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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.


Intracellular lipid accumulation in undifferentiated, 7- and 21-day differentiated ASCs: a Phase contrast images of cells (10x magnification) are presented in the top panel and the corresponding AdipoRed stained images are shown in the bottom panel. These are representative photographs from two independent experiments each performed in duplicate. b Quantification of intracellular lipid showed significantly increased accumulation of lipid droplets in 21-day differentiated ASCs versus undifferentiated and 7-day differentiated ASCs. Values represent mean ± SEM of two independent experiments performed in triplicate. Statistical analysis was performed using Student’s t test and p < 0.05 was considered significant
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Fig1: Intracellular lipid accumulation in undifferentiated, 7- and 21-day differentiated ASCs: a Phase contrast images of cells (10x magnification) are presented in the top panel and the corresponding AdipoRed stained images are shown in the bottom panel. These are representative photographs from two independent experiments each performed in duplicate. b Quantification of intracellular lipid showed significantly increased accumulation of lipid droplets in 21-day differentiated ASCs versus undifferentiated and 7-day differentiated ASCs. Values represent mean ± SEM of two independent experiments performed in triplicate. Statistical analysis was performed using Student’s t test and p < 0.05 was considered significant

Mentions: The AdipoRed assay was employed to monitor accumulation of intracellular lipid at 7 and 21 days of adipogenic cell culture media consistent with a protocol directing differentiation into the adipocyte phenotype. Under the microscope 21-day differentiated cells showed increased fine red-colored lipid droplets bounded by the cell membrane compared to both undifferentiated and 7-day differentiated ASCs (Fig. 1a). Quantitative analysis of intracellular lipid accumulation based on cellular fluorescence confirmed the increased levels of lipid present with time in differentiation media (Fig. 1b).Fig. 1


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)

Intracellular lipid accumulation in undifferentiated, 7- and 21-day differentiated ASCs: a Phase contrast images of cells (10x magnification) are presented in the top panel and the corresponding AdipoRed stained images are shown in the bottom panel. These are representative photographs from two independent experiments each performed in duplicate. b Quantification of intracellular lipid showed significantly increased accumulation of lipid droplets in 21-day differentiated ASCs versus undifferentiated and 7-day differentiated ASCs. Values represent mean ± SEM of two independent experiments performed in triplicate. Statistical analysis was performed using Student’s t test and p < 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

Fig1: Intracellular lipid accumulation in undifferentiated, 7- and 21-day differentiated ASCs: a Phase contrast images of cells (10x magnification) are presented in the top panel and the corresponding AdipoRed stained images are shown in the bottom panel. These are representative photographs from two independent experiments each performed in duplicate. b Quantification of intracellular lipid showed significantly increased accumulation of lipid droplets in 21-day differentiated ASCs versus undifferentiated and 7-day differentiated ASCs. Values represent mean ± SEM of two independent experiments performed in triplicate. Statistical analysis was performed using Student’s t test and p < 0.05 was considered significant
Mentions: The AdipoRed assay was employed to monitor accumulation of intracellular lipid at 7 and 21 days of adipogenic cell culture media consistent with a protocol directing differentiation into the adipocyte phenotype. Under the microscope 21-day differentiated cells showed increased fine red-colored lipid droplets bounded by the cell membrane compared to both undifferentiated and 7-day differentiated ASCs (Fig. 1a). Quantitative analysis of intracellular lipid accumulation based on cellular fluorescence confirmed the increased levels of lipid present with time in differentiation media (Fig. 1b).Fig. 1

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.