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Identification of Common Pathways Mediating Differentiation of Bone Marrow- and

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发表于 2009-3-5 00:56 |显示全部帖子
作者:Tong Ming Liua,b,d, Monique Martinaa,b, Dietmar W. Hutmachera,b,c, James Hoi Po Huia,b, Eng Hin Leea,b, Bing Limd,e
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. Q9 ?/ h* t- d- V          【摘要】
6 j; N0 p, B1 V& p& n$ Z      Mesenchymal stem cells derived from human bone marrow (hBMSCs) and human adipose tissue (hAMSCs) represent a useful source of progenitor cells for cell therapy and tissue engineering. However, it is not clear what the similarities and differences between them are. Like hBMSCs, hAMSCs can differentiate into osteogenic, adipogenic, and chondrogenic cells. Whether MSCs derived from different tissue sources represent fundamentally similar or different cell types is not clear. Given the possible different sources of MSCs for cell therapy, a comprehensive comparison of the different MSCs would be very useful. Here, we compared the transcriptome profile of hAMCS and hBMSCs during directed differentiation into bone, cartilage, and fat. Our data revealed considerable similarities between bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (AMSCs). We uncovered an interesting bifurcation of pathways in both BMSCs and AMSCs, in which osteogenesis and adipogenesis appear to be linked in a differentiation branch separate from chondrogenesis. Our data suggest that although a set of common genes may be needed for early differentiation into all three lineages, a different set of signature genes is associated with maturation into fully differentiated cells. The recruitment of different late differentiation factors explains and supports our conclusion that BMSCs differentiate more efficiently into bone and cartilage, whereas AMSCs differentiate better into adipocytes. This study not only generated a rich database for continuing molecular characterization of various MSCs but also provided a rational basis for assessing qualities of MSCs from different sources for the purpose of cell-based therapy and tissue engineering.   O: H. i% u) o( Y- V- a" L" _
          【关键词】 Gene expression profile Mesenchymal stem cells Adipose tissue Bone marrow FKBP% e8 ^- P; N! k  i2 c% p" l: i
                  INTRODUCTION
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  v8 G3 t8 m0 B9 aMesenchymal stem cells derived from human bone marrow (hBMSCs) represent a source of pluripotent cells that are already in various phases of clinical application . Little is known about similarities and differences between BMSCs and AMSCs at the genetic level and during their differentiation into the three major mesenchymal lineages.9 x, i( m% A7 K, K& O

( w* @6 U9 m6 F  t0 jTo make better use of MSCs for cell-based therapy and tissue engineering, it is useful to understand the process that governs initial commitment and further differentiation into various mesenchymal lineages. In this study, we compared the gene expression profile of human mesenchymal stem cells (hMSCs) derived from adipose tissue and bone marrow during differentiations toward three common mesenchymal lineages. Our data suggest that a set of genes that are upregulated during differentiation is needed for differentiation into all three lineages, whereas late-differentiation genes are essential for terminal differentiation. One of the genes that appear to have a positive role in early differentiation is FKBP5, an immunophilin-binding protein involved in modulating hormone receptor response and transcription regulation.% W6 B) w" n, Z' G& G# {, k6 Z! q3 A
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MATERIALS AND METHODS
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MSC Culture and Osteogenic, Chondrogenic, and Adipogenic Differentiation
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* _0 w" B5 L% C6 C" r! uAfter informed consent was obtained and following institutional review board guidelines from the National University Hospital of Singapore, hAMSCs were cultured as described  was used for comparison in chondrogenesis between BMSCs and AMSCs and functional study of FKBP5 in chondrogenesis. Briefly, 2 x 105 MSCs were placed in a 15-ml polypropylene tube (Falcon; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and centrifuged to pellet. The pellet was cultured at 37¡ãC with 5% CO2 in 500 µl of chondrogenic media that contained 10 ng/ml TGF-¦Â3, 10¨C7 M dexamethasone, 50 µg/ml ascorbate-2-phosphate, 40 µg/ml proline, 100 µg/ml pyruvate, and 50 mg/ml ITS Premix (Becton, Dickinson and Company; 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 µg/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid). The medium was replaced every 3¨C4 days for 21 days. Oil red stain for adipogenesis and Alizarin Red S stain for calcium deposition in osteogenesis were examined by area of positive stain with BioQuant software (BioQuant, Nashville, TN, http://www.bio-quant.com), COLII for chondrogenesis was examined by quantitative polymerase chain reaction.
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cDNA Microarray Analysis
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Total RNA was isolated from MSCs or MSCs induced differentiation to the osteocytes, chondrocytes, and adipocytes using the RNeasy mini-kit (Qiagen, Chatsworth, CA, http://www1.qiagen.com) per the manufacturer's protocol. In brief, 1.5 µg of total RNA was used to synthesize double-strand DNA using one-cycle cDNA synthesis kit. cDNA was purified by using a Sample Cleanup Module (Qiagen). In vitro transcription was performed to produce biotin-labeled cRNA using a GeneChip IVT Labeling Kit. Biotinylated cRNA was cleaned and fragmented to 50¨C200 nucleotides with the Sample Cleanup Module and hybridized for 16 hours at 45¡ãC to Affymetrix HG U133 Plus 2.0 (Santa Clara, CA, http://www.affymetrix.com), containing more than 54,675 human genes. After being washed, the array was stained with streptavidin-phycoerythrin (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). The staining signal was amplified by biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), followed by streptavidin-phycoerythrin stain, and then scanned using GCOS 3000 (Affymetrix). The data were analyzed using GeneSpring software V7.2. A t test on normalized intensity with p  .05 followed by ratio change (ratio of normalized intensity, 2.0 or 0.5) was used to generate the list of genes with significant change in expression profile during three differentiations. In this study, BMSCs from three patients and AMSCs from six patients were used.
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Quantitative Real-Time Polymerase Chain Reaction+ M6 s, c  Q! h7 z0 R2 X/ R
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To confirm the microarray data, real-time polymerase chain reaction (PCR) was performed with the TaqMan expression assay according to the manufacturer's instructions and an ABI 7700 Prism (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com); 0.75 µg of total RNA was converted to cDNA using a high-capacity cDNA archive kit and then diluted to 750 µl. Quantitative real time-PCR was done as follows: initial denaturation for 2 minutes at 50¡ãC and 10 minutes at 95¡ãC, followed by 40 cycles of PCR (95¡ãC for 15 seconds, 60¡ãC for 1 minute) by using 10 µl of 2x Master mix, 1 µl of TaqMan probe, and 9 µl of cDNA. All probes were designed with a 5' fluorogenic, 6-carboxylfluorescein, and a 3' quencher, tetramethyl-6-carboxyrhodamine. The expression of human glyceraldehyde-3-phosphate dehydrogenase was used to normalize gene expression level. Primers used for real-time PCR included CCAAT-enhancer-binding protein- (C/EBP), NOX4, osteomodulin (OMD), and FKBP5.2 E5 Y2 ~+ W' V

+ S4 r" d" o4 n/ S  A5 Y" g, vImmunoblotting Analysis
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Cells were collected by centrifugation, and the cell pellet was resuspended in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS) containing proteinase inhibitors and incubated at 4¡ãC for 30 minutes. Following centrifugation at 16,000g for 15 minutes at 4¡ãC, the supernatant containing total cell extract was collected and kept at ¨C80¡ãC. Protein from cell extract in the gel was electrophoretically transferred onto a Hybond polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). The membrane was incubated for 1 hour at room temperature in Tris-buffered saline and 0.1% Tween 20 (TBS-T) containing 5% skim milk to block nonspecific protein binding and incubated at room temperature for 1 hour with the primary antibody against FKBP5 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) diluted (1:300) in blocking buffer. Following four washes with TBS-T, the membrane was incubated for 1 hour with the horseradish peroxidase-conjugated secondary antibody diluted (1:3000) in blocking buffer for 1 hour. Antibody binding was visualized with an enhanced chemiluminescence Western blotting detection system (Amersham Biosciences).1 w/ Z0 W) \5 U: m0 \

: Q' O/ D$ P9 P8 dRNA Interference" O3 ^- g: U) x& \. _6 @4 O
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Small interfering RNA (siRNA) duplexes (Ambion, Austin, TX, http://www.ambion.com) used in this study consisted of a 21-nucleotide sense strand and a 21-nucleotide antisense strand with a 2-nucleotide T overhang at the 3' end. The sequences were as follows: FKBP5 siRNA sense, GGAGCAACAGUAGAAAUCCTT; antisense, GGAUUUCUACUGUUGCUCCTT. siRNA (FKBP5 100 nM) was introduced into hMSCs using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). MSCs were transfected with cy3-labeled Silence negative control siRNA (Ambion) as an experimental control. At 48 hours post-transfection, specific-siRNA-treated cells and control siRNA cells were analyzed with real-time PCR. To study the long-term effect of FKBP5 knockdown on differentiation of MSCs, lentiviral vector for knockdown was created by cloning short hairpin FKBP5 RNA into pLL3.7. Lentivirus was generated by cotransfecting pLentiviral vector for FKBP5 knockdown and packaging mix (Invitrogen) into 293FT cells, and supernatant was harvested after 48 hours. MSCs were infected with viral supernatant to achieve FKBP5 knockdown, and infected MSCs were induced to undergo differentiation for 14 days to evaluate adipogenesis and osteogenesis and for 21 days to evaluate chondrogenesis. The empty pLL3.7 vector with no insert was used as a control.
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Lentivirus Production and Generation of MSCs Overexpressing Stably Integrated Genes  L# W1 F$ l  c4 g' }
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FKBP5 was amplified from cDNA of BMSCs differentiated into osteogenic differentiation for 14 days, digested with BamHI and EcoRI, and then ligated into pEntry3C (Invitrogen). Via LR (attL and attR) recombination between pEntry3C and pDest6/V5 (Invitrogen), pLentiviral vector for overexpression of FKBP5 was created. Lentivirus was generated by cotransfecting pLentiviral vector for overexpression of FKBP5 and packaging mix (Invitrogen) into 293FT cells, and then MSCs were infected with viral supernatant and were selected with 5 µg/ml blastidin for 7 days. FKBP5-overexpressed MSCs were induced differentiation into three lineages for 2 days and then analyzed with real-time PCR compared with no-insert control.
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1 \( m9 \3 x: g* k& s4 l8 zData and Statistical Analysis
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Data were analyzed using GeneSpring software and using one-way analysis of variance.
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8 r$ f0 K1 b8 S& P! P/ ]! tRESULTS
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MSCs Differentiated into Adipogenic, Osteogenic, and Chondrogenic Lineages( |! p5 l, E* L) J# `; G
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hAMSCs and hBMSCs were isolated from six and three healthy donors, respectively. Their race, age, and sex are described in supplemental online Table 1. MSCs were expanded by subculture every 2¨C3 days at a 1:3 dilution. MSCs at passage 2 cultured in a T75 flask were induced into three mesenchymal lineages as described .4 ~2 \; y! B- b& n
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First, we showed that both mesenchymal stem cells were capable of differentiating into adipogenic, osteogenic, and chondrogenic cells (Fig. 1A), using histochemical staining for lineage-specific markers. Comparison of the degree of tissue-specific staining and expression of lineage-specific markers indicated that AMSCs differentiated less well into chondrocytes by a 170-fold difference in expression of COLII at day 21 and into osteocytes by a 7-fold difference in area of positive Alizarin Red stain for calcium deposition at day 14 (Fig. 1B).
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- @1 r) z8 z0 W! O' e& i; HFigure 1. Comparison of cellular and transcript changes during late adipogenesis, chondrogenesis, and osteogenesis in BMSCs and AMSCs. (A): Histochemical staining of adipocytes (oil red O), chondrocytes (Alcian Blue), and osteocytes (Alizarin Red). BMSCs and AMSCs were induced into adipogenesis for 14 days, chondrogenesis under pellet culture for 21 days, and osteogenesis for 14 days. Note the quantitative differences observable between BMSCs and AMSCs. (B): Comparisons in differentiation between BMSCs and AMSCs were examined by oil red stain for adipogenesis and Alizarin Red S stain for calcium deposition in osteogenesis by area of positive stain with BioQuant software, and COLII for chondrogenesis was examined by quantitative polymerase chain reaction. The probability associated with Student's test was performed. (C): Upregulated genes involved in lipid metabolism during adipogenesis for 14 days. t test p value was generated with GeneSpring software V7.2. Note the higher fold change in all markers in AMSC adipogenesis. (D): Upregulated extracellular matrix genes during chondrogenesis for 14 days. t test p value was generated with GeneSpring software V7.2. Note the higher fold change in the major markers (DPT and Col10A1) of chondrogenesis in BMSCs. (E): Upregulated extracellular matrix genes during osteogenesis for 14 days. t test p value was generated with GeneSpring software V7.2. Abbreviations: AMSC, adipose tissue-derived MSC; BMSC, bone marrow-derived MSC; COL10A1, collagen, type 10, 1; DPT, dermatopontin; FABP4, fatty acid-binding protein 4; FBN2, fibrillin 2; hAMSC, MSC derived from human adipose tissue; hBMSC, MSC derived from human bone marrow; LPL, lipoprotein lipase; OMD, osteomodulin; PDK4, pyruvate dehydrogenase kinase 4, isoenzyme, adipocyte; PLIN, perilipin, LOC283445, acetyl-coenzyme A carboxylase ¦Â; POSTN, periostin; SPON1, spondin 1.
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* x2 s& a9 X& J4 vTo identify genes involved in the commitment of MSCs to the three mesenchymal lineages, total RNA was isolated from undifferentiated MSCs and the MSCs at days 3 and 14 after initiating induction of differentiation into bone, cartilage, and fat cells. cDNAs were labeled and hybridized to a microarray chip (Affymetrix) that contains probes for 54,695 human genes, mostly with known functions. The gene expression profiles were compared over time between AMSCs and BMSCs during differentiation. The results revealed considerable similarity in gene expression profiles during differentiation, especially the top upregulated genes between the two types of cells (supplemental online Table 2).
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1 N# H: F% K/ {/ N0 {Reproducibility of Data Generated from Microarrays0 c/ z- O3 s+ V' k

, @( ~7 Z5 C3 `2 w0 E4 x* |4 qBecause of the genetic variability between different individuals, two methods have been used to filter out this variation. Either RNA samples from different donors can be pooled before use in cDNA array analysis . In our analysis, we applied the latter and computed for mean signal intensity between individuals. To determine the reliability of microarray, we determined the global correlation coefficients between transcriptomes of BMSCs and AMSCs from different patients and from same patients (supplemental online Table 3). We found that the average correlation coefficient between BMSCs was 0.64, and the correlation coefficients were not improved by comparing BMSCs of the same sex. We found that the average correlation coefficient between AMSCs was 0.71, and again, the correlation coefficients were not improved by comparing the same sex. The lowest average correlation coefficient, 0.52, was observed when we compared AMSCs with BMSCs. These data indicated that the microarray data generated was reliable and reproducible enough to detect biological differences between AMSCs and BMSCs and that the differences between AMSCs and BMSCs were not due to culture or technical differences or to variations between individuals. This indicated that data generated from microarrays were reproducible and that culture technique might not underlie differences seen in gene expression.3 s2 l( ]) G% l7 }- V' \* {

) `6 c( i, Q9 Q$ o; N; M! bConfirmation by Real-Time PCR
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2 S$ Z# {1 e8 `, v# n4 R# gTo confirm the data generated from microarray studies, we performed quantitative RT-PCR using TaqMan on the same total RNA samples used in the microarray studies. The average fold change by PCR was compared with average fold change by microarray detection. We selected genes indicative of different lineages, as shown (supplemental online Table 4): C/EBP for adipogenic marker, NOX4 for chondrogenic upregulated gene, OMD for osteogenesis, and FKBP5 for upregulated common gene in all three lineages. The genes found to be differentially expressed in the microarray analysis were confirmed to be differentially expressed by quantitative RT-PCR (supplemental online Table 4). However, the degree of increased or decreased expression differed for some genes, likely as a result of the difference in the sensitivity of the two assays. Nevertheless, the result of this comparison gave us the confidence to use our microarray data to deduce biological meaning.
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Genes Differentially Regulated During Adipogenic Differentiation In Vitro
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/ M0 ~2 @6 j6 J$ uInduced differentiation of MSCs toward adipogenesis resulted in a larger cell morphology and a time-dependent increase in intracellular lipid-filled droplets stained by oil red O (Fig. 1A). Our microarray data showed that approximately 230 common genes were upregulated more than twofold at both day 3 and day 14 after induction of BMSCs and AMSCs. Analyzing the data in a different way, we found that approximately 105 and 320 common genes were upregulated more than twofold at days 3 and 14, respectively. A substantial number of genes have been identified as regulated in a differentiation-dependent manner, including constitutively activated gene peroxisome proliferator adipogenic transcription factors (PPAR), C/EBP, and the entire spectrum of genes associated with lipid metabolism that contribute to the function and phenotype of the mature adipocyte ) were all upregulated (Fig. 1C; supplemental online Table 2A).. f% Z. o9 g9 D

  }  W6 u: k( AAmong upregulated genes for lipid metabolism, genes related to energy reserve metabolism and cholesterol metabolism, such as LEP, LPL, PLIN, SAA1, APOD, and ACDC, were expressed at higher levels in AMSCs than BMSCs, suggesting that AMSCs were superior to BMSCs in adipogenesis. Among upregulated genes for cell cycle, growth, and proliferation, ZNF145, RASD1, and INHBB were expressed more highly in AMSCs, whereas expression of G0S2 and FOX1A was higher in BMSCs. Among signal transduction genes, expression of PDK4, RASD1, and LEP was higher in AMSCs than in BMSCs (supplemental online Table 5). These results together illustrated differences in gene expression between BMSCs and AMSCs during adipogenesis." K# j8 V& U3 Q' ]5 W

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BMSCs cultured in condensate culture under chondrogenic medium, including pellet . COL10A1 significantly increased from 16.79-fold at day 3 to 56.34-fold at day 14 for BMSCs compared with an increase from 5.855-fold at day 3 to 10.07-fold at day 14 for AMSCs, suggesting a difference in gene expression during chondrogenesis between BMSCs and AMSCs (supplemental online Tables 2B, 6).
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: K3 v% [  V5 _4 c3 Z9 hAmong upregulated extracellular matrix genes, in addition to the genes mentioned above, osteoblast-specific factor 2 (fasciclin I-like, POSTN) progressively increased to day 14. Among cell adhesion genes, DPT and POSTN increased, whereas OMD and WISP1 decreased. Among genes for cell cycle, growth, and proliferation, there was no significant change in expression of MAD2L1 and BUB1 (supplemental online Table 6). Among signal transduction genes, WISPI and INPP4B were more highly expressed in AMSCs than in BMSCs.
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Genes Differentially Regulated During Osteogenic Differentiation
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) r6 ^! Y. R4 r' q2 ZCalcium deposition was seen 14 days after induced differentiation toward osteogenesis, as shown by Alizarin red staining. Compared with AMSCs, BMSCs accumulated more calcium during osteogenesis (Fig. 1A). Our microarray data showed that approximately 134 common genes were upregulated more than twofold at both day 3 and day 14 between BMSCs and AMSCs, and approximately 214 and 97 common genes were upregulated more than twofold at days 3 and 14, respectively.
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Genes known to be expressed in osteoblasts were consistently upregulated in osteogenic differentiation cultures compared with undifferentiated MSCs. Expression of osteomodulin, implicated in biomineralization processes , suggesting that growth inhibition occurred. We also found that this growth repressor was upregulated more than twofold during adipogenesis and chondrogenesis (Table 1).5 l3 ]$ a5 E& l2 C: P5 {. j% {* f
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Table 1. Common upregulated genes during differentiation between BMSCs and AMSCs at early and late stages7 [3 s# B; m4 m! L% D; p: v) g

4 o. I( ?5 r6 Q- q. {4 T# hAmong extracellular matrix genes, OMD and tissue inhibitor of metalloproteinase 4 (TIMP4) progressively increased with BMSCs, whereas in AMSCs, they were decreased or did not change. Tissue factor pathway inhibitor 2 (TFPI2) was also upregulated during osteogenesis. Notably, expression of IGFBP2, WISP1, TACSTD2, NEDD, and RGC32 was higher in AMSCs than in BMSCs, whereas expression of FOXO1A and CDD20 was lower in AMSCs (supplemental online Table 7). The expression of FOXO1A and NR2F1 continuously increased in BMSCs but did not change in AMSCs (supplemental online Table 7), further indicating differences in osteogenesis between BMSCs and AMSCs.
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- \2 @# _  a# \6 k8 O7 J0 DComparison of BMSCs and AMSCs in Lineage-Related Genes& ?' V( @8 ?3 x+ e4 p

- E3 N, P" m. }0 m2 ^% ~To obtain a more quantitative comparison of the difference between differentiation of BMSCs and AMSCs into the three lineages, lineage-related genes were chosen for comparison. During adipogenesis (Fig. 1C), lipid metabolism-related genes were upregulated more highly in AMSCs than in BMSCs, including LPL (ratio, 2.04), FABP4 (ratio, 1.47), PDK4 (ratio, 3.41), PLIN (ratio, 1.82), and LOC283445 (ratio, 1.42).
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' k/ h- t* v# m4 E2 _0 z/ U8 eTreatment of MSCs with chondrogenic medium resulted in the expression of genes related to chondrogenesis (Fig. 1D). Compared with AMSCs, differentiated BMSCs showed much higher in expression of extracellular matrix genes DPT (ratio, 3.81) and COL10A1 (ratio, 5.59). Expressions of COL2 and AGC were too low to be detected at days 3 and 14 by RT-PCR; this is consistent with the results of Zuk et al.  and probably resulted from different expression levels at various time points. In osteogenesis (Fig. 1E), differentiated BMSCs expressed much more highly than AMSC levels of extracellular matrix genes, including OMD (ratio, 11.13), DPT (ratio, 2.09), SPON1 (ratio, 3.01), and FBN2 (ratio, 4.68). These results suggested that BMSCs were superior to AMSCs in osteogenesis and chondrogenesis but inferior in their potential for adipogenesis compared with hAMSCs.
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Common Upregulated Genes During Three Differentiations
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0 {0 }0 ^& Q7 S# ?4 @( _% YWe next wanted to look for genes that were upregulated common to both BMSCs and AMSCs. During differentiation of MSCs into three lineages, 11 and 12 common genes between BMSCs and AMSCs were upregulated more than twofold at the early (3 days) and late (14 days) stages, respectively (Table 1). Among these genes, there were six common upregulated genes at both early and late stages, including FKBP5, ZNF145, SAA1, PCDH9, CPM, and DSIPI. FKBP5 has been shown to inhibit the serine/threonine phosphatase activity of calcineurin in the presence of calcium and calmodulin. Zinc finger protein 145 (ZNF145; PLZF) has been shown to be highly expressed during osteoblastic differentiation, playing an important role in early osteoblastic differentiation as an upstream regulator of CBFA1 .
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Our microarray data showed that FKBP5, ZNF145, and CPM expression was upregulated much more highly in adipogenesis and osteogenesis than in chondrogenesis (Table 1), suggesting a differential role of these genes in mesenchymal differentiation and, most interestingly, a linkage between osteogenesis and adipogenesis (Fig. 2). Among common genes at day 3 differentiations to the three mesenchymal lineages, OMD was expressed most highly in osteogenesis. This is consistent with its preferential high expression in osteoblastic lineages . It was also reported that APOD was involved in cellular differentiation and growth arrest.) l& w$ Y; f, U/ o! A+ K

  g1 a, U1 g% O, [% c" e3 k% xFigure 2. Molecular signatures predominantly marking similarities and differences between BMSCs and AMSCs during progression of osteogenesis, adipogenesis, and chondrogenesis. Note the linkage between osteogenesis and adipogenesis and the more robust differentiation of BMSCs and the poorer differentiation of AMSCs to osteocytes and chondrocytes. Abbreviations: ADP, immature adipocytes; AMSC, adipose tissue-derived MSC; BMSC, bone marrow-derived MSC; CHOND, immature chondrocytes; CPM, carboxypeptidase M; DPT, dermatopontin; LPL, lipoprotein lipase; OMD, osteomodulin; OSTEO, immature osteocytes.- N, S9 y- I6 N
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Effect of FKBP5 Knockdown on the Differentiation of MSCs, U3 m$ Q/ M3 m/ G& x

# Z# a- t7 z" C0 k  ?1 A9 [' yAs discussed above, there appears to be a common set of genes upregulated during differentiation to all three lineages. Included in our list is ZNF145, which has been shown to be important for osteogenesis. To further test whether the upregulation of these genes is the cause or effect of differentiation, we chose to investigate FKBP5 by manipulating its expression in MSCs. First, we tested the effect of suppressing the upregulation of FKBP5 by RNA interference-mediated knockdown of the transcript. siRNAs targeting FKBP5 (Ambion) were transfected into hMSCs with Lipofectamine 2000 (Invitrogen). The transfection efficiency of MSCs with cy3-labeled negative control siRNA showed very high transfection efficiency (data not shown). Introduction of siRNA targeting FKBP5 resulted in a downregulation of the lineage markers. Compared with negative control siRNA, FKBP5 siRNA knocked down FKBP5 transcripts by 0.5, 0.35, and 0.3 in mRNA level during adipogenesis, chondrogenesis, and osteogenesis, respectively (Fig. 3A); this was consistent with knockdown in protein level during differentiation into three lineages at day 7 (Fig. 3B). During adipogenesis, C/EBP and PPAR were reduced to 63.7% and 56.7%, respectively, of control cells. For chondrogenesis, expression of Col2A1 and COMP was decreased to 38.2% and 40%, respectively, compared with control cells. For osteogenesis, osteocalcin was decreased by more than osteopontin and alkaline phosphatase (45.7% vs. 70.5% and 69.3%) (Fig. 3A). Consistent with the molecular reduction, FKBP5-knockdown MSCs showed decreased oil red stain, proteoglycan, and calcium deposition at cellular level (Fig. 3C, 3D). These results showed that a knockdown of FKBP5 resulted in suppression of expression of differentiation lineage markers to various degrees, indicating that FKBP5 was involved in differentiation.
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# W  r# |3 c8 O: |- |6 cFigure 3. Effects of small interfering RNA (siRNA)-mediated gene silencing of FKBP5 on the differentiation of MSCs. (A): After 48 hours of transfection with FKBP5-targeted siRNA (100 nM) and differentiation, efficiencies of reduction of FKBP5 and lineage markers siRNA were measured by real-time polymerase chain reaction (PCR) compared with negative control. The probability associated with Student's test was performed. (B): FKBP5 was efficiently knocked down by FKBP5-targeted siRNA (100 nM) in protein level after 7 days (d) of transfection and differentiation. Bone marrow-derived MSCs (BMSCs) were induced into osteogenesis for 7 d. Lane 1, BMSCs transfected with ne' N3 Y( a% p$ a; I* P$ T( f
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