NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells

Abstract

The stromal vascular cell fraction (SVF) of visceral and subcutaneous adipose tissue (VAT and SAT) has increasingly come into focus in stem cell research, since these compartments represent a rich source of multipotent adipose-derived stem cells (ASCs). ASCs exhibit a self-renewal potential and differentiation capacity. Our aim was to study the different expression of the embryonic stem cell markers NANOG (homeobox protein NANOG), SOX2 (SRY (sex determining region Y)-box 2) and OCT4 (octamer-binding transcription factor 4) and to evaluate if there exists a hierarchal role in this network in ASCs derived from both SAT and VAT. ASCs were isolated from SAT and VAT biopsies of 72 consenting patients (23 men, 47 women; age 45 ± 10; BMI between 25 ± 5 and 30 ± 5 range) undergoing elective open-abdominal surgery. Sphere-forming capability was evaluated by plating cells in low adhesion plastic. Stem cell markers CD90, CD105, CD29, CD31, CD45 and CD146 were analyzed by flow cytometry, and the stem cell transcription factors NANOG, SOX2 and OCT4 were detected by immunoblotting and real-time PCR. NANOG, SOX2 and OCT4 interplay was explored by gene silencing. ASCs from VAT and SAT confirmed their mesenchymal stem cell (MSC) phenotype expressing the specific MSC markers CD90, CD105, NANOG, SOX2 and OCT4. NANOG silencing induced a significant OCT4 (70 ± 0.05%) and SOX2 (75 ± 0.03%) downregulation, whereas SOX2 silencing did not affect NANOG gene expression. Adipose tissue is an important source of MSC, and siRNA experiments endorse a hierarchical role of NANOG in the complex transcription network that regulates pluripotency.

Knockdown of NANOG Reduces Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Human Adipose Stem Cells

“…All patients gave their written informed consent. Adipose tissue was processed as previously described [13]. Cells were used at an early passage for all experiments.…”

“…mRNA from ASCs populations isolated from subcutaneous biopsies were isolated using an RNeasy kit (Qiagen, Hamburg, Germany) as previously described [13]. For each gene, mRNA expression was normalized for the housekeeping gene beta-actin (Invitrogen).…”

“…OCT4, SOX2, and NANOG have also been suggested to play a similar role as embryonic stem cells in adult mesenchymal cells, including human Adipose Stem Cells (hASCs). In our previous studies we found that the embryonic stem cell marker NANOG is over-expressed in MSCs derived from adipose tissue and its silencing with a RNA interference technology causes downregulation of OCT4 and SOX2 gene expression [13], confirming recent studies that have demonstrated a central role of NANOG in regulating stem cells' multipotent properties [15,17]. DLK1(PREF1) is a marker used to characterized mouse preadipocyte progenitors which inhibits adipogenesis, suppressing C/EBPβ and C/EBPδ gene expression [18][19][20][21][22][23].…”

“…Adipose stem cells (ASC) represent an alternative source of mesenchymal stem cells that can be easily and safely obtained from adipose tissue, growing under appropriate culture conditions for a long time [8][9][10]. ASCs are characterized by a well-defined cell-surface antigen phenotype consisting of both expression of antigens such as CD29, CD90, CD105 and CD73 and the absence of hematopoietic-endothelial markers such as CD34, CD45, CD117, and HLA-DR [11][12][13][14]. There is still great interest in better understanding the mechanisms underlying the proliferation, differentiation, and heterogeneity of these cells [15,16].…”

Anti-Inflammatory Action of Heterogeneous Nuclear Ribonucleoprotein A2/B1 in Patients with Autoimmune Endocrine Disorders

“…PCR primers for hnRNPA2/B1, IL-6, FAS, IDO, MCP1, CCND1 and p27 were purchased from Qiagen (QuantiTect Primer Assays, Qiagen, Milan, Italy). All reactions were performed with Quantitect Sybr Green PCR Kit (Qiagen) using the Rotor-Gene Q instrument (Qiagen, Milan, Italy) as previously described [20]. The specificity of the amplified products was determined by means of melting peak analysis.…”