Multilineage Differentiation and Characterization of the Human Fetal Osteoblasti - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Men-luh Yena,b, Chih-Cheng Chienc,d, Ing-ming Chiue,f, Hsing-I Huangc,g, Yao-Chang Chenh, Hsin-I Hua,b, B. Linju Yend,e 3 W' @9 T3 K* ?7 Z- f
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      【摘要】
   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 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. + a  M3 W# H! R
      【关键词】 Mesenchymal stem cells Multilineage differentiation Nestin Oct- Osteoprogenitor Cell line) f5 v, d5 [0 _3 o7 k/ c  d( }% L
   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 (. 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.4 B: K" g9 c+ M; k) N6 J

The excitement generated by the transdifferentiation data, however, has not been without controversy , 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 . 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 Cultures
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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 (ts-SV40LTA) gene. At the permissive temperature of 33.5¡ãC, the ts-SV40LTA is active and the hFOB cells proliferate rapidly, whereas at the nonpermissive temperature of 39.5¡ãC, the ts-SV40LTA is inactive, and the cells differentiate and display the phenotype of mature osteoblasts . The cell lines NTERA-2, MCF-7, and HepG2 were obtained from ATCC and cultured according to protocol.3 g$ f  s4 |+ Q5 u' C
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Cell Growth Analysis
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Cells were seeded at 3 x 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+ s% I0 x( p4 R, S, ]; M

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).

Reverse Transcription-Polymerase Chain Reaction" K+ \# N4 L" `$ s
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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.( B& \% z* z8 C: f3 b/ ^

Table 1. Primers used for reverse transcription-polymerase chain reaction* P. G. }, y. _4 D

Differentiation Studies
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All differentiation experiments were conducted at 39.5¡ãC. For adipogenic differentiation, cells were cultured in complete medium with the addition of 0.5 mM isobutyl-methylxanthine, 1 µM dexamethasone, 10 µM insulin, and 60 µM indomethacin (all from Sigma-Aldrich) .
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Alkaline Phosphatase Activity3 l# E( |! O# i9 O6 Q, \

Cells were seeded in six-well plates at a density of 3 x 105 cells per well and cultured in expansion medium at 33.5¡ãC or 39.5¡ãC. 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¡ãC 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 µg/ml of DNA.
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Immunofluorescent, Immunocytochemical, and Cytochemical Staining
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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¡ãC 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 was visualized under a fluorescence microscope (Nikon, Tokyo, http://www.nikon.com).
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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 Sigma-Aldrich, dilution 1:50) at 4¡ãC 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).
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Cytochemistry. Osteoblastic differentiation was evaluated by calcium accumulation with alizarin red stain . All stains were obtained from Sigma-Aldrich.5 W2 _( h+ r4 V, }- U) I; |* \% `* d

RESULTS

hFOB Cells Undergo Spontaneous Differentiation to Mature Osteoblasts When Cultured at 39.5¡ãC
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When cultured at 33.5¡ãC, hFOB cells proliferate. When cultured in 39.5¡ãC, however, hFOB cells spontaneously differentiate into a mature osteoblastic phenotype. Reverse transcription-polymerase chain reaction (RT-PCR) results show that at 39.5¡ãC (Fig. 1A), hFOB cells express increasing amounts of bone-related genes, including Cbfa1 (an early bone-specific transcription factor ).

Figure 1. Osteoblastic differentiation of human fetal osteoblastic 1.19 (hFOB) cells at 39.5¡ãC. hFOB cells were cultured at 33.5¡ãC and 39.5¡ãC 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 x100, scale bar 100 µm), and (D) by spectrophotometric analysis for quantification (see Materials and Methods). *, p

Mature osteoblasts also secrete ALP and deposit calcium. In a time-dependent manner, hFOB cells cultured at 39.5¡ãC show increasing amounts of ALP (Fig. 1B) and calcium deposition, the latter revealed by alizarin red staining (Fig. 1C, 1D).. x$ h+ k& i% c4 Y

hFOB Cells Proliferate at the Permissive Temperature of 33.5¡ãC but Not 39.5¡ãC

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¡ãC and at the differentiating temperature of 39.5¡ãC under expansion medium and under adipocytic-differentiation medium (Fig. 2). Proliferation was seen only for cells grown at 33.5¡ãC; for cells grown at 39.5¡ãC in either expansion medium or adipocytic differentiation medium, a steady decrease in cell numbers 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.
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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¡ãC, ) and nonpermissive conditions in expansion medium (39.5¡ãC, ) and in adipocytic differentiation medium (39.5¡ãC, ). Cells were collected and counted every day for 6 days. Abbreviation: D, day.$ Y3 K# G3 _' B3 S/ d5 R

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¡ãC to 39.5¡ãC, 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¡ãC or 39.5¡ãC (supplemental online Fig. 1).# z7 [7 J2 ~: L5 r

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/endoglin, and CD166/ALCAM . They are negative for multiple hematopoeitic markers, including CD14, 34, 45, CD117/c-kit, and glycophorin A. Immunologically, undifferentiated hFOB cells are positive for HLA-ABC but negative for HLA-DR.) g4 z0 ]) E) t+ I" x
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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.
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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.5 Y- {2 r; t% s# A% _3 @) u

Mesodermal Multilineage Differentiation of hFOB to Adipocytic and Chondrocytic Phenotypes
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When undifferentiated hFOB cells were cultured at 39.5¡ãC 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 x200, scale bar 50 µm) and (B) reverse transcription-polymerase 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 x100, scale bar 100 µm) and (D) RT-PCR analysis for chondrocyte-specific 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.
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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-ß3. 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 .' x! i0 u2 j  f) ]5 |( X5 _  e# V4 u
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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 . 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).2 O* D/ s8 V. t; h
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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¡ãC 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 µm.

Karyotype Analysis of hFOB6 Y) V& D8 q, R3 B

Previous reports show that hFOB cells sustain minimal karyotype damage even after multiple passages . 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).* p3 v0 C! A& P5 P* G7 J
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DISCUSSION( r& V% ^3 ?! l4 E
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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.3 X# p4 D+ ]6 [' O  u% C- M: K5 G5 W  K
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The multipotent differentiation capabilities of BMMSCs have now been well-reported . We found that undifferentiated hFOB cells express similar cell surface markers as BMMSCs, including SH-2/CD105 and SH-3CD/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.- D5 `6 z' ?. @/ c7 h$ h; H
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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 . 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.: T' h* P8 l2 c7 q

The expression of neural-lineage progenitor markers, in particular nestin, has been found in MSCs . 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 primary 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 . 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.

DISCLOSURES$ M1 l( B! f6 ^: c! d  s
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The authors indicate no potential conflicts of interest.7 d1 _, z" ]$ Q7 m/ N; Z9 e
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ACKNOWLEDGMENTS. D/ V$ M8 j$ v; t' [  P+ z! q( I

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).$ T; c# Z2 s6 l. P) d
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