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Expression analysis of human adipose-derived stem cells during in vitro differentiation to an adipocyte lineage.

Satish L, Krill-Burger JM, Gallo PH, Etages SD, Liu F, Philips BJ, Ravuri S, Marra KG, LaFramboise WA, Kathju S, Rubin JP - BMC Med Genomics (2015)

Bottom Line: Setting a cutoff of +/-two-fold change, 1350 transcripts were elevated while 2929 genes were significantly decreased by 7 days.Comparison of early and late stage cultures revealed increased expression of 1107 transcripts while 606 genes showed significantly reduced expression.Quantitative RT-PCR validated the microarray results.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic Surgery, University of Pittsburgh Medical Center, 3550 Terrace Street, 6B Scaife Hall, 15261, Pittsburgh, PA, USA.

ABSTRACT

Background: Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. Although autologous grafting of whole fat has long been practiced, a major clinical limitation of this technique is inconsistent long-term graft retention. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype.

Methods: Microarray analysis was performed on total RNA extracted from separate ACS isolates of six human adult females before and after 7 days (7 days: early stage) and 21 days (21 days: late stage) of adipocyte differentiation in vitro. Differential gene expression profiles were determined using Partek Genomics Suite Version 6.4 for analysis of variance (ANOVA) based on time in culture. We also performed unsupervised hierarchical clustering to test for gene expression patterns among the three cell populations. Ingenuity Pathway Analysis was used to determine biologically significant networks and canonical pathways relevant to adipogenesis.

Results: Cells at each stage showed remarkable intra-group consistency of expression profiles while abundant differences were detected across stages and groups. More than 14,000 transcripts were significantly altered during differentiation while ~6000 transcripts were affected between 7 days and 21 days cultures. Setting a cutoff of +/-two-fold change, 1350 transcripts were elevated while 2929 genes were significantly decreased by 7 days. Comparison of early and late stage cultures revealed increased expression of 1107 transcripts while 606 genes showed significantly reduced expression. In addition to confirming differential expression of known markers of adipogenesis (e.g., FABP4, ADIPOQ, PLIN4), multiple genes and signaling pathways not previously known to be involved in regulating adipogenesis were identified (e.g. POSTN, PPP1R1A, FGF11) as potential novel mediators of adipogenesis. Quantitative RT-PCR validated the microarray results.

Conclusions: ASC maturation into an adipocyte phenotype proceeds from a gene expression program that involves thousands of genes. This is the first study to compare mRNA expression profiles during early and late stage adipogenesis using cultured human primary ASCs from multiple patients.

No MeSH data available.


Related in: MedlinePlus

Relative expression levels of select genes as determined by microarray: Histogram presentations of the relative expression levels of (a) FABP4 (b) ADIPOQ (c) PLIN4 (d) FGF1 (e) FGF11 (f) HES1 (g) RSAD2 (h) PPP1R1A (i) POSTN as quantified by microarray are shown. Signal intensities for each gene product are expressed in arbitrary units after background subtraction. Statistical significance was determined by Student’s t test and p value < 0.05 was considered significant
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Fig3: Relative expression levels of select genes as determined by microarray: Histogram presentations of the relative expression levels of (a) FABP4 (b) ADIPOQ (c) PLIN4 (d) FGF1 (e) FGF11 (f) HES1 (g) RSAD2 (h) PPP1R1A (i) POSTN as quantified by microarray are shown. Signal intensities for each gene product are expressed in arbitrary units after background subtraction. Statistical significance was determined by Student’s t test and p value < 0.05 was considered significant

Mentions: We performed direct quantitation of nine selected gene products by real-time RT-PCR to validate the microarray findings. Genes were selected based on their established relevance to adipocyte biology, or were included to validate their heretofore unknown importance to adipocyte maturation. In every case, a strong correspondence between the microarray and real time RT-PCR data was observed (Figs. 3 and 4).Fig. 3


Expression analysis of human adipose-derived stem cells during in vitro differentiation to an adipocyte lineage.

Satish L, Krill-Burger JM, Gallo PH, Etages SD, Liu F, Philips BJ, Ravuri S, Marra KG, LaFramboise WA, Kathju S, Rubin JP - BMC Med Genomics (2015)

Relative expression levels of select genes as determined by microarray: Histogram presentations of the relative expression levels of (a) FABP4 (b) ADIPOQ (c) PLIN4 (d) FGF1 (e) FGF11 (f) HES1 (g) RSAD2 (h) PPP1R1A (i) POSTN as quantified by microarray are shown. Signal intensities for each gene product are expressed in arbitrary units after background subtraction. Statistical significance was determined by Student’s t test and p value < 0.05 was considered significant
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4513754&req=5

Fig3: Relative expression levels of select genes as determined by microarray: Histogram presentations of the relative expression levels of (a) FABP4 (b) ADIPOQ (c) PLIN4 (d) FGF1 (e) FGF11 (f) HES1 (g) RSAD2 (h) PPP1R1A (i) POSTN as quantified by microarray are shown. Signal intensities for each gene product are expressed in arbitrary units after background subtraction. Statistical significance was determined by Student’s t test and p value < 0.05 was considered significant
Mentions: We performed direct quantitation of nine selected gene products by real-time RT-PCR to validate the microarray findings. Genes were selected based on their established relevance to adipocyte biology, or were included to validate their heretofore unknown importance to adipocyte maturation. In every case, a strong correspondence between the microarray and real time RT-PCR data was observed (Figs. 3 and 4).Fig. 3

Bottom Line: Setting a cutoff of +/-two-fold change, 1350 transcripts were elevated while 2929 genes were significantly decreased by 7 days.Comparison of early and late stage cultures revealed increased expression of 1107 transcripts while 606 genes showed significantly reduced expression.Quantitative RT-PCR validated the microarray results.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic Surgery, University of Pittsburgh Medical Center, 3550 Terrace Street, 6B Scaife Hall, 15261, Pittsburgh, PA, USA.

ABSTRACT

Background: Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. Although autologous grafting of whole fat has long been practiced, a major clinical limitation of this technique is inconsistent long-term graft retention. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype.

Methods: Microarray analysis was performed on total RNA extracted from separate ACS isolates of six human adult females before and after 7 days (7 days: early stage) and 21 days (21 days: late stage) of adipocyte differentiation in vitro. Differential gene expression profiles were determined using Partek Genomics Suite Version 6.4 for analysis of variance (ANOVA) based on time in culture. We also performed unsupervised hierarchical clustering to test for gene expression patterns among the three cell populations. Ingenuity Pathway Analysis was used to determine biologically significant networks and canonical pathways relevant to adipogenesis.

Results: Cells at each stage showed remarkable intra-group consistency of expression profiles while abundant differences were detected across stages and groups. More than 14,000 transcripts were significantly altered during differentiation while ~6000 transcripts were affected between 7 days and 21 days cultures. Setting a cutoff of +/-two-fold change, 1350 transcripts were elevated while 2929 genes were significantly decreased by 7 days. Comparison of early and late stage cultures revealed increased expression of 1107 transcripts while 606 genes showed significantly reduced expression. In addition to confirming differential expression of known markers of adipogenesis (e.g., FABP4, ADIPOQ, PLIN4), multiple genes and signaling pathways not previously known to be involved in regulating adipogenesis were identified (e.g. POSTN, PPP1R1A, FGF11) as potential novel mediators of adipogenesis. Quantitative RT-PCR validated the microarray results.

Conclusions: ASC maturation into an adipocyte phenotype proceeds from a gene expression program that involves thousands of genes. This is the first study to compare mRNA expression profiles during early and late stage adipogenesis using cultured human primary ASCs from multiple patients.

No MeSH data available.


Related in: MedlinePlus