Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Summary

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Abstract

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Introduction

The circadian timing system (from Latin "Circa diem") has emerged in all light-sensitive organisms, as an adaptive mechanism to the rotation of the Earth. In mammals, it is organized in a hierarchical manner, encompassing the central clock, which is situated in the suprachiasmatic nucleus of the ventral hypothalamus, and peripheral (or slave) oscillators that are operative in different organs. Moreover, these cell autonomous self-sustained oscillators are functional in nearly every cell of the body ¹. Photic signals represent a dominant synchronizing cue (Zeitgeber) for the SCN neurons, whereas neural and humoral signals emanating from the SCN reset the peripheral clocks. In addition rest-activity rhythms, that drive in turn feeding-fasting cycles, are further synchronizers for peripheral clocks ². According to our current understanding, the molecular makeup of the core clock is based on transcriptional and translational feedback loops, which are conserved between organisms. This comprises the transcriptional activators BMAL1 and CLOCK, which together activate transcription of the negative core clock PER and CRY genes. High levels of PER and CRY proteins will inhibit their own transcription through inhibition of the BMAL1/CLOCK complex. An auxiliary loop consists of the nuclear receptors REV-ERBs and RORs, which also regulate the transcription of BMAL1 and CLOCK. Furthermore, posttranslational events including phosphorylation, sumoylation, acetylation, O-GlcNAcylation, degradation and nuclear entry of the core clock proteins represent an additional important regulatory layer in establishing the 24 hr oscillation cycle ³.

Accumulating evidence stems from studies in rodent models and highlights the critical role of the circadian system in the coordination of metabolic and endocrine functions ^4-5. A number of large-scale transcriptome analysis suggest that feeding – fasting cycles play a central role in the synchronization of peripheral oscillators ^6-8. In an agreement with these studies, metabolomic and lipidomic analysis in rodents and humans have revealed that a large number of metabolites oscillate in tissue, plasma, and saliva in a circadian manner ^9-11. Importantly, most hormones exhibit circadian rhythms in blood ^5,12-13. Moreover, circadian clocks of the corresponding hormone producing peripheral tissue might regulate hormone secretion locally. Cell-autonomous circadian oscillators have been described in rodent and human pancreatic islet cells ^14-16. These oscillators play an essential role in regulating the pancreatic islet transcriptome and function ^15,17-18. Furthermore, myokine secretion by human skeletal myotubes has been recently demonstrated to exhibit a circadian pattern, which is regulated by cell-autonomous oscillators operative in these cells ¹⁹.

Several approaches for studying circadian rhythms in humans in vivo have been widely used. For instance, plasma melatonin or cortisol levels as well as thoracic skin surface temperature (reviewed in references ^3,20) have been studied to assess endogenous circadian clocks. Although these methods allow studying systemic circadian oscillations in vivo, they are far from providing a reliable assessment of free-running autonomous circadian rhythms in different organs and tissues. Nevertheless, such dissection from the systemic regulation would be an indispensable tool for understanding the specific effect of intracellular molecular clocks on the function of these cells. Therefore, a substantial effort has been undertaken to develop reliable approaches for studying human clocks in immortalized or primary cultured cells synchronized in vitro. Importantly, it has been demonstrated that clock characteristics measured in cultured primary skin fibroblast cells closely reflect the individual clock properties of the whole organism ²¹. The development of fluorescent and bioluminescent circadian reporters has greatly advanced this approach ^22-27. Furthermore, studying primary cell clocks that are derived from different peripheral organs allows for the investigation of the molecular properties of human tissue-specific clocks ^{3,5,16,19-20,28}. Thus, assessment of circadian clocks in in vitro synchronized primary explants or cells, by using bioluminescent reporters, represents a highly useful method to study the molecular makeup of human peripheral clocks and their impact on organ function.

In this article, we will present detailed protocols for assessing circadian gene expression in human primary islet and skeletal muscle cells synchronized in vitro as well as the impact of autonomous cellular clock disruption on the secretory function of these cells.

Protocol

Ethics statement: Manipulations included in this protocol were approved by the Ethics Committee of the Geneva University Hospital and by the Ethical Committee SUD EST IV (Agreement 12/111) ¹⁹. Human islets were isolated from pancreases of brain-dead multi-organ donors in the Islet Transplantation Centre at the University Hospital of Geneva (Switzerland) as described by us in references ^16,18, or obtained from a commercial source.

1. Preparation of Primary Cell Culture

1. Human Pancreatic Islet Isolation, Dissociation and Culture
   NOTE: Coat every tube, plastic tip or pipette with Connaught Medical Research Laboratories (CMRL) medium in order to prevent islets or islet cells from sticking to the plastic surface, which can lead to a significant loss of cell material.
   1. One day prior to islet cell dissociation add 1 ml of laminin-5-rich extracellular matrix (derived from 804G cells as described in reference ²⁹) per 3.5 cm dish. Before plating cells, aspirate the matrix and wash the dish 3 times with sterile bi-distillated water. Allow the dish to dry under the laminar flow cabinet for 5 min.
   2. Inside the laminar flow cabinet, distribute the obtained islets with CMRL medium into 15 ml tube(s). Centrifuge at 272 x g for 5 min.
   3. Aspirate the supernatant, and then resuspend the pellet in 1 ml of sterile Dulbecco's phosphate-buffered saline (DPBS) pre-warmed to 37 °C without calcium and magnesium. Centrifuge at 272 x g for 5 min.
   4. Aspirate the supernatant and resuspend the cell pellet in 1 ml of DPBS.
   5. To count the total number of islets, pipet 10 µl from the 1 ml islet suspension into a new 3.5 cm dish. Count the number of islets in the 10 µl drop under the microscope and from this calculate the total number of islets in the 1 ml islet suspension. Add 14 ml of DPBS and centrifuge the cell suspension one more time at 272 x g for 5 min.
   6. For islet cell dissociation, aspirate the supernatant and add 1 ml of cell detachment solution for a maximum of 1,000 islet equivalents (IEQ). Place the tube in a water bath at 37 °C and gently mix the islets by pipetting up and down several times every minute, during 6-10 min.
      NOTE: To check the digestion quality, pipet a 2 µl drop of suspension on a glass slide and check under the microscope that all cells are well separated, and that no doublets or cell clumps have remained.
   7. Stop the reaction by adding 14 ml of cold CMRL with supplements (10% fetal bovine serum (FBS), 1% of L-alanyl-L-glutamine dipeptide, 1% penicillin-streptomycin (P/S), 1% gentamycin, 1% sodium pyruvate). Centrifuge at 425 x g for 5 min. Aspirate the supernatant and add 15 ml of CMRL medium to the cell pellet.
   8. Resuspend the pellet in a small volume of CMRL with supplements. Count the number of cells under the microscope using a hemocytometer, adjust the CMRL volume in order to obtain a cell concentration of ~650, 000 cells/ml.
   9. Pipet 3 separated drops of 100 µl each from the dispersed cell suspension obtained in step 1.1.8 in a 3.5 cm dish pre-coated with laminin.
      NOTE: Cells attach to the dish in about 24 hr.
   10. Incubate cells (Figure 1A) in a tissue culture incubator at 37 °C in a humid chamber. Change the medium of the cell drops every 2-3 days by aspirating 100 µl from each drop and replacing it with the same volume of fresh medium.
2. Human Primary Myoblast Culture and Differentiation into Myotubes
   1. Tissue biopsy, satellite cell isolation and myoblast culture
      Note: Muscle biopsies were obtained from the group of Etienne Lefai (INSERM, Lyon, France) ¹⁹.
      1. Purify primary skeletal myoblasts according to the previously described procedure ³⁰.
   2. Differentiating primary human myoblasts into myotubes
      1. Take one vial (1 x 10⁶ cells) of human myoblasts stored in liquid nitrogen and thaw cells quickly by putting the vial for 30 sec to 1 min in a water bath at 37 °C with agitation.
      2. Pipet cells (1 ml) into 24 ml of growth medium composed of HAM F-10 supplemented with 20% FBS, 1% P/S, 0.5% gentamycin and 0.2% amphotericin B.
      3. Centrifuge 5 min at 150 x g.
      4. Remove the supernatant and resuspend cells with 15 ml of fresh growth medium per 2.5 x 10⁵ cells.
      5. Plate at least 2.5 x 10⁵ cells per F75 adherent flask. Keep the myoblasts in a cell incubator at 37 °C and 5% CO_2.
      6. Once cells reach 60-80% confluency, dissociate cells with trypsin-EDTA 0.05% for 1-2 min and plate them in 2 ml of growth medium on adherent 3.5 cm petri dishes.
      7. After reaching confluence, remove the growth medium.
      8. Start the differentiation protocol of human myoblasts into myotubes by culturing them in 2 ml of Dulbecco's Modified Eagle Medium (DMEM) containing 1 g/L of glucose, 2% FBS, 1% P/S, 0.5% gentamycin and 0.2% amphotericin B (differentiation medium) in a cell incubator at 37 °C and 5% CO₂. Change the medium every 2 to 3 days.
         NOTE: Myotubes are usually formed within 7-10 days.
      9. Check muscle cell differentiation under the microscope (Figure 2A) by observing the fusion of myoblasts into polynucleated myotubes ¹⁹.

2. Small Interfering RNA (siRNA) Transfection

1. siRNA Transfection of Human Islet Cells
   NOTE: The transfection protocol is performed in drops of 100 µl on the next day after cell dissociation (steps 1.1.1-1.1.10).
   1. Aspirate 100 µl of CMRL medium from each drop and replace it with the same volume of serum-free Minimal Essential Medium (MEM) 2 hr before transfection by pipetting.
   2. Prepare a MEM-based mix of transfection reagent and 50 nM of target siRNA (siClock) or 50 nM of non-targeting siControl according to the manufacturer's instructions.
      1. For one dish with 3 drops prepare two 1.5 ml tubes with 200 µl of MEM each.
      2. Add to one of these tubes 4 µl of transfection reagent.
      3. Add to the second tube 1 µl of the of appropriate siRNA stock solution (20 µM).
      4. Agitate these two tubes slowly on the orbital shaker for 5 min and then mix the content of the tubes together and agitate for 20 more min.
   3. Aspirate 100 µl of MEM from each drop and replace it with the same volume of transfection mix obtained in the previous step by pipetting.
   4. Replace the transfection solution with CMRL medium after 4 hr of incubation at 37 °C. Repeat steps 2.1.1-2.1.3 the following day for cell re-transfection.
2. siRNA Transfection of Human Myotubes
   1. Before transfection, replace the medium (see step 1.2.2.8) with 2 ml of fresh differentiation medium per 3.5 cm petri dish.
   2. In a sterile 1.5 ml tube, prepare a mix of 20 nM siRNA (siControl or siClock), which corresponds to 2.4 µl of a 20 µM siRNA solution, and 12 µl of transfection reagent diluted in 100 µl of differentiation medium. Incubate the solution at room temperature for 15 min with gentle agitation.
   3. Transfect cells with 114.4 µl of the siRNA mix per 3.5 cm petri dish and place cells into a tissue culture incubator at 37 °C and 5% CO₂ for 24 hr.

3. Continuous Long-term Circadian Bioluminescence Recording Performed in Parallel with the Assessment of Hormone Secretion in Living Human Primary Cells

1. Introducing Circadian Bioluminescence Reporters into Human Primary Cells by Lentiviral Transduction
   NOTE: All procedures with lentiviral particles must be performed in a biosafety level 2 facility to take additional precautions for work with agents that pose a moderate potential hazard to personnel and the environment.
   1. Prepare reporter lentiviral particles by co-transfecting the vector of interest pLenti6.4/R4R2/V5-DEST/Bmal1-luc or pLV156-Per2-dLuc (called Bmal1-luc and Per2-luc, respectively,) plasmid ³¹ with lentiviral vectors pMD2G and psPAX into 293T cells using the polyethylenimine method (for detailed procedure see reference ¹⁶).
   2. Titrate the obtained lentiviral particles (details on the titration can be obtained at http://lentilab.unige.ch/). For further experiments, use lentiviruses with titers ranging 10⁴ to 10⁵ transducing units [TU/µl].
   3. Place dishes with human islet cells or human myoblasts (at 30-50% confluency) inside the laminar flow cabinet and replace the medium with 2 ml of fresh supplemented CMRL medium (see step 1.1.7) or growth medium (see step 1.2.2.2), respectively.
   4. Calculate the multiplicity of infection (MOI) (i.e. infectious particles (transducing units)/number of cells).
   5. Transduce the primary cell culture by pipetting lentivirus solution to the dish in order to obtain a MOI = 3 (for example, for 65,000 attached cells add 3 µl of the virus solution with the titer of 6.5 x 10⁴ to 100 µl medium drop).
   6. Incubate overnight in a tissue culture incubator. Change medium the next day.
      NOTE: Transduce human islet cells at least 4 days prior to bioluminescence recording in order to achieve sufficient expression of the reporter construct. Myoblasts are transduced during the expansion phase, then grown to confluence, and subsequently differentiated into myotubes.
2. In Vitro Synchronization of Human Primary cells
   1. Add 10 µM of adenylyl cyclase activator in 2 ml of medium per 3.5 cm petri dish containing the primary cells previously transduced in step 3.1.5.
   2. Incubate for 60 min at 37 °C in a cell culture incubator.
   3. Change the medium containing the adenylyl cyclase activator with 2.5 ml of the recording medium containing 100 µM luciferin.
      NOTE: For human islet cells use CMRL supplemented with 10% FBS, 1% L-alanyl-L-glutamine dipeptide, 1% P/S, 1% gentamycin; for human myotubes use phenol red - free DMEM with 1 g/L glucose supplemented with 2% FBS, 2% L-alanyl-L-glutamine dipeptide, 1% P/S, 0.5% gentamycin and 0.2% amphotericin B.

4. Parallel Assessment of Circadian Bioluminescence Recording and Hormone Secretion Profiles in Synchronized Human Secretory Primary Cells

1. Setting Up Long-term Constant Perifusion and Bioluminescence Recording for Human Primary Cells.
   NOTE: When working outside the laminar flow cabinet, clean all contact surfaces and limit exposure of cultures or medium to the air to avoid contamination.
   1. To prepare the perifusion medium, add 100 µM of luciferin to the medium.
      NOTE: For human islet cells use CMRL supplemented with 10% FBS, 1% L-alanyl-L-glutamine dipeptide, 1% P/S, 1% gentamycin; for human myotubes use phenol red – free DMEM with 1 g/L glucose supplemented with 2% FBS, 2% L-alanyl-L-glutamine dipeptide, 1% P/S, 0.5% gentamycin and 0.2% amphotericin B.
   2. Inside the laminar flow cabinet, open the 3.5 cm dishes containing the transduced, transfected and synchronized primary cell cultures (islet cells or myotubes) as described above. Insert sterile metallic caps (developed in house) (Figure 1B2) into the 3.5 cm dishes that are equipped with silicone influx/efflux connecting tubes (Figure 1B1/B5).
   3. Place the dishes on the measurement platform in the 37 °C light-tight incubator. Fix the dishes to the platform by using a screwable adaptor (Figure 1B3). Insert the influx/efflux tubes of the perifusion system into the appropriate silicone tubes of the cap (Figure 1B1/B5) and set the speed of the pump at a flow rate of ~0.5 ml of medium per 1 hr.
   4. Open the in-house developed Drip-biolumicorder software that records the signals from the photomultiplier tube (PMT) detector. Chose the directory where the data will be stored and start continuous bioluminescence recording from each dish by clicking the "start" icon.
      NOTE: Alternatively to the Drip-biolumicorder software, other programs (e.g. LumiCycle), can be used to record signals from PMT detector.
   5. Place sterile 6-well tissue culture plates in the collection box on ice.
   6. Open the control software that controls the timing of the automated switch among the collection wells. Set up the time window of medium collection (sec). Start collection of the outflow medium every 4 hr (14,400 sec; ~2 ml per time-point) by clicking the "run" icon.
   7. Transfer and measure the outflow medium from each collection well into sterile 2 ml tubes by pipetting. Keep tubes in a -20 °C freezer before starting the next step. Repeat steps 4.1.5-4.1.6 every 24 hr.
   8. Stop the bioluminescence recording and the medium flow by clicking the "stop" icon on the corresponding software. Remove the metallic caps and aspirate the residual medium from the dishes.
   9. In order to normalize the secreted protein values obtained in different experiments, either extract DNA (normalization by genomic DNA content for myotubes ¹⁹), or add 1 ml of lysis acid-ethanol buffer (normalization by total hormone content for islet cells ¹⁸) to the dishes.

5. Measuring Islet Hormone and Myokine Levels in the Outflow Medium Obtained by Continuous Perifusion of Human Primary Endocrine Cells

1. Insulin
   1. Quantify basal insulin levels in the outflow medium from collected time-points by using a human insulin enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer's instructions.
   2. Normalize data to the absolute volume of collected medium in each well and to the total insulin content, extracted from acid-ethanol treated cells at the end of the experiment (step 4.1.9) ¹⁸.
2. Interleukin-6 (IL-6)
   1. Quantify  basal IL-6 levels in the outflow medium from collected time-points by using a human IL-6 ELISA kit following the manufacturer's instructions.
   2. Normalize data to the absolute volume of collected medium in each well and to genomic DNA content at the end of the experiment (step 4.1.9).

6. Circadian Dataset Analyses for Bioluminescence and Hormone Secretion Profiles

1. Bioluminescence Analysis
   1. Analyze bioluminescence profile using the provided software ¹⁹.
2. JTK-cycle Analysis
   1. Analyze hormone secretion profiles and bioluminescence recording results by using the JTK_CYCLE algorithm ³².
   2. Set the circadian period width at 20-24 hr.
      NOTE: In case the experimental conditions were recorded in parallel, a paired statistical analysis can be performed to compare the experiments.
3. CosinorJ Analysis
   1. Alternatively, analyze hormone secretion and circadian bioluminescence profiles using the CosinorJ software ²⁸.

Results

Assessment of Islet Hormone Secretion with Parallel Circadian Bioluminescence Recording from Perifused Human Islet Cells

After providing a first molecular characterization of the circadian clock, operative in human islet cells ¹⁶, we aimed at studying the impact of clock disruption on islet function and transcription ¹⁸. We set up an efficient siClock transfection protocol in dispersed human islet ...

Discussion

The experimental settings described here are composed of lentiviral delivery of circadian bioluminescence reporters into cultured human primary cells, followed by subsequent in vitro synchronization and continuous recording of bioluminescence for several days, and parallel analysis of hormone secretion by the same cells. They represent an efficient approach for exploring molecular mechanisms and functional aspects of circadian clocks in human primary cells.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Trypsin-EDTA	Invitrogen	25300-054	For muscle biopsy digestion
DPBS no calcium no magnesium	Invitrogen	14190-094
HAM F-10	Invitrogen	41550-021	For myoblasts culture
FBS	Invitrogen	10270	Supplement to culture medium
Penicillin-Streptomycin	Sigma	P0781-100	Supplement to culture medium
Gentamycin	Axon	A1492.0001	Supplement to culture medium
Fungizone	Invitrogen	15290-026	Amphotericin B, supplement to culture medium
DMEM 1g/L glucose + Na pyruvate + glutamax	Invitrogen	21885-025	For myotubes culture
DMEM 1g/L glucose -Na Pyruvate - glutamax	Invitrogen	11880-028	Recording medium for LumiCycle
Glutamax	Invitrogen	35050-028	L-alanyl-L-glutamine dipeptide, supplement to recording medium
Accutase	Innovative Cell Technologies	AT-104	Cell detachment solution, for islet cell dissociation
CMRL	Gibco	21530-027	Culture medium for islet cells
Sodium Pyruvate	Gibco	11360-039	Supplement to culture medium
15 ml High-Clarity Polipropylene Conical Tube	Falcon	352096
F75 flask	BD Falcon	353136
3.5 cm Petri dish	BD Falcon	353001
Foskolin	Sigma	F6886	Adenylyl cyclase activator, used for synchronization
Luciferin	Prolume LTD	260150	Supplement to recording medium
OptiMEM	Invitrogen	51985-026	Serum-free Minimal Essential Medium (MEM) used for human islet cells transfection
Lipofectamine RNAiMAX reagent	Invitrogen	13778-150	Transfection reagent
HiPerFect reagent	Qiagen	301705	Transfection reagent
ON-TARGET plus siCLOCK smartpool	Dharmacon	L-008212-00
ON-TARGET plus non targeting siRNA #1 (siControl)	Dharmacon	D-001810-01
DNeasy Blood & Tissue Kit	Qiagen	69504	For myotubes DNA extraction
RNeasy Plus Mini kit	Qiagen	74104	For myotubes RNA extraction
QIAshredder	Qiagen	79654	For myotubes RNA extraction
2 ml collecting tubes	Axygen	311-10-051	To collect the medium with the perifusion
Tissue culture Plate, 6 Well	BD Falcon	353046	To collect the medium with the perifusion
RNeasy Plus Micro kit	Qiagen	74034	For islet RNA extraction
Human IL-6 Instant ELISA kit	eBioscience	88-7066-22
Human Insulin Kit Mercodia	Mercodia	10-1113-01
Hydrochloric acid, min,37%,p.a.	Acros organics	124630010	Used for preparation of lysis buffer (375 ml Ethanol + 7.5%HCl + 117.5% H2O)
Ethanol (>99.8%)	Fluka Analytical	02860-1L	Used for preparation of lysis buffer (375 ml Ethanol + 7.5%HCl + 117.5% H2O)
Human Islets for Research	Prodo Laboratories
Name	Company	Catalog Number	Comments
Equipment:
Centrifuge	Heraeus	Megafuge 1.0R
Water bath	VWR	1112A	at 37 °C
Tissu culture hood	Faster	SafeFastElite
Tissu culture incubator	Heraeus	HeraCell 150	5% CO2 at 37 °C, no water due to the LumiCycle installation
Tissu culture incubator	Heraeus	HeraCell 150	5% CO2 at 37 °C, no water due to the LumiCycle installation
Tissu culture incubator	Thermo Scientific	Hera Cell 150i	5% CO2 at 37 °C
Shaker	Heidolph Instruments	Unimax 1010	For agitation of the siRNA mix
LumiCycle	Actimetrics
LumiCycle software	Actimetrics
CosinorJ software	EPFL		Freely available at: http://bigwww.epfl.ch/algorithms/cosinorj/
Rheodyne titan MX	ERC GmbH		Control software that controls the timing of the automated switch