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TISSUE-SPECIFIC STEM CELLS Multilineage Differentiation and Characterization of the Human Fetal Osteoblastic 1.19 Cell Line: A Possible In Vitro Model of Human Mesenchymal Progenitors MEN-LUH YEN,a,b CHIH-CHENG CHIEN,c,d ING-MING CHIU,e,f HSING-I HUANG,c,g YAO-CHANG CHEN,h HSIN-I HU,a,b B. LINJU YENd,e Departments of aPrimary Care Medicine and bObstetrics/Gynecology, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan; cDepartment of Medicine, School of Medicine, Fu Jen Catholic University, Taipei, Taiwan; dCathay General Hospital, Neihu, Taiwan; eStem Cell Research Center, National Health Research Institutes, Zhunan, Taiwan; fInstitute of Medical Technology, National Chung Hsing University, Taichung; gCathay Medical Research Institute, Cathay General Hospital, Taipei, Taiwan; hDepartment of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Key Words. Mesenchymal stem cells • Multilineage differentiation • Nestin • Oct-4 • Osteoprogenitor • Cell line

ABSTRACT The in vitro study of human bone marrow mesenchymal stromal cells (BMMSCs) has largely depended on the use of primary cultures. Although these are excellent model systems, their scarcity, heterogeneity, and limited lifespan restrict their usefulness. This has led researchers to look for other sources of MSCs, and recently, such a population of progenitor/stem cells has been found in mesodermal tissues, including bone. We therefore hypothesized that a well-studied and commercially available clonal human osteoprogenitor cell line, the fetal osteoblastic 1.19 cell line (hFOB), may have multilineage differentiation potential. We found that undifferentiated hFOB cells possess similar cell surface markers as BMMSCs and also

INTRODUCTION The recent discovery of adult stem cell (ASC) plasticity has overturned the dogma of hierarchical differentiation in stem cell biology and fueled the hope that previously untreatable diseases now may have new therapies ([1, 2] and reviewed in [3]). These transdifferentiation capabilities have been found for progenitor/ stem cells from the bone marrow, including mesenchymal stromal cells (BMMSCs), where differentiation into a number of nonmesodermal lineages has been achieved [4 – 8]. Although embryonic stem cells (ESCs) possess far more differentiation and proliferative capabilities than do BMMSCs and other ASCs, these totipotent stem cells form tumors upon transplantation and therefore are not ideal candidates for therapeutic use. The focus for future clinical use has thus centered on ASCs. The excitement generated by the transdifferentiation data, however, has not been without controversy [9 –11]. Central to many of the problems in ASC plasticity research has been the difficulty in unambiguously identifying and purifying a homogeneous population of ASCs. Even with BMMSCs, which are a well-studied population of ASCs with plasticity data, the heterogenicity of isolated cells is well-documented [12]. Unlike

express the embryonic stem cell-related pluripotency gene, Oct-4, as well as the neural progenitor marker nestin. hFOB cells can also undergo multilineage differentiation into the mesodermal lineages of chondrogenic and adipocytic cell types in addition to its predetermined pathway, the mature osteoblast. Moreover, as with BMMSCs, under neural-inducing conditions, hFOB cells acquire a neural-like phenotype. This human cell line has been a widely used model of normal osteoblast differentiation. Our data suggest that hFOB cells may provide for researchers an easily available, homogeneous, and consistent in vitro model for study of human mesenchymal progenitor cells. STEM CELLS 2007;25:125–131 ESCs, which are capable of apparently unlimited proliferation in vitro without undergoing malignant transformation, BMMSCs have a limited proliferative lifespan, undergoing senescence after approximately 50 population doublings [13], most likely due to the expression of telomerase in ESCs but not BMMSCs [14 –16]. Thus, to have a continuous supply of viable MSCs on hand, it is necessary to periodically obtain tissue samples from subjects to reisolate stem cells. Furthermore, with human-source MSCs, genetic variability cannot be avoided, requiring researchers to obtain cells from a large number of donors in order to achieve statistically powerful results. To overcome these problems, a number of laboratories have immortalized MSCs with ectopic expression of telomerase (human telomerase reverse transcriptase [hTERT]), the immortalization “agent of choice” due to its enhanced chromosomal stability [16 –18]. Although this accomplishes the goal of creating immortal “MSC cell lines,” there are now reports of malignant transformation in these hTERT-expressing MSC cell lines [19, 20], limiting the usefulness of these BMMSCs as in vitro models of normal stem cells. Although the transdifferentiation data on BMMSCs have generated much excitement, the fact remains that these ASCs are rare in numbers [21]. Moreover, with increasing donor age, there is a decrease in differentiation capability [22, 23] as well

Correspondence: B. Linju Yen, M.D., Stem Cell Research Center, National Health Research Institutes, 35 Keyan Road, Zhunan, 350, Taiwan. Telephone: 886-2-2653-4401, ext. 27502; Fax: 886-2-2792-9679; e-mail: [email protected] Received May 19, 2006; accepted for publication September 14, 2006. ©AlphaMed Press 1066-5099/2007/$20.00/0 doi: 10.1634/stemcells.2006-0295

STEM CELLS 2007;25:125–131 www.StemCells.com

hFOB as a Human Mesenchymal Progenitor Cell Line

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Table 1. Primers used for reverse transcription-polymerase chain reaction Gene

Sequence

TA (°C)

Product

Oct-4

F: 5⬘-CGACCATCTGCCGCTTTGAG-3⬘ R: 5⬘-CCCCCTGTCCCCCATTCCTA-3⬘ F: 5⬘-AGCCAGTCTCACCTTCAACCGC-3⬘ R: 5⬘-GGAGTAGCAGAGGGAGGCCG-3⬘ F: 5⬘-GTGGACGAGGCAAGAGTTTCA-3⬘ R: 5⬘-TGGCAGGTAGGTGTGGTAGTG-3⬘ F: 5⬘-ATGAGAGCCCTCACACTCCTC-3⬘ R: 5⬘-CGTAGAAGCGCCGATAGGC-3⬘ F: 5⬘-AGGAACAGATCTTCCTGCTGCA-3⬘ R: 5⬘-TGCATGTGGATGTAGTTGCGCGT-3⬘ F: 5⬘-CGATGGGGATGCTCATAA-3⬘ R: 5⬘-CTTTTGGCATACTCTGTGAT-3⬘ F: 5⬘-TGACACCAAAACCCTCATCA-3⬘ R: 5⬘-GCCAGTGTCTGGTCCATCTT-3⬘ F: 5⬘-TCAGGAACTGAACTCAGTGG-3⬘ R: 5⬘-GCCACTGAGTTCCACAGA-3⬘ F: 5⬘-ACGGCGAGAAGGGAGAAGTTG-3⬘ R: 5⬘-GGGGGTCCAGGGTTGCCATTG-3⬘ F: 5⬘-TGGCACCACCTTCTACAATGAGC-3⬘ R: 5⬘-GCACAGCTTCTCCTTAATGTCACGC-3⬘

63

577 bp

63

272 bp

55

632 bp

55

302 bp

55

571 bp

55

390 bp

60

197 bp

55

441 bp

60

352 bp

55

353 bp

hTERT Cbfa-1 Osteocalcin PTHR PPAR-␥ Leptin Aggrecan Collagen II

␤-Actin

Abbreviations: bp, base pair; F, forward primer; hTERT, human telomerase reverse transcriptase; PPAR-␥ , peroxisome proliferator activated receptor-␥; PTHR, parathyroid hormone receptor; R, reverse primer; TA, annealing temperature.

as stem cell numbers [24]. This has prompted researchers to look for alternative sources of MSCs. Recently, MSCs or MSClike cells from trabecular bone and its adjoining tissues have been isolated [25–27]. Along these lines of thought, we hypothesized that the human fetal osteoblastic 1.19 cell line (hFOB), which has been designated as a preosteoblastic or osteoprogenitor cell line, may have multilineage differentiation potential. The hFOB is an immortalized, clonal human fetal cell line that is well-characterized as a osteoprogenitor with minimal karyotype damage even after multiple passages [28, 29]. We studied the multipotentiality of this cell line and the possibility of this cell line as an in vitro model for human mesenchymal progenitors. We found that hFOB, in addition to undergoing its default osteoblastic differentiation pathway, can be induced to differentiate into adipocytic and chondrogenic phenotypes. We also explored the possibility of nonmesodermal lineage differentiation in terms of a neural phenotype. Cell surface and pluripotency markers, as well as growth characteristics of hFOB, were also studied.

MATERIALS

AND

METHODS

Cell Growth Analysis Cells were seeded at 3 ⫻ 104 per cm2. At selected time intervals, cells were collected by trypsinization and suspended in culture medium. Viable and nonviable cells were then determined by direct counting using a hemocytometer in the presence of 0.5% trypan blue (Sigma-Aldrich) and expressed as percentage increase over initial seeded number at day 0.

Immunophenotyping To detect surface antigens, aliquots of cells were washed with phosphate-buffered saline (PBS) containing 2% FBS after detachment with 0.25% trypsin/EDTA. Antibodies against the human antigens CD14, CD34, CD45, CD90/Thy-1, CD105/SH-2/endoglin, CD117/c-kit, CD166, HLA-ABC, and HLA-DR were purchased from BD Biosciences (San Jose, CA, http://www.bdbiosciences. com). Antibodies against the human antigens glycophorin A, CD13, CD29, and CD44 were purchased from Dako (Glostrup, Denmark, http://www.dako.com). Antibodies against the human antigen SH-3 were purified from the hybridoma cell lines acquired from ATCC. Cells were stained with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies and compared with appropriate isotype controls. Flow cytometry analysis was performed using FACSCalibur (BD Biosciences) with CellQuest software (BD Biosciences).

Cell Cultures

Reverse Transcription-Polymerase Chain Reaction

The conditionally immortalized human fetal osteoblastic cell line hFOB was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, http://www.atcc.org). hFOB was developed by conditionally immortalizing human fetal osteoblasts with a temperature-sensitive mutant of the SV40 large T antigen (tsSV40LTA) gene. At the permissive temperature of 33.5°C, the ts-SV40LTA is active and the hFOB cells proliferate rapidly, whereas at the nonpermissive temperature of 39.5°C, the tsSV40LTA is inactive, and the cells differentiate and display the phenotype of mature osteoblasts [28]. The cells were cultured according to ATCC protocol—which we designate as expansion medium—in a 1:1 mixture of phenol-free Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, http://www.hyclone.com) and geneticin (300 ␮g/ml; Sigma-Aldrich, St. Louis, http://www. sigmaaldrich.com) at either 33.5°C or 39.5°C. For control, BMMSCs were obtained from Cambrex Corporation (East Rutherford, NJ, http://www.cambrex.com) and cultured according to Pittenger et al. [21]. The cell lines NTERA-2, MCF-7, and HepG2 were obtained from ATCC and cultured according to protocol.

RNA was extracted from cells using TRIzol, and mRNA was reverse-transcribed to cDNA using SuperScript III first-strand synthesis system. cDNA was amplified using a Platinum PCR SuperMix. All reagents were obtained from Invitrogen. The amplified products were subjected to electrophoresis in a 2% agarose gel and stained with ethidium bromide (Sigma-Aldrich). Primers used for amplification and the annealing temperatures are listed in Table 1.

Differentiation Studies All differentiation experiments were conducted at 39.5°C. For adipogenic differentiation, cells were cultured in complete medium with the addition of 0.5 mM isobutyl-methylxanthine, 1 ␮M dexamethasone, 10 ␮M insulin, and 60 ␮M indomethacin (all from Sigma-Aldrich) [21]. Chondrogenic differentiation was induced by cell pellet culture as previously described [30]. Briefly, 2 ⫻ 105 cells were suspended in 0.5 ml of medium, pelleted by centrifugation at 500g for 5 minutes in 15-ml conical polypropylene tubes, and cultured in serum-free medium with the addition of 10 ng/ml transforming growth factor-␤3 (TGF-␤3) (R&D Systems, Inc., Minneapolis, http://www.rndsystems.com) in serum-free conditions [21]. Neurogenic differentiation was induced by culturing cells in

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Figure 2. Cell growth characteristics of human fetal osteoblastic 1.19 (hFOB) cells. Cells were cultured under permissive conditions (33.5°C, F) and nonpermissive conditions in expansion medium (39.5°C, E) and in adipocytic differentiation medium (39.5°C, ). Cells were collected and counted every day for 6 days. Abbreviation: D, day.

Figure 1. Osteoblastic differentiation of human fetal osteoblastic 1.19 (hFOB) cells at 39.5°C. hFOB cells were cultured at 33.5°C and 39.5°C for 1 and 3 days and characterized for (A) reverse transcription-polymerase chain reaction analysis of bone-related gene expression of Cbfa1, PTHR, and osteocalcin, (B) ALP secretion, and calcium deposition with alizarin red staining as visualized by (C) phase-contrast microscopy (magnification ⫻100, scale bar 100 ␮m), and (D) by spectrophotometric analysis for quantification (see Materials and Methods). ⴱ, p ⬍ .05. Abbreviations: ALP, alkaline phosphatase; AR-S; alizarin red; D1, day 1; D3, day 3; O, osteoblastic medium; PTHR, parathyroid hormone receptor; UnD, undifferentiated.

serum-free medium with the addition of 50 ng/ml nerve growth factor (NGF) (R&D Systems, Inc.) [31].

Alkaline Phosphatase Activity Cells were seeded in six-well plates at a density of 3 ⫻ 105 cells per well and cultured in expansion medium at 33.5°C or 39.5°C. After 1 or 3 days in culture, the cell layers were rinsed with PBS, scraped into 0.5 ml of buffer (10 mM Tris-HCl), sonicated four times to disrupt cell membranes, and centrifuged (4,000g) at 4°C for 15 minutes. Alkaline phosphatase (ALP) activity was determined by the ALP substrate kit (Bio-Rad Laboratories, Hercules, CA, http:// www.bio-rad.com). Absorbance at 405 nm was measured with a spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA, http://www.moleculardevices.com). ALP activity was corrected for the DNA content determined by the Picogreen dsDNA quantitation kit (Invitrogen) and was expressed as micromoles of paranitrophenol per ␮g/ml of DNA.

Immunofluorescent, Immunocytochemical, and Cytochemical Staining Immunofluorescence. Cultured cells were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 10 minutes at room temperature and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 minutes. Primary antibodies against the human antigens nestin (1:100), glial fibrillary acidic protein (GFAP) (1: 250), microtubule-associated protein 2 (MAP2) (1:250), and NG2 (1:50) were purchased from Chemicon (Temecula, CA, http://www. chemicon.com). Samples were incubated with the primary antibodies at 4°C overnight, rinsed three times with PBS, and incubated for 60 minutes at room temperature with FITC-conjugated secondary antibodies at a dilution of 1:100. All samples were stained with 4⬘,6-diamidino-2-phenylindole (Invitrogen) for 5 minutes. Staining

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was visualized under a fluorescence microscope (Nikon, Tokyo, http://www.nikon.com). Immunocytochemistry. Cultured cells were fixed with 4% PFA for 5 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 20 minutes. Samples were then incubated sequentially first with the primary monoclonal antibodies against the human antigens collagen I and collagen II (both from SigmaAldrich, dilution 1:50) at 4°C overnight. The samples were then stained with biotinylated anti-rabbit antibody and an avidin-biotin conjugate of horseradish peroxidase (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Cytochemistry. Osteoblastic differentiation was evaluated by calcium accumulation with alizarin red stain [32]. The presence of adipocytes was assessed by the cellular accumulation of neutral lipid vacuoles that stain with oil red O [21]. For detection of chondrocytic differentiation, the presence of highly sulfated proteoglycans was assessed by alcian blue stain [33]. All stains were obtained from Sigma-Aldrich.

RESULTS hFOB Cells Undergo Spontaneous Differentiation to Mature Osteoblasts When Cultured at 39.5°C When cultured at 33.5°C, hFOB cells proliferate. When cultured in 39.5°C, however, hFOB cells spontaneously differentiate into a mature osteoblastic phenotype. Reverse transcription-polymerase chain reaction (RT-PCR) results show that at 39.5°C (Fig. 1A), hFOB cells express increasing amounts of bonerelated genes, including Cbfa1 (an early bone-specific transcription factor [34]) and parathyroid hormone receptor (PTHR) and osteocalcin (genes expressed at a later stage of osteoblastic differentiation [35]). Mature osteoblasts also secrete ALP and deposit calcium. In a time-dependent manner, hFOB cells cultured at 39.5°C show increasing amounts of ALP (Fig. 1B) and calcium deposition, the latter revealed by alizarin red staining (Fig. 1C, 1D).

hFOB Cells Proliferate at the Permissive Temperature of 33.5°C but Not 39.5°C Next, we examined the cell growth characteristics in undifferentiated and differentiated hFOB cells. hFOB cells were cultured in three conditions: at the permissive temperature of 33.5°C and at the differentiating temperature of 39.5°C under expansion medium and under adipocytic-differentiation medium (Fig. 2). Proliferation was seen only for cells grown at 33.5°C; for cells grown at 39.5°C in either expansion medium or adipocytic differentiation medium, a steady decrease in cell num-

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Figure 3. Characterization of human fetal osteoblastic 1.19 (hFOB). (A): Flow cytometric analysis for various cell surface markers. (B): Reverse transcription-polymerase chain reaction analysis of hFOB, bone marrow mesenchymal stromal cells, and NTERA-2 (positive control) for hTERT and Oct-4. Abbreviations: Glyco A, glycophorin A; hTERT, human telomerase reverse transcriptase; OD, osteoblastic differentiation day; UnD, undifferentiated.

bers can be seen. At the permissive temperature, daily counts of cell numbers showed an initial lag phase for the first 2 days, with exponential cell growth between days 2 and 3. Counts returned an average doubling time of approximately 25 hours between these two days. From days 3 to 6, cells reached confluency and population growth slowed with cell death occurring thereafter. To ascertain whether the lack of growth was due to decreased cell proliferation or increased cell death, we assayed for cell proliferation with a tetrazolium salt WST-8 (water-soluble tetrazolium-8) assay, as well as cell damage with lactate dehydrogenase release. Results show that when hFOB was moved from 33.5°C to 39.5°C, there was progressive decrease in cell proliferation but that apoptosis, after an initial increase, leveled off after 1 day of culture whether at 33.5°C or 39.5°C (supplemental online Fig. 1).

hFOB Cells Share Multiple Markers with BMMSCs and Express the ESC-Pluripotency Marker of Oct-4 Undifferentiated hFOB cells are positive for a number of BMMSC cell surface markers (Fig. 3A), including CD13, CD29, CD44, CD73/SH3/SH4, CD90/thy-1, CD105/SH2/en-

doglin, and CD166/ALCAM [21]. They are negative for multiple hematopoeitic markers, including CD14, 34, 45, CD117/ckit, and glycophorin A. Immunologically, undifferentiated hFOB cells are positive for HLA-ABC but negative for HLA-DR. By RT-PCR analysis (Fig. 3B), undifferentiated hFOB cells can be seen to express the pluripotency marker Oct-4, and levels decrease with differentiation. No expression of telomerase was detected.

Mesodermal Multilineage Differentiation of hFOB to Adipocytic and Chondrocytic Phenotypes When undifferentiated hFOB cells were cultured at 39.5°C in conditions conducive to adipocytic differentiation, progressive formation of oil droplets could be seen starting at day 1, as visualized by staining with oil red O (Fig. 4A). After 3 days of differentiation, more than 75% of the cells could be seen to harbor oil droplets within the cytoplasm. RT-PCR analysis (Fig. 4B) shows increasing expression of the adipocyte-specific genes PPAR-␥ (peroxisome proliferator activated receptor-␥), a transcriptional factor activated early in adipocytic differentiation


Figure 4. Mesodermal differentiation of human fetal osteoblastic 1.19 (hFOB). Adipocytic differentiation: (A) oil red O stain for lipid droplets (magnification ⫻200, scale bar 50 ␮m) and (B) reverse transcriptionpolymerase chain reaction (RT-PCR) analysis for adipocyte-specific genes PPAR-␥ and leptin. Chondrocytic differentiation: (C) histochemical and immunocytochemical staining for alcian blue (i, ii), collagen I (iii, iv), and collagen II (v, vi) of cells grown in expansion (i, iii, and v) versus chondrocyte-inducing (ii, iv, and vi) medium (magnification ⫻100, scale bar 100 ␮m) and (D) RT-PCR analysis for chondrocytespecific genes aggrecan and collagen II. Abbreviations: A, adipocytic medium; C, chondrocytic medium; D1, day 1; D3, day 3; PPAR-␥, peroxisome proliferator activated receptor-␥; UnD, undifferentiated.

[36], and leptin, which is expressed in more mature adipocytes [37, 38]. For induction of chondrocytic differentiation, undifferentiated hFOB cells were cultured using the micromass culture method in a serum-free medium with the addition of TGF-␤3. After 1 week of induction, positive staining could be seen with alcian blue (Fig. 4Ci, 4Cii), which stains for the highly sulfated proteoglycans found in cartilage [33]. Immunohistochemical staining of the micromass outgrowths for collagen I (Fig. 4Ciii, 4Civ), which is found in bone, was weak, but staining for collagen II (Fig. 4Cv, 4Cvi), a cartilage-specific type of collagen, was strongly positive. RT-PCR analysis (Fig. 4D) shows increasing expression of chondrocyte-specific genes of aggrecan and collagen II [39].

Undifferentiated hFOB Cells Express the Neural Progenitor Marker, Nestin, and Can Be Induced to Express More Mature Neural Lineage Markers In addition to MSC and ESC cell surface markers, undifferentiated hFOB cells stain positive for nestin, which is decreased with differentiation (Fig. 5A, 5B). Undifferentiated hFOB cells also stain weakly positive for the neural markers of GFAP, MAP2, and NG2 (Fig. 5C, 5E, and 5G, respectively), similar to other mesenchymal progenitors and MSCs [40, 41]. When hFOB cells are cultured under serum-free conditions with the addition of NGF, the cells acquire branched processes and show increased staining for GFAP, MAP2, and NG2 (Fig. 5D, 5F, and 5H, respectively). For comparison, undifferentiated BMMSCs stain positive for GFAP, MAP2, and NG2, but a hepatocellular carcinoma cell line (HepG2, data not shown) and a breast cancer cell line (MCF-7) are negative for all three neural markers (supplemental online Fig. 2). www.StemCells.com

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Figure 5. Expression of neural markers in human fetal osteoblastic 1.19 (hFOB) by immunofluorescent staining. Nestin expression in (A) undifferentiated hFOB cells and (B) hFOB cells cultured for 3 days at 39.5°C in neural differentiation medium. Glial fibrillary acidic protein (GFAP) (C, D), NG2 (E, F), and microtubule-associated protein 2 (MAP2) (G, H) expression by hFOB cells after culturing for 3 days in expansion medium (C, E, G) compared with neural differentiation medium (D, F, H). Arrows denote neural-like processes. Scale bars ⫽ 100 ␮m.

Karyotype Analysis of hFOB Previous reports show that hFOB cells sustain minimal karyotype damage even after multiple passages [29]. We also analyzed the chromosomal status of this cell line and found few abnormalities (supplemental online Fig. 3). Although these karyotypic abnormalities are minimal compared with those of cancer cell lines, it may be enough to result in transformation. We therefore also tested hFOB for anchorage-independent growth (AIG) with the soft agar assay; no AIG was seen with hFOB, whereas dramatic growth was seen with MCF-7 (supplemental online Fig. 4).

DISCUSSION In this study, we show that the preosteoblastic hFOB cell line is capable of multilineage differentiation into the mesodermal lineages of chondrogenic and adipocytic cell types in addition to its predetermined pathway, the mature osteoblast. Moreover, as with BMMSCs, under neural-inducing conditions, hFOB cells acquire a neural-like phenotype. Undifferentiated hFOB cells possess similar cell surface markers as BMMSCs and also express the ESC-related pluripotency gene, Oct-4, as well as the neural progenitor marker nestin.


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The multipotent differentiation capabilities of BMMSCs have now been well-reported [21, 42– 45]. Found in the stromal cell compartment of the bone marrow, MSCs are able to differentiate into a number of mesodermal lineages as well as transdifferentiate into some ectodermal and endodermal lineages [1– 8]. More recently, it was found that other mesodermal tissues/organs also harbor MSCs or at least MSC-like progenitor cells, including adipose tissue [45– 47], muscle [48, 49], synovium [50], and bone [25–27]. These findings have led us to speculate that hFOB, a well-studied preosteoblastic cell line, may be able to differentiate into lineages other than bone. Isolated from fetal ribs, the hFOB cell line is well-characterized for its ability to differentiate into mature osteoblast [28, 29]. We found that undifferentiated hFOB cells express similar cell surface markers as BMMSCs, including SH-2/CD105 and SH3CD/73, and are negative for hematopoietic markers. Moreover, the ability of hFOB to differentiate into multiple lineages other than its default osteoblastic pathway and into an extramesodermal lineage strongly suggests that hFOB is not only an osteoprogenitor but may in fact represent an earlier stage of mesenchymal progenitor. We also found expression of Oct-4, an ESC pluripotency marker, in undifferentiated hFOB. Oct-4, besides being expressed by ESCs, is also expressed by various fetal-source progenitor/stem cells, including those from amniotic fluid and placenta [51–53]. Although Oct-4 is not generally detected in BMMSCs, serum deprivation appears to select for a more primitive subpopulation of BMMSCs capable of expressing Oct-4 [54]. Thus, it appears the expression of Oct-4 in hFOB may be indicative of its fetal origin as well as to its potential as a multilineage progenitor. The expression of neural-lineage progenitor markers, in particular nestin, has been found in MSCs [55]. Our results show that undifferentiated hFOB cells express high levels of nestin, which decrease upon differentiation. An intermediate filament, nestin is detected in neural precursors, including neurospheres and neuron- and glial-restricted precursors [56, 57]. It has also been found in developing cells such as muscle and myocardial cells [58] and in more immature MSC populations [55]. Recent data also show that nestin is found in ESC-derived embryoid bodies and also at specific time points during differentiation before it is downregulated with terminal differentiation [59]. Although it is still unclear how nestin is regulated, it appears to be a marker of multilineage progenitor cells [59]. The expression pattern of nestin in hFOB, therefore, is consistent with our finding of its multilineage differentiation capabilities. In vitro cell culture systems are an important tool in our understanding of cellular and molecular mechanisms, and pri-

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mary cultures have been the mainstay for stem cell researchers focusing on human BMMSCs. Although these are excellent model systems, their limited lifespan in culture and heterogeneity of phenotype and differentiation stage restrict their usefulness. Cell lines, on the other hand, offer a homogeneous and essentially infinite system. However, because the majority of cell lines are derived from cancerous outgrowths, this has posed a problem in the study of normal mechanisms. Of the various nonmalignant adult progenitor/stem cell lines commercially available, all are murine [60, 61]. The commercially available bipotential/tripotential murine mesenchymal progenitor cell lines have been invaluable models; the results obtained with these cell lines have provided reproducible data and strong support for the existence of MSCs [62– 64]. Although there are human BMMSC lines immortalized with telomerase, these lines are not available commercially, and the recent reports of malignant transformation of these cell lines limit their usefulness in studying normal stem cell biology [19, 20]. The use of tsSV40LTA for immortalization of hFOB, on the other hand, has not resulted in malignant transformation by many criteria; hFOB does not proliferate in low serum conditions or show AIG (supplemental online Fig. 4); rather, it exhibits cell-cell contact inhibition even after multiple passages [28, 29]. Furthermore, when transplanted into SCID (severe-combined immunodeficient) mice, it is not tumorigenic but rather is able to recapitulate its normal developmental endpoint of bone formation, unlike osteosarcoma cell lines [29]. The fact that the ts-SV40LTA is a modulatable oncogene means its oncogenic effects are negated at the nonpermissive temperature of 39.5°C, allowing normal cellular processes to proceed [65– 67]. These characteristics coupled with our current findings support the view that hFOB may also be feasible as an in vitro model for human early mesenchymal progenitor cells, providing for researchers a consistent, reliable, and easily accessible experimental model.

ACKNOWLEDGMENTS This work was supported in part by a National Science Council grant (Taiwan, NSC 94-2314-B-002-212 and a grant from Cathay General Hospital (CGH-MR-9405).

DISCLOSURES The authors indicate no potential conflicts of interest.

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