Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation.
Pluripotent stem cells within the adipose stromal compartment, termed adipose-derived stromal cells (ASCs), have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. Imaging with expression of exogenous or endogenous green fluorescent protein (GFP) reporters facilitates the detailed research on ASCs' physiological behavior during differentiation in vivo. This study was aimed to confirm whether ASCs expressing GFP still could be induced to chondrogenesis, and to compare the expression of exogenous or endogenous GFP in ASCs during chondrogenic differentiation. ASCs were harvested from inguinal fat pads of normal nude mice or GFP transgenic mice. Monolayer cultures of ASCs from normal mice were passaged three times and then infected with replication-incompetent adenoviral vectors carrying GFP genes. Allowed to recover for 5 days, Ad/GFP infected ASCs were transferred to chondrogenic medium as well as the ASCs from transgenic mice cultured in vitro over the same passages. The level of GFP in transgenic ASCs maintained stable till 3 months after chondrogenic induction. Whereas, high level of GFP expression in Ad/GFP infected ASCs could last for only 8 weeks and then declined stepwise. Important cartilaginous molecules such as SOX9, collagen type I, collagen type II, aggrecan, collagen type X were assessed using immunocytochemistry, RT-PCR, and Western Blot. The results indicated that no matter the GFP was exogenous or endogenous, it did not influence the chondrogenic potential of ASCs in comparison with the normal controls. Moreover, chondrogenic lineages from ASCs also underwent phenotypic modulation called dedifferentiation as a result of long-term culture in monolayers similar to normal chondrocytes.
Lin
Yunfeng
Y
Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, Chengdu, P.R. China.
Tian
Weidong
W
Chen
Xizhe
X
Yan
Zhengbin
Z
Li
Zhiyong
Z
Qiao
Ju
J
Liu
Lei
L
Tang
Wei
W
Zheng
Xiaohui
X
D000273:Adipose Tissue / Q000166:cytology* / Q000378:metabolism*; D053674:Aggrecans; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D020219:Chondrogenesis / Q000502:physiology*; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis; D015870:Gene Expression; D017930:Genes, Reporter; D006025:Glycosaminoglycans / Q000096:biosynthesis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D037181:Lectins, C-Type / Q000096:biosynthesis; D051379:Mice; D008819:Mice, Nude; D008822:Mice, Transgenic; D010641:Phenotype; D011509:Proteoglycans / Q000096:biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055758:SOX9 Transcription Factor; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C508320:Acan protein, mouse; D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D006609:High Mobility Group Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D012333:RNA, Messenger; D011994:Recombinant Proteins; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D003094:Collagen; D004247:DNA