTERT-cbMSC could keep a normal fi broblast morphology until PD201.

Establishment of immortalized MSC with RFP expression 459

Figure 1 to http://www.informahealthcare.com/cyt/ (DOI: 10.3109/14653240903555827)). The optimal selection concentration of Hygromycin B was deter-mined as 30 μg/mL as a result of the appropriate selection of cbMSC without containing pGRN145 vector.

Analyzes of growth kinetics, telomerase gene expression, telomerase activity and morphologies of primary cbMSC and hTERT-cbMSC

PD, PDT, hTERT gene expression and telomerase activity of primary cbMSC and hTERT-cbMSC were analyzed. Primary cbMSC had no obvious hTERT expression (PD5 and PD10) and telomerase activity (PD14 and PD30) and had almost stopped growth in culture ex vivo within 43 days (Figure 1A). After hTERT transfection, the hTERT-cbMSC maintained

Results

Generation of hTERT-immortalized cbMSC by non-viral transfection

Different ratios of transfection reagent to DNA were compared to establish an effi cient non-viral transfection protocol for cbMSC. Our prelimi-nary experimental results showed that the highest transfection effi ciency and cell survival rate could be obtained when the ratio of jetPEI™ to DNA was 1:2 and the treatment time 5 h. For effi cient selection of cells transfected with pGRN145 vec-tor that expressed both hTERT and Hygromy-cin B resistance gene, the sensitivity of cbMSC to different concentrations of drug was tested by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) assay (Supplementary

Figure 3. Cell-surface marker profi les of cbMSC and hTERT-cbMSC. The surface markers were analyzed by fl ow cytometry with antibodies against the indicated antigens on (A) primary cbMSC at P18, (B) hTERT-cbMSC at P16 and (C) hTERT-cbMSC at P201. The representative isotype control is shown as a dotted line.

460 C.-J. Hung et al.

gradually grew slower during PD15–24 (PDT 54.2 � 7.4 h) and then virtually stopped growth at PD25–26 (PDT 142.8 � 2.0 h) and had signifi -cant senescent and aging morphology at PD29. In contrast, hTERT-cbMSC proliferated with a stable PDT (23.8 � 1.9 h) and maintained fi broblast-like morphology for 200 PD, as primary cbMSC had with early PD (before PD14).

Comparison of cell-surface antigen expression between primary cbMSC and hTERT-cbMSC

The surface antigen analysis of hTERT-cbMSC (at PD16 and PD201) and primary cbMSC (at PD18) was detected by fl ow cytometry (Figure 3). All cells mentioned above had similar profi les of surface

stable growth over 190 days. It should be noted that hTERT gene expression was obviously high in cbMSC at PD15 but decreased continuously with PD increase (PD138 and PD201) (Figure 1C). The trend of telomerase activity in hTERT-cbMSC was consistent with the trend of hTERT gene expression (Figure 1D). These results supported the fact that the transgene was kept in plasmid and did not insert into the genome. The telomerase activity as TPG units at PD8, PD129 and PD201 was 267.9 � 38.0, 73.4 � 18.4 and 17.0 � 1.7, respectively. The down-regulation of hTERT expression may reduce the risk of malignant transformation. Growth kinetics and morphologies of cbMSC were also monitored (Figure 1B and Figure 2). Primary cbMSC main-tained stable growth before PD14 (PDT 28 � 3 h),

Figure 4. Differentiation ability of cbMSC and hTERT-cbMSC. Adipogenic (A – D, I – K) and osteogenic (E – H, L – N) differentiation abilities of primary cbMSC at PD5, hTERT-cbMSC at PD201 and hTERT/RFP-cbMSC at PD32. The adipogenic capability was detected by lipid droplet accumulation by Oil red O stain after a 30-day induction (B, D, J). Osteogenic capability was characterized with mineral nodules by Alizarin red S stain after a 30-day induction (F, H, M). The adipocytes (K) and osteocytes (N) that were induced from hTERT/RFP-cbMSC were imaged under a fl uorescence microscope and expressed red fl uorescence.

Establishment of immortalized MSC with RFP expression 461

Generation of RFP expressing hTERT-cbMSC for TBI experiments and in vivo detection

In order to detect hTERT-cbMSC easily in vivo , RFP-expressing hTERT-cbMSC (hTERT/RFP-cbMSC) were established. The results showed that in vitro osteogenic and adipogenic differentiation abilities were maintained in the hTERT/RFP-cbMSC (Figure 4I, J, L and M) and both hTERT/RFP-cbMSC and their induced osteoblasts and adipocytes could be detected by expression of red fl uorescence under a fl uorescence microscope (Figure 4K, N). We also transplanted hTERT/RFP-cbMSC (PD35) into rat models with TBI for in vivo detection. After transplantation, the rats were killed at days 2, 7 and 14, and the brain tissues of the rats were cryosectioned at 10-μm thickness for observation under a fl uorescence microscope (Fig-ure 5). At day 2 after transplantation, the distri-bution of hTERT/RFP-cbMSC could be detected easily under the fl uorescence microscope and were found in the lateral ventricle (Figure 5A–C). Subsequently, these cells had moved up the right

antigens. They were all negative for CD14, CD19, CD31, HLA-DR, CD117, CD34 and CD45, and positive for CD105, CD73, CD29 (adherent mol-ecules), CD44 and HLA-ABC.

Comparison of in vitro differentiation capacity between primary cbMSC and hTERT-cbMSC

Primary cbMSC and hTERT-cbMSC were induced in the osteogenic and adipogenic induction media for differentiation potential assays. After adipogenic induction, the results showed that primary cbMSC (PD5) and hTERT-cbMSC (PD201) had a compa-rable ability to differentiate into adipocytes within 30 days (Figure 4A–D), although the formation of lipid droplets was rarely observed. This was because cbMSC has a low ability to differentiate into adipocytes [25]. After osteogenic induction, both primary cbMSC (PD5) and hTERT-cbMSC (PD201) maintained a similar ability to differenti-ate into osteoblasts within 30 days and generated a large amount of mineral nodules (Figure 4E–H).

Figure 5. In vivo detection of hTERT/RFP-cbMSC in the rat model with TBI. After 3 days of brain contusion, TBI was induced in rats. The rats were transplanted with 1 � 10 5 hTERT/RFP cbMSC into the SVZ and then were killed at 2 days (A – C), 7 days (D – F) and 14 days (G – I) after transplantation, and the brain slices examined under a fl uorescence microscope. Schematic diagrams of brain slices are shown at (A) day 2, (D) day 7 and (G) day 14; (B, E, H) (light phase) and (C, F, I) (fl uorescence phase) were observed under a fl uorescence microscope and zoomed in from the area by squaring of the red line in (A, D and G), respectively. The red fl uorescent-expressing cells were found in the lateral ventricle zone (LVZ) at day 2 after transplantation and were found in the upper right of LVZ at day 7. The RFP-expressing hTERT-cbMSC could be observed proliferating at the damaged region of LVZ at day 14.

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some numbers of both genetically modifi ed cbMSC were found to be 46. Array CGH analysis of hTERT-cbMSC (PD132) and hTERT/RFP-cbMSC (PD35) was also performed (Figure 7) and no genomic deletions or amplifi cations were detected.

Discussion

hMSC have been demonstrated that can be induced to differentiate into cells of mesodermal lineage, such as osteoblasts, chondrocytes and adipocytes [25]. Therefore, hMSC play a crucial role in tissue engineering and regenerative medicine, and have important clinical potential for cell therapy. How-ever, hMSC studies have been restricted by limited cell numbers and diffi cult detection in vivo . Cell num-ber is the main issue for stem cell study and trans-lational research. Most in vivo studies have shown that MSC typically exhibit low levels of engraft-ment within injured tissue as a result of limited cell numbers, and therefore do not contribute physi-cally to tissue regeneration to a signifi cant extent (26). How to trace transplanted stem cells in vivo is another critical issue. Thus it is important to establish immortalized hMSC with (or without) a label marker as a cell-based tool for stem cell study and translational research.

The most effi cient DNA delivery system for primary cells uses viral-based techniques (27). However, random insertion mediated by retro-viral vectors may result in mutation in the host, leading to carcinogenesis (28). Adenoviral vectors have already been called into question because of the potential for severe patient reactions (29). Although recent evidence suggests that lentiviral vectors could be less likely to activate unwanted gene expression in hMSC (18), safety issues are still a concern because of their high similarity to retroviral vectors (30).

Non-viral gene transfer is dependent on chemical or physical delivery of the genetic mate-rial into the host cell. Most non-viral methods are considered relatively low in immunogenicity and toxicity compared with viral vectors. However, a disadvantage of non-viral gene transfer is usually low-transfection effi ciency. We have successfully established two immortalized cell lines, hTERT-cbMSC and hTERT/RFP-cbMSC, to express hTERT with or without RFP by non-viral polymer transfection. The hTERT-cbMSC and hTERT/RFP-cbMSC could grow over 200 OD with a stable doubling time (Figure 1), maintaining a fi broblast-like morphology (Figure 2), correct expression of MSC markers (Figure 3) and similar differentia-tion capacities (Figure 4) as primary cbMSC with early PD.

of the lateral ventricle at day 7 after transplanta-tion (Figure 5D–F) and expanded in this region at day 14 after transplantation (Figure 5G–I). The results showed that hTERT/RFP-cbMSC were effi ciently attracted to the injury area and signifi cantly proliferated within 2 weeks in the rat model of TBI.

Evaluation of potential neoplastic transformation

In this study, the tumorigenic potential of hTERT-cbMSC (PD132) and hTERT/RFP-cbMSC (PD35) was evaluated by injection of these cells into SCID mice (6–8-week-old, female, BALB/c strain). After 6 months of observation, no tumor formation was found (data not shown).

Genomic stability of hTERT-cbMSC and hTERT/RFP-cbMSC

Karyotypic analysis of hTERT-cbMSC (PD132) and hTERT/RFP-cbMSC (PD35) was performed (Figure 6). These cells were both diploid and did not exhibit signifi cant chromosomal abnormalities. The chromo-

Figure 6. G-banded chromosome analysis of hTERT-cbMSC and hTERT/RFP-cbMSC. Both hTERT-cbMSC at PD132 (A) and hTERT/RFP-cbMSC at PD35 (B) had a normal diploid karyotype.

Establishment of immortalized MSC with RFP expression 463

useful tool and can provide useful information for in vivo detection in the animal model.

Long-term safety is a big issue for gene-modifi ed cells (37). However, it is still controversial regarding whether telomerase is an oncogene. Although some studies have shown that telomerase can be considered a candidate for neoplastic transformation because of a strong link between telomerase expression and many cancer cell types (38), it has also been shown that telomerase-immortalized cells have no malignant transformation and genetic abnormalities (14,39). In addition, some studies have shown that hTERT is not an oncoprotein and does not induce tumor formation (39). The roles of telomerase in cancer development still need to be clarifi ed. In our stud-ies, the high hTERT gene expression and enzyme activity in hTERT-cbMSC gradually decreased as PD increased, and this may minimize the risk of malignant transformation. In addition, our analysis showed that the established cells had no tumor for-mation in SCID mice and there were no signifi cant chromosome abnormalities. These results show that hTERT-cbMSC and hTERT/RFP-cbMSC could served as excellent cell-based tools for stem cell research and translational study.

Acknowledgments

The authors gratefully acknowledge the fi nancial sup-port of this study by the Food Industry Research and Development Institute, Taiwan (07G291-04) and the Ministry of Economic Affairs, Taiwan (96-EC-17- A-99-R1-0643).

Many studies have tried to assess the fate of MSC administered in vivo and their effects on disease progression in experimental animal mod-els by specifi c marker detection, such as in situ hybridization (31), ectopic lacZ gene expres-sion by X-gal staining (32), iron fl uorophore particle (IFP) by magnetic resonance imag-ing (MRI) (33) and fl uorescent protein by fl uo-rescence microscope observation (34). In situ hybridization and X-gal staining need complex procedures to analyze. Although the IFP-labeled cells have a high tracing sensitivity in vivo , it is not practical because of high costs. In order to detect cells easily at a low cost, we selected RFP to label hTERT-cbMSC for in vivo detection.

TBI, predominantly caused by falls and motor vehicle accidents, is the leading cause of death and severe disability in people under 45 years of age (35). Although TBI is a problem of major medi-cal and socioeconomic signifi cance (36), it is often diffi cult to reconstruct the events leading to pri-mary and secondary lesions of varying severity and regional distribution that constitute TBI. We believe that rat with TBI is a good animal model for stem cell research and translational research, especially regarding stem cell transplantation and in vivo detection. In our study, we injected hTERT/RFP-cbMSC into rat with TBI. After transplanta-tion, hTERT/RFP-cbMSC could be detected easily under a fl uorescence microscope and were found in the lateral ventricle initially. Subsequently, these cells moved and proliferated in the region of injury. These results show that hTERT/RFP-cbMSC is a

Figure 7. Microarray profi ling of hTERT-cbMSC and hTERT/RFP-cbMSC by array CGH analysis. Both hTERT-cbMSC at PD132 (A) and hTERT/RFP-cbMSC at PD35 (B) were analyzed by array CGH using Affymetrix 10K mapping chips, and their whole genome from chromosomes 1 to X was not detected with any genomic deletion or amplifi cation.

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Disclosure of interest: The authors report no potential confl icts of interest. We have already deposited both cell lines in an open-access cell bank of the Bioresource Collection and Research Center, Taiwan, and made them available to public.

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Supplementary material available online

Supplementary Figure 1.

Blumbergs PC, Jones NR, North JB. Diffuse axonal injury in 36. head trauma. J Neurol Neurosurg Psychiatry. 1989;52:838–41. Aluigi M, Fogli M, Curti A, Isidori A, Gruppioni E, 37. Chiodoni C, et al. Nucleofection is an effi cient nonviral transfection technique for human bone marrow-derived mes-enchymal stem cells. Stem Cells. 2006;24:454–61.

Greider CW. Telomerase activity, cell proliferation, and can-38. cer. Proc Natl Acad Sci USA. 1998;95:90–2. Huang Q, Chen M, Liang S, Acha V, Liu D, Yuan F, et al. 39. Improving cell therapy: experiments using transplanted tel-omerase-immortalized cells in immunodefi cient mice. Mech Ageing Dev. 2007;128:25–30.

Establishment of immortalized MSC with RFP expression 465