Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids
Summary
This protocol reports a unique method of using a streaming cytometer and multiple antibodies for simultaneous assessment of multiple mitochondrial functional parameters, including changes in mitochondrial volume, amounts of the mitochondrial respiratory chain (MRC) complex subunits, and mitochondrial DNA (mtDNA) replication.
Abstract
Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain's three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.
Introduction
Mitochondria are essential cellular organelles and the major site of the adenosine triphosphate (ATP) production. In addition to providing energy for cells, mitochondria also participate in multiple cellular processes, including cell information transmission, cell differentiation, and apoptosis, and have the ability to regulate cell growth and cell cycle. Changes in mitochondrial function have been identified in various neurodegenerative diseases, including Parkinson's disease (PD)1,2, Alzheimer's disease (AD)3, and amyotrophic lateral sclerosis (ALS)4. Mitochondrial dysfunction plays a role in the aging process, accumulating somatic mtDNA mutations and declining respiratory chain function5.
Various types of mitochondrial dysfunction occur in neurodegeneration, and the ability to measure such changes is extremely useful when studying disease mechanisms and testing potential treatments. Furthermore, establishing suitable in vitro model systems that recapitulate disease in human brain cells is vital for better understanding disease mechanisms and developing new therapies. iPSCs from patients with neurodegenerative diseases have been used to generate diverse brain cells that manifest mitochondrial damage6,7,8,9. The development of 3D brain organoids derived from iPSCs is a major step in disease modeling. These iPSC-derived brain organoids provide complexity and contain the patient´s own genetic background, thus providing a disease model that more accurately reflects pathology in the patient's brain.
While some research has been conducted on mitochondrial studies using iPSC-derived brain organoids10,11,12, convenient and reliable techniques for determining multiple mitochondrial functional parameters in iPSC-derived brain organoids remain limited. Flow cytometry provides a powerful tool to measure mitochondrial parameters at the single-cell level, as we have demonstrated previously13. This study provides a detailed protocol for generating cortical organoids from iPSCs, combined with a novel flow cytometry-based approach to simultaneously measure multiple mitochondrial parameters, including mitochondrial mass, respiratory chain complex subunits, and mtDNA copy number (Figure 1). Importantly, by using mitochondrial mass as a denominator, these protocols allow one to measure both the total and specific levels per mitochondrial unit.
Protocol
Representative Results
Discussion
A protocol is presented for generating cortical brain organoids from human iPSCs and for performing the flow cytometric analysis of mitochondrial parameters in single cells isolated from these organoids. The cellular composition of the organoids was verified by confocal microscopy with immunohistochemical staining for neuronal and glial cell markers. The flow cytometry-based strategy co-staining with anti-NDUFB10, VDAC 1, and TFAM has been shown to allow the measurement of specific levels of complex I and mtDNA relative …
Divulgations
The authors have nothing to disclose.
Acknowledgements
We extend our sincere gratitude to Gareth John Sullivan from the Institute of Basic Medical Sciences at the University of Oslo, Norway, for generously providing us with the AG05836 (RRID:CVCL_2B58) cell line. We kindly thank the Molecular Imaging Centre, Flow Cytometry Core Facility at the University of Bergen in Norway. This work was supported by the following funding: K.L was partly supported by the University of Bergen Meltzers Høyskolefonds (project number:103517133) and Gerda Meyer Nyquist Guldbrandson og Gerdt Meyer Nyquists legat (project number: 103816102). L.A.B was supported by the Norwegian Research Council (project number: 229652), Rakel og Otto Kr.Bruuns legat and Gerda Meyer Nyquist Guldbrandson og Gerdt Meyer Nyquists legat.
Materials
| Antibodies using in flow cytometry | |||
| anti-DUFB10 Alexa Fluor 405 | NOVUS biologicals | NBP2-72915AF405 | |
| anti-VDAC1 Alexa Fluor 647 | Santa cruz technology | sc-390996 | |
| anti-TFAM Alexa Fluor 488 | Abcam | ab198308 | |
| L/D fixable near-IR dead cell stain kit | Life technologies | L10119 | |
| Antibodies using in immunofluorence staining | |||
| anti-Tuj1 | Abcam | ab78078 | |
| anti-SOX2 | Abcam | ab97959 | |
| anti-Alexa Flour 488 | Thermo Fisher Scientific | A28175 | |
| anti-Alexa Flour 594 | Thermo Fisher Scientific | A-21442 | |
| Commercial cells | |||
| AG05836 (RRID:CVCL_2B58) | Provided by Gareth John Sullivan from the Institute of Basic Medical Sciences at the University of Oslo, Norway | ||
| Essential 8 Medium (iPSC culture medium) | |||
| Essential 8 Basal Medium | Thermo Fisher Scientific | A1516901 | |
| Essential 8 Supplement (50x) 2% (v/v) | Thermo Fisher Scientific | A1517101 | |
| Store at 4 °C and warm up to RT before use. | |||
| Instruments | |||
| Heracell 150i CO2 Incubators | Fisher Scientific, USA | ||
| Orbital shakers – SSM1, SSL1 | Stuart Equipment, UK | ||
| CCD Microscope Camera Leica DFC3000 G | Leica Microsystems, Germany | ||
| Water Bath Jb Academy Basic Jba5 JBA5 Grant Instruments | Grant Instruments, USA | ||
| Fluid aspiration system BVC control | Vacuubrand, Germany | ||
| Leica TCS SP8 STED confocal microscope | Leica Microsystems, Germany | ||
| 50 mL falcon tube | Sigma-Aldrich | CLS430828 | |
| BD LSR Fortessa | BD Biosciences, USA | ||
| Flowjo Sampler Analysis | FlowJo LLC, USA | ||
| 10 mL pipette | Sigma-Aldrich | SIAL1100 | |
| 1, 10, 100, 1000 mL pipette | Sigma-Aldrich | ||
| 40 µm Cell stariner | Sigma-Aldrich | CLS431750 | |
| ultra-low attachment 96-well plate | S-BIO | MS-9096UZ | |
| Countess II automated cell counter | Thermo Fisher Scientific | ||
| Neural differentiation medium (NDM+) | |||
| DMEM/F12 | Life technologies | 11330032 | |
| Neurobasal medium | Life technologies | 2110349 | |
| Insulin 0.025% (v/v) | Roche | 11376497001 | |
| MEM-NEAA 0.5% (v/v) | Life technologies | 11140050 | |
| Glutamax supplement 1% (v/v) | Life technologies | 35050 | |
| Penicilin/Streptomycin 1% (v/v) | Life technologies | 15140-122 | |
| N2 supplement 0.5% (v/v) | Life technologies | 17502-048 | |
| B27 supplement 1% (v/v) | Life technologies | 17504-044 | |
| β-Mercaptoethanol 50 µM | Sigma-aldrich | M3148 | |
| BDNF 20 ng/mL | Peprotech | 450-02 | |
| Ascorbic acid 200 µM | Sigma-Aldrich | A92902 | |
| Store at 4° C for upto 2 weeks | |||
| Neural differentiation medium minus viatmin A (NDM-) | |||
| DMEM/F12 | Life technologies | 11330032 | |
| Neurobasal medium | Life technologies | 2110349 | |
| Insulin 0.025% (v/v) | Roche | 11376497001 | |
| MEM-NEAA 0.5% (v/v) | Life technologies | 11140050 | |
| Glutamax supplement 1% (v/v) | Life technologies | 35050 | |
| Penicilin/Streptomycin 1% (v/v) | Life technologies (recheck) | 15140-122 | |
| N2 supplement 0.5% (v/v) | Life technologies | 17502-048 | |
| B27 supplement W/O vit. A 1% (v/v) | Life technologies | 12587010 | |
| β-Mercaptoethanol 50 µM | Sigma-aldrich | M3148 | |
| Store at 4° C for upto 8 days | |||
| Neural Induction Medium (NIM) | |||
| DMEM/F12 | Life technologies | 11330032 | |
| Knockout serum replacement 15% (v/v) | Life technologies | 10828028 | |
| MEM-NEAA 1% (v/v) | Life technologies | 11140050 | |
| Glutamax supplement 1% (v/v) | Life technologies | 35050 | |
| β-Mercaptoethanol 100 µM | Sigma-Aldrich | M3148 | |
| LDN-193189 100 nM | Stemgent/Reprocell | 04-0074 | |
| SB431542 10 µM | Tocris | 1614 | |
| XAV939 2 µM | Sigma-Aldrich | X3004 | |
| Store at 4° C for upto 10 days | |||
| Neutralisation medium | |||
| IMDM | Life technologies | 21980032 | |
| FBS 10% | Sigma-Aldrich | 12103C | |
| Other reagents | |||
| DPBS (Ca2+/Mg2+ free) | Thermo Fisher Scientific | 14190250 | |
| Bovine Serum Albumin | Europa Bioproducts | EQBAH62-1000 | |
| Accutase | Life technologies | A11105-01 | |
| Geltrex | Life technologies | A1413302 | |
| EDTA | Life technologies | 15575038 | |
| Advanced DMEM / F12 | Life technologies | 12634010 | |
| Neural tissue dissociation kit | Miltenyi biotec | 130-092-628 | |
| Y-27632 dihydrochloride Rock Inhibitor | Biotechne Tocris | 1254 | |
| Fluoromount-G™ Mounting Medium | SouthernBiotech | 0100-20 | |
| PFA | Thermo Fisher Scientific | 28908 |
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