Identification and Functional Analysis of Candidate Genes Regulating Mesenchymal - 《stemcells》干细胞学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 29 下一页

返回列表

查看: 547384\|回复: 282	go Identification and Functional Analysis of Candidate Genes Regulating Mesenchymal [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 00:04 |只看该作者 |倒序浏览 |打印

作者：Lin Song, Nicole E. Webb, Yingjie Song, Rocky S. Tuan作者单位：Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, USA , r: a: N  U0 r% w# E& f* \7 P5 C
   - F4 i. v0 L7 |% m  o. y# Q


                     5 m( \: c6 l* B, R
         : r1 [5 K3 w' C& ]( S

         . o4 W$ k! U* A9 `
         2 D& C6 ]/ n' J

      2 k) p8 W- k8 J


      【摘要】
   Adult human mesenchymal stem cells (hMSCs) possess multilineage differentiation potential, and differentiated hMSCs have recently been shown to have the ability to transdifferentiate into other lineages. However, the molecular signature of hMSCs is not well-known, and the mechanisms regulating their self-renewal, differentiation, and transdifferentiation are not completely understood. In this study, we demonstrate that fully differentiated hMSCs could dedifferentiate, a likely critical step for transdifferentiation. By comparing the global gene expression profiles of undifferentiated, differentiated, and dedifferentiation cells in three mesenchymal lineages (osteogenesis, chondrogenesis, and adipogenesis), we identified a number of "stemness" and "differentiation" genes that might be essential to maintain adult stem cell multipotency as well as to drive lineage-specific commitment. These genes include those that encode cell surface molecules, as well as components of signaling pathways. These genes may be valuable for developing methods to isolate, enrich, and purify homogeneous population of hMSCs and/or maintain and propagate hMSCs as well as guide or regulate their differentiation for gene and cell-based therapy. Using small interfering RNA gene inactivation, we demonstrate that five genes (actin filament-associated protein, frizzled 7, dickkopf 3, protein tyrosine phosphatase receptor F, and RAB3B) promote cell survival without altering cell proliferation, as well as exhibiting different effects on the commitment of hMSCs into multiple mesenchymal lineages. ( w: m( u$ s; ?- M; `
      【关键词】 Mesenchymal stem cells Transdifferentiation Self-renewal Microarray, J3 t, O! ?; [5 ^
   INTRODUCTION# t1 c# p% _1 l3 i! w, e
  Z. C* r/ X( j& k; O8 a3 ^# B
Stem cells that reside in adult tissues and organs are capable of self-renewal and multipotential differentiation. Adult stem cells remain in a nonproliferative, quiescent state during most of their lifetime until stimulated by the signals triggered by tissue damage and remodeling . However, the downstream intracellular effectors in these processes are still largely unknown." `& e* k' C8 N1 O
! |$ A7 p4 o1 {' {% j; ]
In addition to self-renewal and multipotentiality, adult stem cells possess the ability to transdifferentiate, that is, to switch their specific developmental lineage into another cell type of a different lineage, sometimes across embryonic germ layers. For example, mesenchymal stem cells (MSCs) can be induced to become nonmesodermal cells, including functional neurons, astrocytes, oligodendrocytes, and endothelial cells, by appropriate extrinsic stimuli in vitro , osteoblasts, chondrocytes, and adipocytes differentiated from human mesenchymal stem cells (hMSCs) can transdifferentiate into other cell types in response to extrinsic factors, likely through genetic reprogramming. However, the molecular mechanisms behind the transdifferentiation process are poorly understood. Do stem cells switch their phenotype directly from one cell type to another? Do differentiated cells dedifferentiate first to a primitive cell type before committing to a different lineage? Are common signaling pathways employed by the different stem cells, or are there unique mechanisms controlling individual stem cells? Answers to these questions will undoubtedly enhance our understanding of how stem cells both maintain their multipotentiality and control their commitment and differentiation.

Global gene expression profiling has been widely used to identify transcriptional signatures of specific stem cells and to gain insights into the signaling mechanisms regulating their differentiation program in embryonic stem cells (ESCs) , using serial analysis of gene expression (SAGE) and microarray analysis. Several genes have been identified to be highly expressed in undifferentiated hMSCs, including vimentin, connective tissue growth factor, collagen type I 1, and eukaryotic translation elongation factor 1 1. Although these genes are expressed in hMSCs and thus might be considered as their molecular signature for purification and enrichment, little evidence is available on their functionality in hMSC maintenance and self-renewal, and it is not known whether they are merely housekeeping genes or actually play critical roles in preventing cells from differentiating. Another issue that remains unresolved is the regulatory mechanism controlling MSC multilineage differentiation capabilities, particularly the possibility of common signaling pathways that are shared by more than one differentiation pathways. Given the heterogeneous nature of in vitro-expanded MSCs as well as the complexity of culture conditions used in individual studies, it is almost impossible to perform comparative gene expression analysis to generate common gene lists from published data. Furthermore, the lack of functional analysis of genes in these studies has delayed in-depth assessment of the role of these reported genes.

In this study, we used an in vitro differentiation and dedifferentiation culture system using human MSCs and performed global gene expression profiling on undifferentiated hMSCs, differentiated osteoblasts, chondrocytes, and adipocytes, as well as dedifferentiated cells derived from these three distinct mesenchymal lineages. Our results demonstrated for the first time that differentiated cells could dedifferentiate into a primitive stem cell-like stage before transdifferentiating into another cell type. By comparing differentially expressed genes during differentiation and dedifferentiation processes in all three lineages, we identified a list of genes that are candidate markers of hMSCs and may function to maintain stem cells at an uncommitted state or initiate their differentiation process. We have further explored the function of five genes (actin filament-associated protein , and RAB3B) in stem cell proliferation, survival, and multilineage differentiation by inactivating their expression using small interfering RNA (siRNA) technology. Our results demonstrate that all five genes promote cell survival but exhibit different effects on the commitment of hMSCs into multiple mesenchymal lineages.& c6 x6 f0 |; M

MATERIALS AND METHODS
; P3 r! b$ {' F  o. U
Reagents7 @# `: v- ]+ E/ i

Cell culture media, reagents, and antibodies were purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). All chemicals were obtained from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com) unless specified otherwise.$ r3 b/ }( ?- I; o9 a4 ]! W

Isolation, Culture Expansion, Differentiation, and Dedifferentiation of hMSCs5 `" ]/ f3 h- k# Y* q

hMSCs were isolated from bone marrow aspirate obtained with Institutional Review Board (George Washington University, Washington, DC) from patients (aged 45¨C84 years) undergoing elective total hip arthroplasty. Cells were culture-expanded in basal medium (BM), containing Dulbecco¡¯s modified Eagle¡¯s medium, 10% fetal bovine serum, and antibiotics . hMSCs that underwent fewer than 15 population doublings were used.

Differentiation of hMSCs into three mesenchymal lineages was induced as described previously ) for 14 or 21 days. To induce dedifferentiation process, cells were removed from the induction medium, replated on monolayer, and cultured in BM for 20 days further.

To examine the effects of specific gene knockdown on hMSCs, cells were transfected with gene-specific siRNA, collected 24 hours post-transfection, and cultured accordingly as described above.

Analysis of hMSC Differentiation! i7 x- s  `# c- [0 t) a" A0 s
. H0 r2 t1 Q  R' F' p+ f9 A
Osteogenesis was detected by histochemical staining of alkaline phosphatase (ALP) activity using an enzyme kit from Sigma-Aldrich and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of expression levels of ALP and osteocalcin (OC). Adipogenesis was detected by the presence of neutral lipids in the cytoplasm stained with Oil Red O. To analyze chondrogenic differentiation, high-density cell pellets were fixed with 4% paraformaldehyde and cryosectioned at 8-µm thickness for histological and immunochemical staining as described previously . Sulfated matrix proteoglycan was stained with Alcian Blue (pH 1.0). Collagen type II was detected by immunohistochemistry. For crystal violet staining, cells were fixed, incubated with 0.2% crystal violet in 2.5% acetic acid for 5 minutes, and rinsed with water.

Global Gene Expression Profiling6 r% E8 a% [: r! p+ H, p* n. w

hMSCs isolated from the following patients were used for lineage-specific induction to obtain undifferentiated, differentiated, and dedifferentiated cells: three female patients aged 59, 67, and 70 for osteogenesis; four female patients aged 59, 67, 70, and 71 for adipogenesis; and three female patients aged 62, 67, and 70 for chondrogenesis.

All of the following procedures were performed according to the manufacturers¡¯ instructions. Reagents were used at the recommended concentrations. Total RNA was isolated using TRIzol reagent and cleaned up with RNeasy Mini kit (Qiagen, Valencia, CA, http://www1.qiagen.com). First-strand cDNA was synthesized from 8 µg of total RNA using T7-oligo(dT) primer and SuperScript II reverse transcriptase at 42¡ãC for 1 hour. Second-strand cDNA was then synthesized using Escherichia coli DNA ligase, DNA polymerase I, and RNase H. Double-stranded cDNA was cleaned up with the GeneChip cDNA cleanup module (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). Biotin-labeled cRNA was synthesized using Enzo BioArray HighYield RNA transcript labeling kit followed by RNA cleanup. Fifteen µg of cRNA was then fragmented in 1x fragmentation buffer at 94¡ãC for 35 minutes and kept at ¨C20¡ãC. Because of the limited amount of total RNA obtained from chondrogenic samples, 2 µg of total RNA was first amplified to produce aRNA using RiboAmp OA RNA amplification kit (Acturus), which was converted to double-stranded cDNA before being used for generating biotin-labeled cRNA. To make the hybridization cocktail, fragmented cRNA was mixed with control oligonucleotide B2, eukaryotic hybridization controls, herring sperm DNA, and acetylated bovine serum albumin (BSA) in 1x hybridization buffer; heated at 99¡ãC for 5 minutes; and equilibrated to 45¡ãC before being hybridized to the GeneChip arrays for 16 hours at 45¡ãC. Arrays were then washed and stained using the Affymetrix Fluidics Station 450 following the user¡¯s manual. The stained arrays were scanned using the Affymetrix GeneChip scanner 3000, controlled by Affymetrix microarray suite software. Each sample was hybridized to two arrays: human genomes U133A and U133B.
1 R# N2 f( X( m: N# h2 J+ _
Data Analysis
' h: m. D2 Y( V- l
For GeneChip arrays, the raw intensity of individual samples was normalized and scaled among the samples using Microarray Data Management & Analysis System (http://bdmtest.niams.nih.gov/bdm_mysql_new/login.php). Principal component analysis (PCA) was performed using Partek Pro software (Partek, Inc., St. Charles, MO). Gene expression levels in each differentiation lineage (osteogenesis, adipogenesis, and chondrogenesis) were compared between undifferentiated and differentiated samples, as well as dedifferentiated and differentiated samples. Paired Student¡¯s t tests were performed to compare the expression change between undifferentiated and differentiated samples, as well as between dedifferentiated and differentiated samples, with a statistical significance level set at p  .05. Genes that exhibited a 2.0-fold change at p  .05 were filtered. Only the genes that exhibited similar expression pattern in cells obtained from all patients were selected for further analysis. Selected genes were annotated using open source DAVID 2.0 (http://apps1.niaid.nih.gov/david). Selected genes were also analyzed using Ingenuity pathways analysis application (Ingenuity Systems, Mountain View, CA).

A paired Student¡¯s t test was performed with a significance level of p  .05 for other sample analysis.0 a! q& z8 ]! b. G6 i

Quantitative RT-PCR; \; Z) f' ^( w$ _# L/ N

RNA was isolated using TRIzol reagent and cleaned up with RNeasy Mini kit (Qiagen). First-strand cDNA was synthesized using SuperScript first-strand synthesis system (Invitrogen). Five to 10 ng of cDNA was amplified and detected by using iQ SYBR Green supermix kit in an iCycler iQ real-time PCR detection system (Bio-Rad, Hercules, CA, http://www.bio-rad.com). The amount of transcript was normalized to an internal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control and averaged from triplicate samples. The following primers were used. Bone sialoprotein (BSP): 5'-ggtctctgtggtgccttctg-3', 5'-tgctacaacactgggctatgg-3'; OC: 5'-gcctttgtgtccaagc-3', 5'-ggaccccacatccatag-3'; lipoprotein lipase (LPL): 5'-gagatttctctgtatggcacc-3', 5'-ctgcaaatgagacactttctc-3'; fatty acid binding protein 4 (FABP4): 5'-tgggccaggaatttgacgaagt-3', 5'-tcaacgtcccttggcttatgct-3'; cartilage oligomeric matrix protein (COMP): 5'-tgtccccagaagagcaaccc-3', 5'-attgtcgtcgtcgtcgtcgc-3'; metalloproteinase 13 (MMP13): 5'-aacgccagacaaatgtgaccc-3', 5'-tccgcatcaacctgctgagg-3'; PTPRF: 5'-cgagcaaggcggagaggag-3', 5'-tcaaggaggcaagcacaaagc-3'; AFAP: 5'-aagctgcttgagtccacctgaa-3', 5'-actatttgacccgaaggcagca-3'; RAB3B: 5'-caagtgtgacatggaggaagag-3', 5'-agggagagtgggctgagag-3'; FZD7: 5'-aacacgacggcaccaagacc-3', 5'-ggcagggcacggcatagc-3'; DKK3: 5'-gctgaccaggcttcttcctacatc-3', 5'-gcagggcactcttctccacatttc-3'; ALP: 5'-tggagcttcagaagctcaacacca-3', 5'-atctcgttgtctgagtaccagtcc-3'; GAPDH: 5'-agggggcagagatgatgacc-3', 5'-caaggctgagaacgggaagc-3'.
" w4 v% f" v& `" U9 ?. k% D) Z. l
siRNA Transfection" `& g: {8 I- }8 ?# L( J2 o6 p; X+ Z
2 e& C( F$ s7 x
hMSCs were seeded at 6.5 x 103 cells per cm2 (approximately 50% confluence) in antibiotic-free basal medium 24 hours prior to transfection. siRNA transfection was performed following the manufacturer¡¯s protocol. Briefly, 10 µM gene-specific siRNA oligomers (Ambion Inc.) were diluted in Opti-MEM I Reduced Serum Medium and mixed with Lipofectamine 2000 (Invitrogen). After a 20-minute incubation at room temperature, the complexes were added to the cells at a final siRNA concentration of 33 nM. The medium was replenished with antibiotic-containing medium 24 hours post-transfection. Culture medium was then changed every 3 days for the duration of the experiment. hMSCs treated with Lipofectamine 2000 only (untransfected control) and hMSCs transfected with a Silencer negative control siRNA (transfection control) were used as experimental controls.
! j" f4 |4 ?/ A' p5 z& H! G! I
Immunofluorescence Microscopy
: X' v8 y6 c* P% D0 u2 ]
Transfected hMSCs were cultured in monolayer on coverslips for 7 or 14 days before fixation in 4% paraformaldehyde for 15 minutes at room temperature. Fixed cells were rinsed several times with phosphate-buffered saline (PBS), permeabilized with 0.5% Triton X-100 in deionized H2O, and rinsed in PBS. Following a 30-minute incubation with 1% BSA in PBS at room temperature, cells were then incubated in 10 µg/ml primary antibody for 1 hour at 37¡ãC. The following antibodies were used: monoclonal mouse anti-AFAP IgG (BD Biosciences, San Diego, http://www.bdbiosciences.com), goat anti-human Dkk-3 IgG (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), monoclonal rat anti-human Frizzled-7 IgG (R&D Systems), polyclonal rabbit anti-PTPRF IgG (Orbigen, Inc), and polyclonal rabbit anti-Rab3B IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). Cells were rinsed several times in PBS and then incubated in 2 µg/ml conjugated secondary antibody for 1 hour at 37¡ãC. The secondary antibodies used were as follows: Alexa Fluor 488-conjugated goat anti-mouse IgG (A-11029), fluorescein isothiocyanate-conjugated anti-goat IgG (F-2016) (Sigma-Aldrich), Alexa Fluor 488-conjugated goat anti-rat IgG (A-11006), and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (A-21206). Following secondary antibody incubation, cells were rinsed in PBS, and nuclei were counterstained with 1 µg/ml 4',6-diamidino-2-phenylindole, dihydrochloride (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.co) and rinsed again. Coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), sealed with nail polish, allowed to dry overnight at room temperature, and stored in dark at ¨C20¡ãC until analysis.

Western Analysis

Cells were collected by incubation with trypsin-EDTA followed by centrifugation. The cell pellet was resuspended in lysis buffer (20 mM Tris, pH7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, and 0.1% SDS) containing proteinase inhibitors, freeze-thawed three times, and incubated at 4¡ãC for 30 minutes. Following centrifugation at 18,000g for 15 minutes at 4¡ãC, the supernatant containing total cell extract was collected and kept at ¨C80¡ãC. Protein concentration was determined by Bradford assay. Total cell protein aliquots (80 µg) were mixed with Laemmli sample buffer, boiled for 5 minutes, and separated in a 4%¨C20% Tris-HCl gel (Bio-Rad). Proteins were then transferred to nitrocellulose membranes using a mini Trans-blot electrophoretic transfer cell apparatus (Bio-Rad).$ ~. M; H  y. x8 _- f. n1 g

For immunoblotting, the nitrocellulose membrane was first incubated with the blocking buffer containing 1% fish gelatin and 0.05% Tween 20 in Tris-buffered saline (TBS) for 1 hour at room temperature. The membrane was then incubated with primary antibodies (as described above) diluted (1:100) in blocking buffer for 2 hours and rinsed three times in wash buffer (0.05% Tween 20 in TBS). Horseradish peroxidase-conjugated secondary antibodies diluted (1:1,000) in blocking buffer were added to the membrane, incubated for 1 hour, and rinsed several times with wash buffer. The membrane was then incubated with SuperSignal West Pico peroxide and luminal enhancer solutions (Pierce) for 5 minutes, exposed to the film, and developed. Films were scanned using an UMAX PowerLookIII scanner, saved as digital images, and processed using Adobe Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA).

Cell Proliferation and Apoptosis Assay/ d% j0 Q7 i( o# Z# z/ U
" j2 Z8 e1 z0 t/ B
Transfected cells were cultured as monolayers. At day 2, 5, 7, 9, 12, or 14, cultures were rinsed with PBS and detached with trypsin-EDTA; cells were collected by centrifugation and resuspended in BM; duplicate aliquots placed into 96-well plates; and 10 µl of Cell Counting Kit-8 solution (Dojindo Laboratories) was added to each well. After incubation for 3 hours and 15 minutes at 37¡ãC, A450 was measured using a Victor5 Light Luminescence Counter (PerkinElmer Life Sciences, Boston, http://www.perkinelmer.com), with standards of known cell numbers prepared in the same manner used to determine cell numbers for the experimental conditions.

To detect apoptotic cells, cultures were fixed 3 days post-transfection and stained with a fluorescence-based BD ApoAlert DNA fragmentation assay kit (BD Biosciences) following the manufacturer¡¯s protocol.; Q/ v+ r8 N$ E7 h7 c& P$ A
$ e+ [1 c4 g+ a  @* Q8 V+ S
Image Analysis

Light and epifluorescence microscopy were done using a Leica DM RX/E microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com) with appropriate filters and captured with an ORCA-ER CCD digital camera (Hamamatsu Photonics) using Openlab software (Improvision, Inc.). All images were processed using Adobe Photoshop 7.0.
' H! c2 y  T2 k: \7 i
RESULTS  p/ F9 L+ ^- N& a7 ^" y

Committed Adult hMSCs Regain Their Multipotency Following Dedifferentiation
) c% i1 D+ w: b
Previously, we demonstrated that adult hMSCs were capable of being induced to acquire the characteristics of other cell types after they were fully committed, a phenomenon termed transdifferentiation . In this study, to assess how differentiated stem cells maintain their capability to acquire a completely new phenotype, we first assessed whether differentiated hMSCs could dedifferentiate prior to transdifferentiation. As shown in Figure 1A, undifferentiated hMSCs induced in vitro for 21 days acquired the typical characteristics of mature osteoblasts, adipocytes, and chondrocytes. However, upon withdrawal of the induction stimuli, these fully differentiated cells gradually lost their characteristic markers (Fig. 1A) and appeared spindle-shaped, similar to undifferentiated hMSCs (Fig. 1B), suggesting that mature cells might have dedifferentiated and regained the properties of primitive hMSCs, that is, self-renewal and multipotency.

Figure 1. Histochemical and immunohistochemical analyses of undifferentiated, differentiated, and subsequently dedifferentiated human mesenchymal stem cells (hMSCs). (A): Osteoblast formation was induced by culturing hMSCs with dexamethasone (Dex), ascorbate, ß-glycerophosphate, and VD3 and assessed on the basis of histochemically detectable, membrane-bound ALP activity (a¨Cc). Adipocytes were induced by culturing hMSCs with BM supplemented with 1 µM dexamethasone, 1 µg/ml insulin and 0.5 mM 3-isobutyl-1-methylxanthine and identified by the presence of Oil Red O-stained neutral lipids in the cytoplasm (d¨Cf). Chondrocyte formation of hMSCs was induced in a transforming growth factor-ß3-supplemented, serum-free, high-density pellet culture and determined by the secretion of Alcian Blue-stained sulfated proteoglycan (g¨Ci) and collagen type II-stained extracellular matrix (j¨Cl). Differentiated cells exhibited typical phenotypes as evidenced by the specific staining, whereas dedifferentiated cells, similar to undifferentiated hMSCs, lacked these phenotypic markers. (B): Crystal violet staining of undifferentiated hMSCs (a) and dedifferentiated cells from osteoblasts (b), adipocytes (c) and chondrocytes (d). Dedifferentiated cells exhibited similar fibroblastic morphology as undifferentiated hMSCs. Bar = 10 µm. Abbreviation: COL II, collagen type II.' f0 R& A; H$ b

Global Gene Expression Analysis During Stem Cell Differentiation and Dedifferentiation
# n4 G8 Y: P3 z- W5 D5 G  M" i2 G
If the dedifferentiated cells had regained the potential of hMSCs, it was reasonable to predict that they would share a similar gene expression profile to primitive stem cells. Thus, the differentiation and dedifferentiation culture system employed here provided a useful platform to identify genes that control stem cell self-renewal and differentiation. Global gene expression levels were determined and compared in undifferentiated hMSCs, differentiated osteoblasts, chondrocytes, and adipocytes, as well as dedifferentiated cells from osteoblasts, chondrocytes, and adipocytes. Genes were categorized into two groups according to their expression patterns (Fig. 2A). "Differentiation genes" included genes that showed increased expression during differentiation but decreased expression during dedifferentiation. On the other hand, "stemness genes" included genes that were downregulated during differentiation but upregulated during dedifferentiation. As expected, dedifferentiated cells exhibited a gene expression profile similar to that of undifferentiated hMSCs, and both groups of cells were distinct from differentiated cells, as analyzed by PCA (Fig. 2B). Within the differentiated groups, cells from the individual lineages exhibited different expression profiles, indicating the intrinsic uniqueness of each cell type.

Figure 2. Identification of genes regulating stem cell self-renewal and multilineage differentiation. (A): Schematic diagram depicting the experimental design. Genes were selected based on their expression change during differentiation and dedifferentiation processes. (B): Principal component analysis of global gene expression data. Differentiated cells (i.e., AD, OS, and CH) exhibited a significantly different global gene expression profile compared with undifferentiated human mesenchymal stem cells (hMSCs) and dedifferentiated cells (De-AD, De-CH, and De-OS). On the other hand, undifferentiated and dedifferentiated cells shared a similar global gene expression profile. (C): Venn diagrams showing the genes that changed their expression levels during differentiation and dedifferentiation processes by at least twofold (p 4 y' y5 S  h- ^% s) Y
! F: [/ c, d( `% Y  ^: T
As shown in Figure 2, there are 460 genes in the stemness genes group, with more than twofold significant decrease during differentiation and increase during dedifferentiation, in at least one lineage. Among these, 62 genes exhibited similar expression patterns in two lineages, and 11 genes in all three lineages (supplemental online Table 2). In the differentiation genes group, 456 genes were upregulated during differentiation and downregulated during dedifferentiation significantly, by more than twofold in at least one lineage, with 12 genes shared by all three lineages and 40 genes by two lineages (supplemental online Table 1). Six genes from differentiation genes group that are typical markers of individual lineage were selected for quantitative RT-PCR analysis, including BSP and OC for osteoblasts, LPL and FABP4 for adipocytes, and COMP and MMP13 for chondrocytes. Although they confirmed the GeneChip data, the quantitative PCR results revealed much higher fold changes in gene expression during both differentiation and dedifferentiation processes in all six genes (supplemental online Table 3). In addition, the fold change between dedifferentiated and undifferentiated cells was close to one, suggesting that the two cell populations are similar at the transcription level as indicated by PCA. Taken together, these results confirmed our hypothesis and proved the effectiveness of our system to identify candidate genes controlling stem cell self-renewal and multipotency.  T4 [# \! B$ l) `* k
; ?. w. W( ~! e1 s8 @! P8 \* ?
In general, 37% of differentially expressed genes belong to one of eight major canonical pathways: integrin signaling (RHOJ, ITGA10, FN1, RAP2B, ITGA7, RHOU, COL4A1, ITGA11, LAMB3, LAMA4, PIK3R1, AKT3, MAPK3, COL4A2, LAMA2), IGF-1 signaling (YWHAH, IGF1, IGFBP5, IGFBP4, IGFBP3, IGFBP7, PIK3R1, AKT3, PRKAR2B, MAPK3, FOXO1A), G-protein-coupled receptor signaling (NFKBIA, PDE3A, PDE1C, RGS2, EDNRB, PIK3R1, AKT3, PRKAR2B, RGS4, MAPK3, AGTR1), IL-6 signaling (NFKBIA, HSPB1, IL6, IL1R1, interleukin 1 receptor type II , LBP, JAK2, TNFAIP6, MAPK3, MAP2K3), insulin receptor signaling (PPP1CB, JAK2, PIK3R1, PTPRF, AKT3, PRKAR2B, MAPK3, SGK, FOXO1A, NCK1), pyrimidine metabolism (UPP1, TXNRD1, POLE4, REV3L, ENTPD1, NME1, TRUB2, NT5E, DUT, DPYSL3), nuclear factor-B signaling (NFKBIA, TNFSF11, NGFB, IL1R2, IRAK3, PIK3R1, AKT3, IL1R1, TLR2), and Wnt/ß-catenin signaling (SFRP4, ACVR2, FZD1, FZD7, WNT5A, LEF1, TCF3, AKT3, CDH2, DKK3).
1 Q4 F* r, n. S" O7 k
The identified stemness genes and differentiation genes are involved in different cellular processes and functions. For example, a large percentage of stemness genes are involved in signaling pathways, such as IGF-1 signaling (YWHAH, IGF1, IGFBP4, IGFBP5, IGFBP3, AKT3, MAPK3), JAK/Stat signaling (STAT1, STAT4, SOCS2, SOCS5), TGF-ß signaling (INHBA, SMURF2, SMAD3, SERPINE1, ACVR2), and Wnt/ß-catenin signaling (SFRP4, WNT5A, FZD7, CDH2, TCF3, DKK3). On the other hand, the differentiation genes group contains genes involved largely in metabolism. For instance, GCLC, GLUL, and GSS in glutamate metabolism and GPX3, ANPEP, GCLC, and GSS in glutathione metabolism. Genes in nuclear factor B (NF-B) signaling (NFKBIA, IL1R2, IRAK3, PIK3R1, TLR2) and death receptor signaling (TNFSF1, BIRC3) are also significantly represented in the differentiation genes group. Among the genes that shared expression pattern in more than two lineages, those involved in organ morphology, renal and urological disease, amino acid metabolism, dental disease, organismal survival, and free radical scavenging are highly represented in the differentiation genes group, whereas the stemness genes group contains genes that are primarily involved in cell morphology, cancer, cell-to-cell signaling and interaction, cellular growth and proliferation, nervous system development and function, tissue development, and tumor morphology.0 E& C( Y2 B- T) {# v2 T
: _! @6 U$ |* ^1 @: A0 U  S
Ninety-one genes in the stemness genes group encode proteins that are cell surface proteins and/or receptors, including 20 genes shared by at least two lineages (Table 1). Except for a few genes whose functions are unknown, the majority of these genes function in defined cellular processes, such as metabolism (ATPase and solute carrier proteins), carcinogenesis and metastasis (Tetraspanin family members), cell growth and survival and senescence (AXL, TNFRSF10D), development (NOTCH2, NUMB, JAG1), and signal transduction (PTPRF, FZD7, ICAM1).
6 f1 J! E" E$ I& `, w
Table 1. Selected cell surface proteins and receptors- U$ c/ l" I, I: |4 \

Functional Analysis of Genes Involved in Stem Cell Self-Renewal and Multipotency
8 M9 W- N5 m) ]2 T$ F4 J' [
As proof of principle, five genes from the stemness genes group were selected based on their unique expression pattern in individual lineage and their cellular function. By reducing their expression level using siRNA, their effects on hMSC expansion and multilineage differentiation were accessed. Initially identified from GeneChip data, PTPRF exhibited similar expression patterns in all three lineages, AFAP and RAB3B in two lineages, and FZD7 and DKK3 in only one lineage. However, quantitative RT-PCR analysis demonstrated that AFAP and RAB3B also shared a pattern similar to that of PTPRF in all three lineages (Fig. 2D; supplemental online Table 3), whereas FZD7 and DKK3 appeared to behave differently in chondrogenic lineage than in osteogenic and adipogenic lineages (Fig. 2D; supplemental online Table 3). Using gene-specific siRNA, we successfully reduced the gene expression level by 60%¨C80% compared with transfection controls (Fig. 3A). Protein levels were also reduced for all five genes as confirmed by Western analysis (Fig. 3B) and immunofluorescence staining (Fig. 3C), and inactivation of these genes was sustained for at least 7 days post-siRNA transfection.

Figure 3. Reduction of gene expression using siRNA transfection. (A): Real-time reverse transcription-polymerase chain reaction analysis of transcript levels of five selected genes 7 days post-transfection. On average, expression of selected genes was significantly reduced by 60%¨C80% of untransfected and transfection controls. Values are mean ¡À SD (n = 3). *, p
  T9 Y( G& `* g1 w1 K
When cultured in basal medium, hMSCs with reduced level of AFAP, PTPRF, RAB3B, FZD7, or DKK3 exhibited a slower growth rate up to 9 days post-transfection, with a more dramatic difference observed in cells with inactivated DKK3 and PTPRF, but little difference after 9 days, except in DKK3-inactivated cells, compared with controls (Fig. 4A). These results suggest that these genes have little effect on stem cell proliferation when functioning alone. In addition, reduction of each of these five genes dramatically increased the percentage of cells undergoing apoptosis (Fig. 4C), suggesting that these genes function as cell survival protectors.
! h; M' J- l% m
Figure 4. Effect of gene reduction by siRNA transfection on human mesenchymal stem cell proliferation and cellular viability. (A): Growth curves showing cell proliferation over 14 days post-siRNA transfection. Cells exhibited a slightly slower rate of proliferation from day 1 to day 8 but a similar rate after day 8, compared with both untransfected control and transfection control. Values are mean ¡À SD (n = 3). (B): Gene knockdown by siRNA transfection increased the percentage of apoptotic cells 3 days post-transfection. Mean values are presented (n = 2). Abbreviations: AFAP, actin filament-associated protein; DKK3, dickkopf 3; FZD7, frizzled 7; PTPRF, protein tyrosine phosphatase receptor F.' C( K" I+ h! v6 K- H' r+ t; ~5 e" }
) c2 h! o* V6 Q: A- K8 ]
hMSCs with reduced expression of these five genes were challenged to undergo differentiation into three mesenchymal lineages. As shown in Figure 5, cells with inactivated AFAP, DKK3, FZD7, PTPRF, or RAB3B were able to differentiate into osteoblasts with enhanced ALP activity (Fig. 5A). Reduction of AFAP, FZD7, or RAB3B enhanced ALP transcription significantly (Fig. 5B). Furthermore, inactivation of FZD7 significantly increased OC expression, whereas others appeared to have little difference compared with controls (Fig. 5C). Unlike the uniform enhancement on osteogenesis, inactivation of each of these five genes exhibited a unique impact on chondrogenesis. As shown in Figure 6, reduction of DKK3 increased the production of Alcian Blue-stained sulfated proteoglycan but decreased the collagen type II. Inactivation of AFAP and RAB3B had an indiscernible effect on sulfated proteoglycan but a dramatic increase on collagen type II deposition. On the other hand, reduction of either FZD7 or PTPRF decreased both proteoglycan and collagen type II synthesis. Contrary to the enhancement of osteogenesis and varied effects on chondrogenesis, inactivation of any of these five genes suppressed adipogenesis, demonstrated by the reduction in the number of Oil Red O-positive adipocytes (Fig. 6).
, P; f& G6 E. c* a* ]5 |
Figure 5. Osteogenic differentiation potential of human mesenchymal stem cells was altered by siRNA-mediated reduction of gene expression. (A): Alkaline phosphatase (ALP) staining of transfected cells cultured in the absence or presence of osteogenic induction medium for 14 days. Compared with controls, transfected cells showed enhanced ALP staining at the cellular level in osteogenic medium (bottom row). In addition, the number of ALP-positive cells increased in four transfected cell populations (AFAP, FZD7, PTPRF, and RAB3B), even in the absence of osteogenic stimuli (top row). (B): ALP expression levels analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Reduction of AFAP, FZD7, and RAB3B significantly increased ALP transcription. (C): Osteocalcin (OC) expression levels analyzed by quantitative RT-PCR. Reduction of FZD7 expression level increased OC transcription significantly, whereas other genes had little effect on OC level. For (B) and (C), all values are mean ¡À SD (n = 3). *, p ) Z% B4 z7 X' s3 c$ z" o
3 |  e0 _, T; j6 @, p* U/ n
Figure 6. Chondrogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs) was altered by siRNA gene reduction. Chondrogenesis of hMSCs was assessed on the basis of Alcian Blue staining of sulfated proteoglycan matrix and collagen type II immunostaining after cells were induced in chondrogenic medium for 21 days post-transfection. Sections from two individual pellets are shown for each staining. The level of sulfated proteoglycan was increased in cells transfected with DKK3 siRNA and decreased in those transfected with FZD7 or PTPRF siRNA. The sulfated proteoglycan level was not significantly changed in cells transfected with AFAP or RAB3B siRNA. On the other hand, reduction of AFAP or RAB3B increased collagen type II production, whereas reduction of DKK3, FZD7, or PTPRF decreased it. Adipogenesis was detected by the presence of Oil Red O-stained neutral lipids in the cytoplasm. All transfected cells exhibited fewer adipocytes after a 21-day induction. Bar = 10 µm. Abbreviations: AFAP, actin filament-associated protein; DKK3, dickkopf 3; FZD7, frizzled 7; PTPRF, protein tyrosine phosphatase receptor F; TC, transfected control; UC, untransfected control.

DISCUSSION

Transdifferentiation refers to the process in which stem cells of a certain lineage differentiate into cell types of a different lineage across embryonic germ layers or the process in which fully differentiated cells switch their phenotype and acquire characteristics of other cell types within or beyond their original lineages. For example, Park et al. . Similarly, differentiated cells could dedifferentiate, losing their committed phenotype by suppressing the lineage-specific genes while activating those that are responsible for stem cell maintenance. Since the dedifferentiation process is a functional reversal of differentiation, we can reasonably argue that genes maintaining the stemness of cells would be downregulated during differentiation and elevated during dedifferentiation.9 H  Y7 q1 Q+ D# L# x$ y
- R3 i* Y6 h" I8 H& U) z
The in vitro differentiation and dedifferentiation system developed here provides a useful platform to identify genes that might play crucial roles in stem cell self-renewal, maintenance, and multilineage differentiation. Particularly, by comparing the differentially expressed genes in three mesenchymal lineages, we were able to select a group of genes common in multiple cell types, which likely serve as markers of uncommitted hMSCs or function as regulatory factors for differentiation. One interesting gene in the differentiation genes group is IL1R2, which is involved in multiple signaling pathways (NF-B, p38 MAPK, PPAR, and IL-6 signaling) . At present, the exact molecular mechanism of NF-B action in stem cell differentiation is not known and requires further investigation.- L8 r5 [3 S0 V; \; d
# c# n7 g% m7 Y+ g7 }6 E7 R3 s
Our culturing and screening system generated a list of genes that were identified previously to be enriched in undifferentiated hMSCs, including Thy-1, epithelial protein lost in neoplasm ß, biglycan, dickkopf 3, decorin, thrombospondin1, steroid-sensitive gene 1, CD73, and inhibin ß A . This implies that similar regulatory mechanisms might exist to control stem cell maintenance and commitment regardless of their origin or pluripotency. However, we did not detect the typical ESC markers (e.g., OCT-4, NANOG, SOX4, or FGF4) in MSCs. Lack of these markers in MSCs could be due to their subdetection expression levels or might reflect their difference from pluripotent ESCs.

Adult hMSCs are usually isolated based on their adhesion to plastic, which results in a morphologically, phenotypically, and functionally heterogeneous population of cells . However, very few cell surface markers were identified. In contrast, in this study, 91 genes were identified to encode surface antigens. Some genes encode well-known cell surface receptors for stem cells, such as THY-1 and CD151, whereas others appear unique in MSCs. Because of their kinetic expression profiles during differentiation and dedifferentiation, cell surface receptors identified from at least two independent lineages are likely the alternative candidate markers to enrich or purify a homogeneous population of human MSCs. Further investigation is required to confirm the feasibility of using these markers.

Stem cells in adult connective tissues, such as MSCs, are assumed to maintain a mitotically quiescent state in their native environment. Even upon stimulation by injury or remodeling, MSCs are expected to undergo tightly controlled proliferation and differentiation, as extensive cell division or differentiation may result in oncogenic conditions or excess tissue that interferes with normal physiological function, respectively. Similar regulation may also operate in cultured MSCs in vitro. The complexity of balancing cell survival, proliferation, and differentiation naturally requires the functioning and cross- talk of multiple signaling pathways. We have identified here several signaling factors that are highly expressed in undifferentiated hMSCs, including those in phosphatidylinositol-3-kinase (PI3K) signaling (PTPRF, AFAP, and RAB3B) and Wnt/ß-catenin signaling (FZD7 and DKK3). As expected, all five genes exhibited similar effects on cell apoptosis and proliferation, protecting MSCs from extensive cell division as well as from cell death. PTPRF, AFAP, and RAB3B likely function through PI3K and AKT or ERK1/2 to balance cell growth or apoptosis, whereas ß-catenin/LEF/TCF mediate the effects of FZD7 and DKK3. On the other hand, these five genes play different roles in lineage-specific commitment. Consistent with their expression pattern during differentiation and dedifferentiation, all five genes suppress osteogenesis. In addition, as expected, AFAP and RAB3B inhibit chondrogenesis, whereas DKK3 and FZD7 promote chondrocyte formation. However, PTPRF appears to enhance chondrogenesis, and all five genes seem to stimulate adipogenic commitment, which is inconsistent with their reduced expression during differentiation and our prediction. The mechanisms by which these genes act to effect these changes are not known, nor is the relationship of gene expression level and its normal function. Since adipogenesis is a cell-density dependent process, one possible explanation is that increased cell death caused by gene inactivation could reduce cell density, which in turn suppresses adipocyte formation. Furthermore, it is very likely that more than one signaling pathways are required during chondrogenesis of hMSCs, which cross-talk and function in a collaborative and temporal matter.

In conclusion, our study demonstrates the self-renewal, maintenance, and multilineage commitment of adult MSCs are tightly regulated by a variety of signaling pathways in a collaborative matter. The cell surface molecules identified here provide candidate markers for the enrichment and purification of genuine MSCs, as well as their identification in situ. A homogeneous population of MSCs will greatly facilitate the functional analysis of the molecular mechanisms controlling these cells. Moreover, the elucidation of the signaling pathways will enhance our ability to maintain, propagate, and expand functional MSCs in vitro; to obtain multipotential cells by inducing dedifferentiation in committed cells; and to guide MSC differentiation into specific lineage(s) for cell therapy and tissue engineering applications.

Disclosures
3 A( \$ N: y  V& F* z
The authors indicate no potential conflicts of interest.
- j: n* _$ B% \# Y% A
ACKNOWLEDGMENTS. T3 K/ O$ m- f' n. M: f9 I3 x

We thank Drs. Hong-wei Sun and Shen Huang for assistance in GeneChip data analysis. The research was funded in part by the Intramural Research Program of National Institute of Arthritis & Musculoskeletal & Skin Diseases, NIH (Z01 AR41113).
      【参考文献】
6 u- d% x$ M6 A" m4 ?( R

Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: Stem cells and their niche. Cell 2004;116:769¨C778.
- c% a& n7 }" r; K
Hirao A, Arai F, Suda T. Regulation of cell cycle in hematopoietic stem cells by the niche. Cell Cycle 2004;3:1481¨C1483.
' R! G  C$ m: W+ e/ T4 X
Roelen BA, Dijke P. Controlling mesenchymal stem cell differentiation by TGFBeta family members. J Orthop Sci 2003;8:740¨C748.
: q) e  q" I& ?" c: @  D. D
Kratchmarova I, Blagoev B, Haack-Sorensen M et al. Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. Science 2005;308:1472¨C1477.: ^7 X" M; S2 E7 p/ H. m  f
( [( q0 y, I  X5 Z" u) m3 S) f
Mannello F, Tonti GA, Bagnara GP et al. Role and function of matrix metalloproteinases in the differentiation and biological characterization of mesenchymal stem cells. STEM CELLS 2006;24:475¨C481." M: A% ?# E% `9 M; r
7 l! x9 N  D9 n% @
Gregory CA, Prockop DJ, Spees JL. Non-hematopoietic bone marrow stem cells: Molecular control of expansion and differentiation. Exp Cell Res 2005;306:330¨C335.
; m$ _6 W, C) P1 N7 U
Kortesidis A, Zannettino A, Isenmann S et al. Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells. Blood 2005;105:3793¨C3801.6 f( X5 A2 X4 a( p' s. s. I

Cho KJ, Trzaska KA, Greco SJ et al. Neurons derived from human mesenchymal stem cells show synaptic transmission and can be induced to produce the neurotransmitter substance P by interleukin-1 alpha. STEM CELLS 2005;23:383¨C391.
8 ~! V/ k  S: k' D2 u! C
Wislet-Gendebien S, Hans G, Leprince P et al. Plasticity of cultured mesenchymal stem cells: Switch from nestin-positive to excitable neuron-like phenotype. STEM CELLS 2005;23:392¨C402.$ ~+ c, v* o5 k9 A

Keene CD, Ortiz-Gonzalez XR, Jiang Y et al. Neural differentiation and incorporation of bone marrow-derived multipotent adult progenitor cells after single cell transplantation into blastocyst stage mouse embryos. Cell Transplant 2003;12:201¨C213.& D0 T! O- @# ~. L

Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41¨C49.9 Z6 E9 ^3 d' U! l2 Q1 f

Sato Y, Araki H, Kato J et al. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 2005;106:756¨C763.: i$ ^8 X/ ^- `
/ a' L- Q0 w8 K7 l6 x
Song L, Tuan RS. Transdifferentiation potential of human mesenchymal stem cells derived from bone marrow. FASEB J 2004;18:980¨C982.( H! M2 T3 [  |7 P
7 M1 x& L8 o  o$ f' n# F' S$ V. m
Kelly DL, Rizzino A. DNA microarray analyses of genes regulated during the differentiation of embryonic stem cells. Mol Reprod Dev 2000;56:113¨C123.

Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": Transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597¨C600.! t& |* @# |  v% S, M

Tanaka TS, Kunath T, Kimber WL et al. Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity. Genome Res 2002;12:1921¨C1928.8 p: Z1 A! w8 Q

Sato N, Sanjuan IM, Heke M et al. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 2003;260:404¨C413.
/ Q0 N4 A  M* H6 t) I
Bhattacharya B, Miura T, Brandenberger R et al. Gene expression in human embryonic stem cell lines: Unique molecular signature. Blood 2004;103:2956¨C2964.( o1 b7 ]' o6 {& c. f/ I* R
& b# I# H- H. \1 A
Sperger JM, Chen X, Draper JS et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci U S A 2003;100:13350¨C13355.0 ]$ T6 `" b& Y8 w
- \! b2 k8 P; P5 K4 H0 _
Gallardo TD, Hammer RE, Garry DJ. RNA amplification and transcriptional profiling for analysis of stem cell populations. Genesis 2003;37:57¨C63.

Rao RR, Calhoun JD, Qin X et al. Comparative transcriptional profiling of two human embryonic stem cell lines. Biotechnol Bioeng 2004;88:273¨C286.
& Y4 u/ R( R& r, E: d. n
Golan-Mashiach M, Dazard JE, Gerecht-Nir S et al. Design principle of gene expression used by human stem cells: Implication for pluripotency. FASEB J 2005;19:147¨C149.' B- A# t1 m) o7 }
3 O7 }7 ]: [3 X; J% G
Wei CL, Miura T, Robson P et al. Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. STEM CELLS 2005;23:166¨C185.- c8 z8 K  k: Z" a. ^: a

Ivanova NB, Dimos JT, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601¨C604.
3 ]* l# {) b) ^/ H( G4 A5 x
Wen T, Gu P, Minning TA et al. Microarray analysis of neural stem cell differentiation in the striatum of the fetal rat. Cell Mol Neurobiol 2002;22:407¨C416.
7 w9 i/ X  B2 p% t& g5 U
Tumbar T, Guasch G, Greco V et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359¨C363.* p* z. s3 Z; {& R

Blanpain C, Lowry WE, Geoghegan A et al. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 2004;118:635¨C648.
  k' B3 c8 d  ~3 U$ r) b
Suarez-Farinas M, Noggle S, Heke M et al. Comparing independent microarray studies: The case of human embryonic stem cells. BMC Genomics 2005;6:99.( k8 C8 C. D, N$ r# c

Short B, Brouard N, Occhiodoro-Scott T et al. Mesenchymal stem cells. Arch Med Res 2003;34:565¨C571., s# T, b( |. Q, }* R& c
/ I: m- @# i( @, x1 ]
Phinney DG. Building a consensus regarding the nature and origin of mesenchymal stem cells. J Cell Biochem Suppl 2002;38:7¨C12.4 @6 q+ F+ |% `" s
0 ]* \9 Z) z0 D+ j
Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med (Maywood) 2001;226:507¨C520.4 m& d1 \2 q: w2 ~

Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: Characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004;8:301¨C316.2 {" @7 O; i( z' C$ n- d4 j

Shi S, Robey PG, Gronthos S. Comparison of human dental pulp and bone marrow stromal stem cells by cDNA microarray analysis. Bone 2001;29:532¨C539.
5 X# H1 ]3 x3 r
Tremain N, Korkko J, Ibberson D et al. MicroSAGE analysis of 2,353 expressed genes in a single cell-derived colony of undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages. STEM CELLS 2001;19:408¨C418.
( O4 w& t$ [$ H, R% M
Jia L, Young MF, Powell J et al. Gene expression profile of human bone marrow stromal cells: High-throughput expressed sequence tag sequencing analysis. Genomics 2002;79:7¨C17./ Y6 D, l* G8 a' N/ L! H

Jeong JA, Hong SH, Gang EJ et al. Differential gene expression profiling of human umbilical cord blood-derived mesenchymal stem cells by DNA microarray. STEM CELLS 2005;23:584¨C593.5 R" S4 p( ^& a/ n  o( ~) ~

Silva WA Jr., Covas DT, Panepucci RA et al. The profile of gene expression of human marrow mesenchymal stem cells. STEM CELLS 2003;21:661¨C669.
4 y6 g/ C6 h2 y
Wagner W, Wein F, Seckinger A et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 2005;33:1402¨C1416.
' A# O" R& ^& L
Gotherstrom C, West A, Liden J et al. Difference in gene expression between human fetal liver and adult bone marrow mesenchymal stem cells. Haematologica 2005;90:1017¨C1026.
( Q. l! I/ t: B/ ^; h  Y! B+ C, Y
Djouad F, Bony C, Haupl T et al. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res Ther 2005;7:R1304¨CR1315.
/ Z: e* {% _5 s. l. Z- y3 T- ?
Qi H, Aguiar DJ, Williams SM et al. Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc Natl Acad Sci U S A 2003;100:3305¨C3310.2 d* w- s5 m1 D% r  r3 P! K
+ [% X! s" U/ a- S, y, v) u
Doi M, Nagano A, Nakamura Y. Genome-wide screening by cDNA microarray of genes associated with matrix mineralization by human mesenchymal stem cells in vitro. Biochem Biophys Res Commun 2002;290:381¨C390./ D1 C+ D6 S6 }0 B, k1 @! d( s$ [
& b* e& D# H8 ?
Sekiya I, Vuoristo JT, Larson BL et al. In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A 2002;99:4397¨C4402.

Sekiya I, Larson BL, Vuoristo JT et al. Adipogenic differentiation of human adult stem cells from bone marrow stroma (MSCs). J Bone Miner Res 2004;19:256¨C264.: f9 }6 Z& [' a, ~8 m

Park SR, Oreffo RO, Triffitt JT. Interconversion potential of cloned human marrow adipocytes in vitro. Bone 1999;24:549¨C554.+ M( y$ v% u; W7 r
7 J/ J9 z4 o( p# `- I2 {
Tagami M, Ichinose S, Yamagata K et al. Genetic and ultrastructural demonstration of strong reversibility in human mesenchymal stem cell. Cell Tissue Res 2003;312:31¨C40.- ~9 E5 X, S3 J) }1 S
4 @- U- d) `" Q( c. w) V2 Z
Tsonis PA. Regeneration in vertebrates. Dev Biol 2000;221:273¨C284.

Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res 2002;69:908¨C917.
7 P# ?/ x! ?3 J- Z) M
Muzio M, Ni J, Feng P et al. IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 1997;278:1612¨C1615.( F) u3 y+ F! H) Z1 F( u
# {- {+ }2 h& q
Daun JM, Fenton MJ. Interleukin-1/Toll receptor family members: Receptor structure and signal transduction pathways. J Interferon Cytokine Res 2000;20:843¨C855.
- T0 O: Z6 h4 {" H  P3 P3 l$ O5 C) P
Pinteaux E, Parker LC, Rothwell NJ et al. Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells. J Neurochem 2002;83:754¨C763.
( Z" z$ W- U6 s+ @4 h9 A
Re F, Sironi M, Muzio M et al. Inhibition of interleukin-1 responsiveness by type II receptor gene transfer: A surface "receptor" with anti-interleukin-1 function. J Exp Med 1996;183:1841¨C1850.* D& C- S4 M6 f/ Y
  x- u3 w- X, G$ |
Mracek T, Cannon B, Houstek J. IL-1 and LPS but not IL-6 inhibit differentiation and downregulate PPAR gamma in brown adipocytes. Cytokine 2004;26:9¨C15.5 ^) L6 b; g& g5 o9 Y+ j7 y. _  p

Yamasaki S, Nakashima T, Kawakami A et al. Cytokines regulate fibroblast-like synovial cell differentiation to adipocyte-like cells. Rheumatology (Oxford) 2004;43:448¨C452.
  @$ s9 p; n7 b9 Z
Suzawa M, Takada I, Yanagisawa J et al. Cytokines suppress adipogenesis and PPAR-gamma function through the TAK1/TAB1/NIK cascade. Nat Cell Biol 2003;5:224¨C230.$ G; b% ]- s% [

Francois M, Richette P, Tsagris L et al. Peroxisome proliferator-activated receptor-gamma down-regulates chondrocyte matrix metalloproteinase-1 via a novel composite element. J Biol Chem 2004;279:28411¨C28418., H7 z8 _5 n: e, W, o+ |; j

Gronthos S, Zannettino AC, Hay SJ et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci 2003;116:1827¨C1835.

收藏0 分享0 顶0 踩0