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


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2015-07-24

AUTHORS

Latha Satish, J. Michael Krill-Burger, Phillip H. Gallo, Shelley Des Etages, Fang Liu, Brian J. Philips, Sudheer Ravuri, Kacey G. Marra, William A. LaFramboise, Sandeep Kathju, J. Peter Rubin

ABSTRACT

BackgroundAdipose 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.MethodsMicroarray 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.ResultsCells 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.ConclusionsASC 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. More... »

PAGES

41

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s12920-015-0119-8

DOI

http://dx.doi.org/10.1186/s12920-015-0119-8

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PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/26205789


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24 schema:description BackgroundAdipose 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.MethodsMicroarray 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.ResultsCells 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.ConclusionsASC 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.
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31 schema:keywords ASC
32 Ingenuity Pathway Analysis
33 MethodsMicroarray analysis
34 Primary ASCs
35 RNA
36 RT-PCR
37 ResultsCells
38 Unsupervised hierarchical clustering
39 abundant differences
40 acquisition
41 addition
42 adipocyte differentiation
43 adipocyte lineage
44 adipocyte phenotype
45 adipogenesis
46 adipose-derived stem cells
47 adult females
48 analysis
49 analysis of variance
50 autologous grafting
51 canonical pathways
52 cell function
53 cell populations
54 cells
55 changes
56 clinical limitations
57 clustering
58 comparison
59 conditions
60 consistency
61 culture
62 cutoff
63 days
64 days culture
65 differences
66 differential expression
67 differential gene expression profiles
68 differentiation
69 expression
70 expression analysis
71 expression patterns
72 expression profiles
73 expression programs
74 fat
75 fat cells
76 females
77 first study
78 function
79 gene expression patterns
80 gene expression profiles
81 gene expression programs
82 genes
83 graft retention
84 grafting
85 group
86 hierarchical clustering
87 human adipose-derived stem cells
88 human adult females
89 intra-group consistency
90 isolates
91 late-stage cultures
92 limitations
93 lineages
94 mRNA expression profiles
95 major clinical limitation
96 markers
97 markers of adipogenesis
98 maturation
99 mature adipocyte phenotype
100 mature fat cells
101 mediators
102 microarray results
103 multiple genes
104 multiple patients
105 network
106 novel mediator
107 pathway
108 pathway analysis
109 patients
110 patterns
111 phenotype
112 population
113 potential novel mediator
114 proceeds
115 profile
116 program
117 quantitative RT-PCR
118 reconstruction
119 regenerative resources
120 resources
121 results
122 retention
123 significant network
124 soft tissue reconstruction
125 stage
126 stage cultures
127 stem cells
128 stromal stem cells
129 study
130 technique
131 thousands
132 thousands of genes
133 time
134 tissue reconstruction
135 total RNA
136 transcriptome profiles
137 transcripts
138 transition
139 two-fold change
140 variance
141 version 6.4
142 whole fat
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