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Cell Surface and Transcriptional Characterization of Human Adipose-Derived Adher

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发表于 2009-3-5 10:51 |显示全部帖子
Department of Plastic and Reconstructive Surgery, University of Virginia, Charlottesville, VA, USA6 W, F2 x& K- u" |4 s6 X  g$ ^3 d6 D
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Key Words. Adult stem cells ? Flow cytometry ? Microarray ? Integrins: T& X, V$ M% x8 C0 ]. A( p

( f4 g: M6 H, I9 _1 bCorrespondence: Adam J. Katz, M.D., P.O. Box 800376, Department of Plastic and Reconstructive Surgery, University of Virginia, Charlottesville, VA 22908, USA. Telephone: 434-924-8042; Fax: 434-924-1333; e-mail: ajk2f@virginia.edu
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0 X9 d: j6 ?' s  f1 JABSTRACT. Y* [  C. T) P7 Q6 ]

: ?' J( u7 L6 Z' AAdult stem cells hold great promise for use in tissue repair and regeneration. In recent years, interest has rapidly grown in the developmental plasticity and therapeutic potential of stromal cells that have been isolated from human subcutaneous adipose tissue. Adipose tissue represents an abundant, practical, and appealing source of donor tissue for autologous cell replacement.2 i7 \5 w2 [* H+ T5 A  \* W
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Several groups have demonstrated that mesenchymal cells within the stromal-vascular fraction (SVF) of subcutaneous adipose tissue display multilineage developmental plasticity in vitro and in vivo . These cells have alternatively been referred to as processed lipoaspirate cells (PLA), adipose-derived stem cells, adipose-derived stromal cells, and adipose-derived mesenchymal progenitor cells. It is also likely that cells previously considered preadipocytes are essentially the same cell population. These many names reflect a lack of consensus and an evolving knowledge base with regard to the anatomic origin, phenotype, and function of these cells. Our lab has chosen to refer to these cells as adipose-derived adherent stromal (ADAS) cells. This is a purely descriptive name that also provides some distinction from stromal vascular fraction (SVF) cells, which have not been further separated based on adherence to tissue culture plastic.
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There are obvious similarities between hADAS cells and mesenchymal stem cells (MSCs). Both represent the stromal cell fraction isolated from an adipose depot (subcutaneous tissue for the former, bone marrow for the latter) on the basis of adherence to tissue culture plastic. While an extensive body of work exists pertaining to the phenotypic characterization of MSCs, the phenotypic characterization of hADAS cells is in its infancy . The plasticity observed of cultured hADAS cells is intriguing and includes cell lineages thought of as ectodermal and mesodermal.6 j9 P' H9 ^8 _; P- N

5 O; Q+ b$ q, u* oThere are no known stem cell–specific markers for the prospective identification of putative stem cells or progenitor cells within adipose tissue, bone marrow, or any other mesodermally derived adult tissue. Based on extensive work with other stem cell populations, however, several proteins have emerged as candidate markers associated with a primitive, stem cell phenotype; these include telomerase, CD133, and ABCG2) . The purpose of this study was threefold: (1) to further characterize early passage, undifferentiated hADAS cells on a transcriptional and cell surface level, (2) to determine the degree of variability of cell populations isolated from different donors, and (3) to determine whether hADAS cells specifically exhibit markers that are associated with other stem cell populations.
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MATERIALS AND METHODS
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Qualitative Confirmation of Developmental Plasticity
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Using the qualitative assays described, the hADAS cell populations used in these studies differentiated in vitro into adipogenic, osteogenic, and neurogenic lineages (data not included). Adipogenic differentiation was considered positive based on a rounded morphology with intracellular lipid accumulation that stained with Oil Red O. Osteogenic differentiation was considered positive based on the appearance of nodules that stained positive for calcified matrix using von Kossa staining. Neurogenic differentiation was considered positive based on cell morphology and positive staining for neurofilament ?-tubulin.9 D5 U* P, ~# @+ [

" X7 d2 H, f3 x0 Z+ F1 TGene Array Analysis
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6 N) v" l* O1 q. X, @, a1 i3 i3 J) I/ pTranscriptional characterization of three different hADAS cell populations was performed using microarrays for human extracellular matrix or adhesion molecules (Super-Array, cat. no. HS-010N) and human angiogenesis-related factors (cat. no. HS-009N). A representative array result is shown in Figure 2. Expected results were observed for internal controls on each array. Out of a total of 172 unique genes screened, only 7 genes were not transcribed by any of the hADAS cell populations. These unexpressed genes include the integrins L, M, ?6, ?7, selectin-L, and selectin-P and the apoptosis-related cysteine protease caspase 9. Of the 165 genes transcribed by at least one population, 114 (69%) were unanimously transcribed by all three hADAS cell populations, and a total of 143 (87%) were transcribed by at least two of the three populations (Table 2; Fig. 3). Some of the most highly transcribed genes include endoglin; FGFs 2, 6, and 7; FGF receptor 3; neuropilin-1; integrins 5 and 11; integrin ?1; TGF-? receptors 2 and 3; SPARC (osteonectin); osteopontin; fibronectin-1; VEGF-D; TNF-; and (MMP2) gelatinase A (Table 2). Twenty-two (13%) of the transcribed genes were transcribed by only one hADAS cell population, and all correspond to a single donor (see "Patient A," Fig. 3).6 u5 B# L0 t2 \9 ]
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Figure 2. Representative example of a gene array expression profile of extracellular matrix and adhesion molecules for a human adipose-derived adherent stromal cell population. The bottom two rows include positive and negative controls.! j4 b7 A, b7 p! G% r  u: G
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Table 2. Transcription profile of extracellular matrix and angiogenesis-related genes in early-passage, undifferentiated human adipose-derived adherent stromal (hADAS) cells (n = 3)* B% E/ H0 A. M" s  L
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Figure 3. Venn diagram summarizing gene transcripts of all three human adipose-derived adherent stromal cell populations tested, as shown in Figure 2 (see also Table 2).
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6 V4 @( h9 ^- [Flow Analysis+ s" Q9 Y# @- ]& F6 X; ~) q

/ V+ }6 o* o. sCells from seven separate donors were evaluated by flow cytometry for the expression of over 24 cell surface proteins that relate primarily to extracellular matrix interactions. Flow results are summarized in Table 3. Of the 26 proteins screened, 21 were associated with highly consistent patterns of expression among all cell populations tested. Proteins that were consistently expressed by a majority of hADAS cells (average 97% or more of cells) included HLA-ABC and CD29 (integrin ?1), CD49e (integrin 5/VLA-5), CD51 (integrin V), and CD90 (Thy-1). Proteins that showed a positive, but more variable, expression across the seven donor groups include CD49b (integrin 2/VLA-2), CD49d (integrin 4/VLA-4), CD61 (integrin ?3), CD138 (syndecan-1), and CD140a (PDGFR-?). The remainder of screened proteins were consistently absent or expressed on only negligible numbers of cells, including HLA-DR, CD4, CD8a, CD11a (integrin L), CD11b (integrin M), CD11c (integrin X), CD18 (integrin ?2), CD41a (gpIIb), CD49f (integrin ?6/VLA-6), CD62L (L-selectin), CD62P (P-selectin), CD106 (VCAM-1), CD117 (c-kit), CD133, CD243 (MDR-1), and ABCG2 (Table 3). Many of these cell surface proteins were also evaluated on a transcriptional level by microarray. Data summarizing both the transcriptional levels and the expression levels of these proteins are shown in Figure 4 and Table 3.0 B0 n) X  S$ M4 w" y8 A

* K5 e* h8 K7 y9 ?Table 3. Gene transcription and protein expression levels of select markers by early-passage, undifferentiated human adipose-derived adherent stromal cells
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Figure 4. Representative gene array and flow cytometry data for integrin ?1 (CD29), integrin ?L (CD11a), and integrin 6 (CD49f). Pixels were quantitated using the GEArray Analyzer for each square present on the gene array. Genes were considered positive if signal intensity was greater than that of the negative control gene PUC18. Flow array charts show the percentage of cells expressing corresponding protein. The numbers in the dot plots represent raw counts and do not reflect nonspecific binding related to isotype-matched controls.
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2 N0 w' V* M! {& i( FRT-PCR Analysis
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0 ]( b* T/ G/ J2 r, Y! b7 S! G( `% eMultiple hADAS cell populations were assayed by PCR for transcriptional evidence of two genes associated with a stem cell phenotype: telomerase and ABCG2. Our results revealed no evidence of either of these genes in hADAS cells that were cultured according to our methods (Fig. 5). The negative result for ABCG2 transcript correlates with a lack of detection of ABCG2 protein by flow cytometry (Table 3).
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Figure 5. Gel results of ABCG2 reverse transcriptase poly-merase chain reaction of human adipose-derived adherent stromal (hADAS) cells. The left lane is the A549 cell line (positive control) with a band between 300 and 400 bp (expected: 366 bp). The next four lanes are different hADAS cell populations. The right lane is a reference ladder in 100-bp increments., W5 ~" [6 j1 w, y. P

4 c; \6 X: i* {( N8 m  d; EDISCUSSION
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Early-passage, undifferentiated hADAS cells transcribe many genes that are related to the extracellular matrix and angiogenesis and which are implicated in matrix remodeling, inflammation, morphogenesis, and tissue repair. Highly transcribed genes include endoglin; FGFs2, 6 and 7; FGFR3; neuropilin-1; osteonectin; fibronectin; VEGF-D; TGF-? R2 and R3; and integrins 5, 11, and ?1. Our findings suggest remarkably good consistency in the transcriptional profile of hADAS cells isolated from different donors, as well as many similarities with published profiles for marrow-derived stromal cells. This study also confirms and expands on the cell surface profile of hADAS cells and confirms many cell surface similarities with marrow-derived stromal cells. More specifically, our results demonstrate good donor-to-donor consistency and reproducibility, both internally and when compared to previously published data from other groups. This uniformity is remarkable, given the inconsistent nomenclature and nonstandardized cell isolation and culture protocols. Of note, emerging evidence suggests that both adherence to plastic and time in culture appear to alter the cell surface phenotype. Our findings support the notion that adipose-derived stromal cells isolated by adherence to tissue culture plastic have a remarkably consistent molecular and cell surface profile, yet lack an easily definable phenotype. In the near future, it is hoped that a consensus on nomenclature and hADAS cell isolation and culture protocols will emerge, so as to allow more efficient and meaningful communication and interpretation of published research. Continued characterization of these cells will also help clarify phenotypic, developmental, and regenerative differences between them and freshly isolated SVF cells, and it may ultimately enable the prospective isolation of specific, purified subpopulations of putative multipotential stem cells and lineage-committed progenitor cells.
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- S; d8 O/ [$ OACKNOWLEDGMENTS
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Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–228.- |) E* z6 A4 A3 j! z

9 B. b3 e7 d9 I) `9 kZuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279–4295.: p6 d5 s; ]: o! P; I4 v7 A, C

" L! _/ _& K0 q$ o9 l( R9 aGimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 2003;5:362–369.
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8 s' ^5 A4 g$ o: BErickson GR, Gimble JM, Franklin DM et al. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun 2002;290:763–769.( n$ Q3 a5 @/ E

  Q! U* |! y( ^! _, xSafford KM, Hicok KC, Safford SD et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 2002;294:371–379.) I6 b  x' A$ M# `. h9 D
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Halvorsen YD, Franklin D, Bond AL et al. Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng 2001;7:729–741.9 `1 X0 }, j0 O, A) s2 C

% c4 n. ~" H: U& G* z, ?Baddoo M, Hill K, Wilkinson R et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem 2003;89:1235–1249.3 v4 a7 Y% J5 u/ c. ~" \
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Phinney DG, Kopen G, Righter W et al. Donor variation in the growth properties and osteogenic potential of human marrow stromal cells. J Cell Biochem 1999;75:424–436.
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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;19:408–418.
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Ibberson D, Tremain N, Gray A et al. What is in a name? Defining the molecular phenotype of marrow stromal cells and their relationship to other stem/progenitor cells. Cytotherapy 2001;3:409–411.$ F1 t- I. h! n  W  ^& l" ~

/ V7 S: Y. r2 h5 e6 k& X8 oGronthos S, Franklin DM, Leddy HA et al. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol 2001;189:54–63.
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De Ugarte DA, Alfonso Z, Zuk PA et al. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunol Lett 2003;89:267–270.; S, h( q1 I: }

, J/ u5 v. [7 u" pFu WY, Lu YM, Piao YJ. Differentiation and telomerase activity of human mesenchymal stem cells. Di Yi Jun Yi Da Xue Xue Bao 2001;21:801–805.: [, k. L0 R; X

; V, @" ?: ]  g& ^1 qShi S, Gronthos S, Chen S et al. Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression. Nat Biotechnol 2002;20:587–591.6 e) t$ \3 E7 g1 Q" O/ S
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Kobari L, Giarratana MC, Pflumio F et al. CD133  cell selection is an alternative to CD34  cell selection for ex vivo expansion of hematopoietic stem cells. J Hematother Stem Cell Res 2001;10:273–281.3 Q( k: ^& l4 E. Q7 [, ]

0 {2 f7 P' ]9 U: [' j: uKatz AJ. Mesenchymal cell culture: adipose tissue. In: Atala A, Lanza RP, eds. Methods of Tissue Engineering. Academic Press, NY;2002:277–286.
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Tholpady SS, Katz AJ, Ogle RC. Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro. Anat Rec 2003;272A:398–402.5 y& {$ _2 c2 k! M3 K- o

& n) H  ]6 H8 vScharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507–512.
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