ABSTRACT
BACKGROUND: Effective stem cell therapy is dependent on the stem cell quality that is determined by their differentiation potential, impairment of which leads to poor engraftment and survival into the target cells. However, limitations in our understanding and the lack of reliable markers that can predict their maturation efficacies have hindered the development of stem cells as an effective therapeutic strategy. Our previous study identified CD10, a pro-adipogenic, depot-specific prospective cell surface marker of human adipose-derived stem cells (ASCs). Here, we aim to determine if CD10 can be used as a prospective marker to predict mature adipocyte quality and play a direct role in adipocyte maturation. METHODS: We first generated 14 primary human subject-derived ASCs and stable immortalized CD10 knockdown and overexpression lines for 4 subjects by the lentiviral transduction system. To evaluate the role of CD10 in adipogenesis, the adipogenic potential of the human subject samples were scored against their respective CD10 transcript levels. Assessment of UCP1 expression levels was performed to correlate CD10 levels to the browning potential of mature ASCs. Quantitative polymerase chain reaction (qPCR) and Western blot analysis were performed to determine CD10-dependent regulation of various targets. Seahorse analysis of oxidative metabolism and lipolysis assay were studied. Lastly, as a proof-of-concept study, we used CD10 as a prospective marker for screening nuclear receptor ligands library. RESULTS: We identified intrinsic CD10 levels as a positive determinant of adipocyte maturation as well as browning potential of ASCs. Interestingly, CD10 regulates ASC's adipogenic maturation non-canonically by modulating endogenous lipolysis without affecting the classical peroxisome proliferator-activated receptor gamma (PPARγ)-dependent adipogenic pathways. Furthermore, our CD10-mediated screening analysis identified dexamethasone and retinoic acid as stimulator and inhibitor of adipogenesis, respectively, indicating CD10 as a useful biomarker for pro-adipogenic drug screening. CONCLUSION: Overall, we establish CD10 as a functionally relevant ASC biomarker, which may be a prerequisite to identify high-quality cell populations for improving metabolic diseases.
Subject(s)
Adipocytes , PPAR gamma , Adipogenesis , Cell Differentiation , Cells, Cultured , Humans , Neprilysin , PPAR gamma/genetics , Prospective Studies , Stem CellsABSTRACT
Autophagy is an evolutionarily conserved intracellular catabolic process that is essential for cellular housekeeping and nutrient homeostasis. Recently, we provided evidence that thyroid hormone (TH) is a major inducer of autophagy in mammalian cells. Here, we describe a method for detecting TH-induced autophagic flux in hepatic, muscle, and brown adipocyte cells using lysosomal inhibitor bafilomycin A1 (BafA1) and conventional Western blot techniques.
Subject(s)
Autophagy/drug effects , Lysosomes/drug effects , Lysosomes/metabolism , Thyroid Hormones/metabolism , Adipocytes, Brown/drug effects , Adipocytes, Brown/metabolism , Animals , Blotting, Western , Cell Line , Cells, Cultured , Humans , Macrolides/pharmacology , Microtubule-Associated Proteins/metabolism , Myoblasts/metabolism , Protein Isoforms , Receptors, Thyroid Hormone/metabolismABSTRACT
Thyroid hormone (TH) and autophagy share similar functions in regulating skeletal muscle growth, regeneration, and differentiation. Although TH recently has been shown to increase autophagy in liver, the regulation and role of autophagy by this hormone in skeletal muscle is not known. Here, using both in vitro and in vivo models, we demonstrated that TH induces autophagy in a dose- and time-dependent manner in skeletal muscle. TH induction of autophagy involved reactive oxygen species (ROS) stimulation of 5'adenosine monophosphate-activated protein kinase (AMPK)-Mammalian target of rapamycin (mTOR)-Unc-51-like kinase 1 (Ulk1) signaling. TH also increased mRNA and protein expression of key autophagy genes, microtubule-associated protein light chain 3 (LC3), Sequestosome 1 (p62), and Ulk1, as well as genes that modulated autophagy and Forkhead box O (FOXO) 1/3a. TH increased mitochondrial protein synthesis and number as well as basal mitochondrial O2 consumption, ATP turnover, and maximal respiratory capacity. Surprisingly, mitochondrial activity and biogenesis were blunted when autophagy was blocked in muscle cells by Autophagy-related gene (Atg)5 short hairpin RNA (shRNA). Induction of ROS and 5'adenosine monophosphate-activated protein kinase (AMPK) by TH played a significant role in the up-regulation of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), the key regulator of mitochondrial synthesis. In summary, our findings showed that TH-mediated autophagy was essential for stimulation of mitochondrial biogenesis and activity in skeletal muscle. Moreover, autophagy and mitochondrial biogenesis were coupled in skeletal muscle via TH induction of mitochondrial activity and ROS generation.
