EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS CD38 Is Required for Neural Differentiation of Mouse Embryonic Stem Cells by Modulating Reactive Oxygen Species WENJIE WEI,a YINGYING LU,a BAIXIA HAO,b KEHUI ZHANG,c QIAN WANG,a ANDREW L. MILLER,b LIANG-REN ZHANG,c LI-HE ZHANG,c JIANBO YUEa Key Words. Calcium flux • Cell signaling • Neural differentiation • Embryonic stem cells • Signal transduction
a
Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China; bDivision of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China; cState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China Correspondence: Jianbo Yue, Ph.D., Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China. Telephone: 852– 3442-2812; Fax: 852–3442-0549; e-mail: [email protected] Received November 18, 2014; accepted for publication April 27, 2015; first published online in STEM CELLS EXPRESS June 23, 2015. C AlphaMed Press V
ABSTRACT CD38 is a multifunctional membrane enzyme and the main mammalian ADP-ribosyl cyclase, which catalyzes the synthesis and hydrolysis of cADPR, a potent endogenous Ca21 mobilizing messenger. Here, we explored the role of CD38 in the neural differentiation of mouse embryonic stem cells (ESCs). We found that the expression of CD38 was decreased during the differentiation of mouse ESCs initiated by adherent monoculture. Perturbing the CD38/cADPR signaling by either CD38 knockdown or treatment of cADPR antagonists inhibited the neural commitment of mouse ESCs, whereas overexpression of CD38 promoted it. Moreover, CD38 knockdown dampened reactive oxygen species (ROS) production during neural differentiation of ESCs by inhibiting NADPH oxidase activity, while CD38 overexpression enhanced it. Similarly, application of hydrogen peroxide mitigated the inhibitory effects of CD38 knockdown on neural differentiation of ESCs. Taken together, our data indicate that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating ROS production. STEM CELLS 2015; 33:2664—2673
SIGNIFICANCE STATEMENT The in vitro generation of neural lineage cells from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. The functions regulated by the CD38 signaling are diverse. Here we found that the CD38 signaling pathway is required for neural differentiation of mouse ES cells by modulating ROS production. Our findings not only increase our understanding of ES cell differentiation and embryonic neural development, but also encourage the application of therapies based on ES cells in the treatment of neurological and psychiatric diseases.
INTRODUCTION
1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.2057
The in vitro generation of neural lineage cells from ESCs is a promising approach to produce cells suitable for neural tissue repair and cellbased replacement therapies of the nervous system [1, 2]. ESCs can give rise to neural lineages following formation of embryoid bodies (EBs) and treatment with all-trans retinoic acid (RA) or small molecules [3, 4]. Conditions have also been established for monolayer differentiation of ESCs, reducing the complexity compared with multicellular EB. Upon withdrawal of self-renewal stimuli, ESCs generate neural progenitors in adherent monoculture [1, 5]. However, under the adherent monoculture in which all cells are in defined conditions, some cells still adopt nonneural fates [1]. To achieve a high efficiency of directed neural differentia-
STEM CELLS 2015;33:2664–2673 www.StemCells.com
tion of ESCs and make it suitable for cell replacement therapy, it is important to identify novel signaling events that play a role in the commitment of the neural lineages. The Ca21-signaling pathway mediated by cADPR is ubiquitous and the functions it regulates are equally diverse [6, 7], for example, the circadian clock in plants and long-term synaptic depression in hippocampus. Understanding the molecular mechanisms involved in this novel signaling pathway is important not only for scientific reasons but also has clinical relevance. The latter has been demonstrated in CD38 knockout mice. CD38 is a membrane-bound protein and the main mammalian ADP-ribosyl cyclase for synthesizing cADPR from NAD. Besides cADPR, CD38 also catalyzes the hydrolysis of cADPR or NAD to ADPR, a primary activator of TRPM2 channel. C AlphaMed Press 2015 V
Wei, Lu, Hao et al. CD38 knockout mice exhibit multiple physiological defects, for example, impaired immune responses, metabolic disturbances, and social behavioral modifications [6]. The role of cADPR as a Ca21 signaling molecule in the nervous system has been extensively studied [8]. cADPR synthesis by CD38 in neuronal cells is stimulated or modulated via multiple pathways [9]. cADPR can either directly elicit Ca21 increase or potentiate Ca21 releasing primed by other stimuli, such as depolarizing, on neurons and glial cells [10]. Functionally, Ca21 releasing from presynaptic and postsynaptic intracellular stores is required in synaptic transmission. cADPR has also been shown to play a key role in long-term synaptic depression in hippocampus, possibly through cGMP pathway [11]. Recently, it has been shown that CD38 knockout female mice displayed disrupted maternal behavior due to defect in oxytocin releasing from hypothalamic cell dendrites and nerve terminals in the posterior pituitary, suggesting that dysfunctional CD38/cADPR signaling leads to human diseases associated with abnormal social behavior, for example, autism [12]. Therefore, pharmacological manipulation of intracellular cADPR signaling by CD38 or cADPR agonists might provide new therapeutic opportunities for treating neural developmental disorders. Recently, we demonstrated that the CD38 signaling plays an important role in regulating cell proliferation and neuronal differentiation in PC12 cells [13], and the CD38 signaling also inhibits the cardiomyocyte differentiation of mouse ESCs [14]. Therefore, it is of interest to explore the involvement of the CD38 signaling in the neural differentiation of mouse ESCs. By adherent monoculture of mouse ESCs, we efficiently induced the neural differentiation of ESCs and found that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating reactive oxygen species (ROS) production.
MATERIALS
AND
METHODS
Mouse ESC Culture and Neural Differentiation Induction Mouse ESCs, Sox1-green fluorescent protein (GFP) 46C, were maintained in Millipore ESGRO Complete Clonal Grade Medium (Millipore, MA, Cat No. SF001–500, http://www. merckmillipore.com) and passaged every 2 days. ESCs neural differentiation was induced according to an established protocol [1].
CD38 shRNA and CD38-Flag Lentivirus Production and Infection
2665
text to be retained; PI, PLL were used only once and thus have been deleted.
Western Blot Analysis Western blot analysis was performed as described previously [13]. The primary antibodies used were: Nestin, #NES, Aves lab, Oregon, 1:1,000, http://www.aveslab.com; Tuj1, #NB100– 57388, Novus, CO, 1:1,000, http://www.novusbio.com; glial fibrillary acidic protein (GFAP), sc-6170, Santa Cruz, Texas, 1:1,000, http://www.scbt.com.
Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction Quantitative real-time reverse transcriptase polymerase chain reaction was performed as described previously [15]. The primers for detecting CD38, Sox1, Marsh1, S100beta, p47phox, gp91phox, NADPH oxidase (NOX)24, and GAPDH mRNAs are listed in Supporting Information Table S1. Relative gene expression was normalized to GAPDH expression.
Immunohistochemistry Immunohistochemistry was performed as described previously [16]. The primary antibodies used were: Tuj1, #NB100–57388, Novus, CO, 1:500, http://www.scbt.com; GFAP, #G3893, sigma, MO, 1:500, http://www.sigmaaldrich.com; p22phox, Santa Cruz, Texas, #sc20781, 1:500, http://www.scbt.com; FLAG, #F1804, sigma, MO, 1:500, http://www.sigmaaldrich.com.
Flow Cytometry Analysis of Sox1 Expression, ROS, and Mitochondrial Membrane Potential Cells at indicated differentiation days were digested by 0.05% trypsin, centrifuged down at 1,000 rpm 5 minutes, and washed with 13 phosphate-buffered saline (PBS) once. For Sox1-GFP expression assessment, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, centrifuged down again, and resuspended in 13 PBS. For ROS measurement, cells were washed with 13 PBS twice, and then loaded with 1 mM carboxymethyl-H2 -dichlorofluorescein diacetate (CM-H2DCFDA) (Lifetech, NY, #C6827, https://www. lifetechnologies.com) in 13 HBSS for 30 minutes at 378C. For the measurement of mitochondrial membrane potential, the cells were incubated with 100 nM tetramethylrhodamine methyl ester (TMRM, Lifetech, NY, #T668, https://www.lifetechnologies.com) for 20 minutes at 308C. All the fluorescence measurements were performed by BD FACS Canto II analytic flow cytometer (BD biosciences, CA, http://www.bdbiosciences.com/).
Ca21 Measurement
The production and infection of CD38-short hairpin RNA (shRNA) and CD38-Flag lentivirus were performed as described previously [13].
Cytosolic Ca21 in differentiated ESCs was performed as described previously [17].
Propidium Iodide Staining
AMP and ATP Measurement
For the analysis of cell-cycle distribution, cells were fixed by 70% ethanol for 15 minutes on ice, incubated with 40 mg/ml propidium iodide and 0.1 mg/ml RNase for 30 minutes, and analyzed in a BD FACSCanto II cell analyzer (BD biosciences, http://www.bdbiosciences.com/).
a
Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China; bDivision of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China; cState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China Correspondence: Jianbo Yue, Ph.D., Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China. Telephone: 852– 3442-2812; Fax: 852–3442-0549; e-mail: [email protected] Received November 18, 2014; accepted for publication April 27, 2015; first published online in STEM CELLS EXPRESS June 23, 2015. C AlphaMed Press V
ABSTRACT CD38 is a multifunctional membrane enzyme and the main mammalian ADP-ribosyl cyclase, which catalyzes the synthesis and hydrolysis of cADPR, a potent endogenous Ca21 mobilizing messenger. Here, we explored the role of CD38 in the neural differentiation of mouse embryonic stem cells (ESCs). We found that the expression of CD38 was decreased during the differentiation of mouse ESCs initiated by adherent monoculture. Perturbing the CD38/cADPR signaling by either CD38 knockdown or treatment of cADPR antagonists inhibited the neural commitment of mouse ESCs, whereas overexpression of CD38 promoted it. Moreover, CD38 knockdown dampened reactive oxygen species (ROS) production during neural differentiation of ESCs by inhibiting NADPH oxidase activity, while CD38 overexpression enhanced it. Similarly, application of hydrogen peroxide mitigated the inhibitory effects of CD38 knockdown on neural differentiation of ESCs. Taken together, our data indicate that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating ROS production. STEM CELLS 2015; 33:2664—2673
SIGNIFICANCE STATEMENT The in vitro generation of neural lineage cells from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. The functions regulated by the CD38 signaling are diverse. Here we found that the CD38 signaling pathway is required for neural differentiation of mouse ES cells by modulating ROS production. Our findings not only increase our understanding of ES cell differentiation and embryonic neural development, but also encourage the application of therapies based on ES cells in the treatment of neurological and psychiatric diseases.
INTRODUCTION
1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.2057
The in vitro generation of neural lineage cells from ESCs is a promising approach to produce cells suitable for neural tissue repair and cellbased replacement therapies of the nervous system [1, 2]. ESCs can give rise to neural lineages following formation of embryoid bodies (EBs) and treatment with all-trans retinoic acid (RA) or small molecules [3, 4]. Conditions have also been established for monolayer differentiation of ESCs, reducing the complexity compared with multicellular EB. Upon withdrawal of self-renewal stimuli, ESCs generate neural progenitors in adherent monoculture [1, 5]. However, under the adherent monoculture in which all cells are in defined conditions, some cells still adopt nonneural fates [1]. To achieve a high efficiency of directed neural differentia-
STEM CELLS 2015;33:2664–2673 www.StemCells.com
tion of ESCs and make it suitable for cell replacement therapy, it is important to identify novel signaling events that play a role in the commitment of the neural lineages. The Ca21-signaling pathway mediated by cADPR is ubiquitous and the functions it regulates are equally diverse [6, 7], for example, the circadian clock in plants and long-term synaptic depression in hippocampus. Understanding the molecular mechanisms involved in this novel signaling pathway is important not only for scientific reasons but also has clinical relevance. The latter has been demonstrated in CD38 knockout mice. CD38 is a membrane-bound protein and the main mammalian ADP-ribosyl cyclase for synthesizing cADPR from NAD. Besides cADPR, CD38 also catalyzes the hydrolysis of cADPR or NAD to ADPR, a primary activator of TRPM2 channel. C AlphaMed Press 2015 V
Wei, Lu, Hao et al. CD38 knockout mice exhibit multiple physiological defects, for example, impaired immune responses, metabolic disturbances, and social behavioral modifications [6]. The role of cADPR as a Ca21 signaling molecule in the nervous system has been extensively studied [8]. cADPR synthesis by CD38 in neuronal cells is stimulated or modulated via multiple pathways [9]. cADPR can either directly elicit Ca21 increase or potentiate Ca21 releasing primed by other stimuli, such as depolarizing, on neurons and glial cells [10]. Functionally, Ca21 releasing from presynaptic and postsynaptic intracellular stores is required in synaptic transmission. cADPR has also been shown to play a key role in long-term synaptic depression in hippocampus, possibly through cGMP pathway [11]. Recently, it has been shown that CD38 knockout female mice displayed disrupted maternal behavior due to defect in oxytocin releasing from hypothalamic cell dendrites and nerve terminals in the posterior pituitary, suggesting that dysfunctional CD38/cADPR signaling leads to human diseases associated with abnormal social behavior, for example, autism [12]. Therefore, pharmacological manipulation of intracellular cADPR signaling by CD38 or cADPR agonists might provide new therapeutic opportunities for treating neural developmental disorders. Recently, we demonstrated that the CD38 signaling plays an important role in regulating cell proliferation and neuronal differentiation in PC12 cells [13], and the CD38 signaling also inhibits the cardiomyocyte differentiation of mouse ESCs [14]. Therefore, it is of interest to explore the involvement of the CD38 signaling in the neural differentiation of mouse ESCs. By adherent monoculture of mouse ESCs, we efficiently induced the neural differentiation of ESCs and found that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating reactive oxygen species (ROS) production.
MATERIALS
AND
METHODS
Mouse ESC Culture and Neural Differentiation Induction Mouse ESCs, Sox1-green fluorescent protein (GFP) 46C, were maintained in Millipore ESGRO Complete Clonal Grade Medium (Millipore, MA, Cat No. SF001–500, http://www. merckmillipore.com) and passaged every 2 days. ESCs neural differentiation was induced according to an established protocol [1].
CD38 shRNA and CD38-Flag Lentivirus Production and Infection
2665
text to be retained; PI, PLL were used only once and thus have been deleted.
Western Blot Analysis Western blot analysis was performed as described previously [13]. The primary antibodies used were: Nestin, #NES, Aves lab, Oregon, 1:1,000, http://www.aveslab.com; Tuj1, #NB100– 57388, Novus, CO, 1:1,000, http://www.novusbio.com; glial fibrillary acidic protein (GFAP), sc-6170, Santa Cruz, Texas, 1:1,000, http://www.scbt.com.
Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction Quantitative real-time reverse transcriptase polymerase chain reaction was performed as described previously [15]. The primers for detecting CD38, Sox1, Marsh1, S100beta, p47phox, gp91phox, NADPH oxidase (NOX)24, and GAPDH mRNAs are listed in Supporting Information Table S1. Relative gene expression was normalized to GAPDH expression.
Immunohistochemistry Immunohistochemistry was performed as described previously [16]. The primary antibodies used were: Tuj1, #NB100–57388, Novus, CO, 1:500, http://www.scbt.com; GFAP, #G3893, sigma, MO, 1:500, http://www.sigmaaldrich.com; p22phox, Santa Cruz, Texas, #sc20781, 1:500, http://www.scbt.com; FLAG, #F1804, sigma, MO, 1:500, http://www.sigmaaldrich.com.
Flow Cytometry Analysis of Sox1 Expression, ROS, and Mitochondrial Membrane Potential Cells at indicated differentiation days were digested by 0.05% trypsin, centrifuged down at 1,000 rpm 5 minutes, and washed with 13 phosphate-buffered saline (PBS) once. For Sox1-GFP expression assessment, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, centrifuged down again, and resuspended in 13 PBS. For ROS measurement, cells were washed with 13 PBS twice, and then loaded with 1 mM carboxymethyl-H2 -dichlorofluorescein diacetate (CM-H2DCFDA) (Lifetech, NY, #C6827, https://www. lifetechnologies.com) in 13 HBSS for 30 minutes at 378C. For the measurement of mitochondrial membrane potential, the cells were incubated with 100 nM tetramethylrhodamine methyl ester (TMRM, Lifetech, NY, #T668, https://www.lifetechnologies.com) for 20 minutes at 308C. All the fluorescence measurements were performed by BD FACS Canto II analytic flow cytometer (BD biosciences, CA, http://www.bdbiosciences.com/).
Ca21 Measurement
The production and infection of CD38-short hairpin RNA (shRNA) and CD38-Flag lentivirus were performed as described previously [13].
Cytosolic Ca21 in differentiated ESCs was performed as described previously [17].
Propidium Iodide Staining
AMP and ATP Measurement
For the analysis of cell-cycle distribution, cells were fixed by 70% ethanol for 15 minutes on ice, incubated with 40 mg/ml propidium iodide and 0.1 mg/ml RNase for 30 minutes, and analyzed in a BD FACSCanto II cell analyzer (BD biosciences, http://www.bdbiosciences.com/).
