Optimized Calcium-Phosphate based co-transfection of TAU and tdTomato into human iPSC-derived neurons for the study of intracellular distribution of wildtype and mutant human TAU.

doi:10.21203/rs.3.pex-2142/v1

Method Article

Optimized Calcium-Phosphate based co-transfection of TAU and tdTomato into human iPSC-derived neurons for the study of intracellular distribution of wildtype and mutant human TAU.

https://doi.org/10.21203/rs.3.pex-2142/v1

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The study of TAU protein in disease-relevant neuronal cells in culture requires efficient delivery systems for transfection of exogenous TAU and also modulators and interactions of TAU. Transfection of cultivated cells using calcium phosphate precipitation is a simple and cost-effective approach, also for difficult-to-transfect and sensitive cells such as primary neurons. Because of its low cell toxicity and ease of use, the Ca²⁺-phosphate transfection method is one of the most widely used gene transfer procedures in neuroscience. However, Ca²⁺-phosphate transfection efficacy in neurons is poor, often in the range of 1–5%, limiting its use in functional investigations. Here, we outline our improved Ca²⁺-phosphate transfection methodology for human iPSC-derived neurons that yields a reasonable efficiency (20-30% for bright volume markers) without apparent effects on cell health. We have used it to introduce wildtype and mutant human TAU with and without co-transfection of a volume marker (used here: tdTomato). In sum, our procedure can deliver neuronal genes (e.g. MAPT) using typical eukaryotic expression vectors (e.g. using CMV promoter), and is optimized for transfection of human iPSC-derived neurons.

TAU

Primary Neuronal Cell Culture

hiPSC-derived neurons

Calcium phosphate transfection

eukaryotic expression vectors

Because primary neurons are postmitotic and sensitive to microenvironmental alterations, they are among the most difficult cell types to transfect ^[1,2,4,7]. In comparison to viral vectors, the Ca²⁺-phosphate transfection approach permits transfection of plasmid DNA regardless of its size, and is labor- and cost-effective^[2,4,5]. Graham and Van Der Eb (1973) were the first to employ this approach to transport adenovirus DNA into mammalian cells^[8]. Transfection with Ca²⁺-phosphate causes the formation of DNA crystals that are complexed with the calcium ions in the phosphate buffer ^[2,4]. This permits DNA/Ca²⁺-phosphate precipitates to develop, which attach to the cell surface (when gradually deposited over a monolayer of cells), are taken up by endocytosis, and eventually reach the nucleus ^[13]. Ca²⁺-phosphate transfection efficiency typically ranges from 0.5 to 5%^[1,4,5,7,9]. It is feasible though to achieve a transfection efficiency of about 30-40% with thorough tuning and consistent execution of the experimental methodology^[4]. The formation of optimally sized precipitate particles and the subsequent dissolution of the Ca²⁺-phosphate precipitate are crucial components in obtaining this high rate^[4,5]. The development of homogenous and tiny Ca²⁺-phosphate precipitates, as well as their removal following transfection, were crucial in the prior work ^[3,4].

Here, we have improved the former approach^[4] by using completely defined components and no commercial kits. For consistent tiny precipitates, we optimized the quantity of DNA and calcium chloride concentration. Moreover, we developed a washing buffer that dissolves all Ca²⁺-phosphate precipitates after transfection. The approach presented here may be used to transfect neurons at practically any stage of differentiation in culture, with the exception of extremely early stages, and with decreasing efficiency when neurons become more mature.

In sum, our procedure can i) deliver neuronal genes (e.g. MAPT) using typical eukaryotic expression vectors (e.g. using CMV promoter), ii) be used for cotransfection, iii) be adapted for efficiency and expression duration, and iv) is optimized for transfection of human iPSC-derived neurons.

- Tissue culture sterile water (nuclease-free water)

- Cell culture media (500ml MEM, 5% FBS, 10ml B27 supplement, 100mg NaHCO3, 20mM D-glucose, 0.5 mM L-glutamine)^[4,5]
- Transfection medium: Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), without supplements.

- 1X PBS solution

- 2X Hepes Buffered Saline (HBS), pH = 7.05 - 7.12

- 2.5M CaCl₂

- vector DNA (plasmids), check DNA concentration and purity before use. Plasmids used here are: pShuttleCMV/td-Tomato, pDendra2c-hTau40, pShuttleCMV/Dendra2c/ht40/P301L.

Preparation of buffers

2X Hepes Buffered Saline (HBS) solution ^[6],

pH = 7.05 - 7.12

- 16.4 g NaCl (0.28 M final)

- 11.9 g HEPES (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid; 0.05 M final)

- 0.21 g Na₂HPO4 (1.5 M final)

- 800 ml H20

Titrate to pH 7.05 - 7.12 with NaOH or HCl

- Add H₂O to 1 Liter

Filter sterilize through a 0.45-um nitrocellulose filter.

Check for transfection efficiency.

Store at -20°C in 10-ml aliquots.

2,5M CaCl₂

- 183.7 g CaCl2. 2H₂0 (powder for cell culture)

- H₂O to 500 ml

Filter sterilize through a 0.45-um nitrocellulose filter.

Store at -20°C in 10-ml aliquots.

This solution can be frozen and thawed repeatedly.

- standard Laminar flow hood

- 5% CO₂ incubator at 37°C

- 10% CO₂ incubator at 37°C

- Vortex machine

- Centrifuge

- Water bath (37 °C)

- pHmeter

- Sterile 24-well tissue culture plates

This protocol is optimized for human iPSC-derived neurons and for a 24-well plate (WP).

We have experienced that upscaling (e.g. to standard 12 or 24 well plates) results in improved viability and transfection efficiency.

Day 0: Prepare Cultured cells for Transfection

- Seed cells in a 24WP with coverslips (approximately 50.000 cells per well) and incubate with appropriate media (0.5ml) at 37°C, 5% CO₂ incubator for at least 10 days (for details on how to cultivate and differentiate these cells refer to Bachmann et al, 2021^[14]). There is no need for adaptation of the iPSC culture nor the differentiation procedure (see also Bachmann et al., 2022)

Day 10 after differentiation: Transfection (optimal between Day 8 and Day 15 for the study of axodendritic proteins)^[4]

Transfer selected coverslips to be transfected containing cultured neurons from their original well to a new 24-well plate with 0.5 ml pre-warmed transfection medium (see Buffers and Reagents). Return both the original and the new plate to the incubator (5% CO₂).^[4]
Prepare DNA for transfection (make sure it is pure and clean).
Prepare DNA-CaCl₂ solution (A) in a tube, in the final volume of 1/20th (25 ul) of total growth medium volume in which cells are plated. Add water first, then the DNA, mix well, and finally the CaCl₂ solution and mix again.
Prepare 2X HBS solution (B) in another tube, in the final volume of 1/20th (25ul) of total growth medium volume in which cells are plated.
_____________________________________________________________________________
Solution A            plasmid DNA                  1 ug (optimal concentration)
                            2.5 M CaCl2                   2.48 ul
                            Sterile H2O                     Adjust the total volume of solution A to 25 ul

Solution B            2X HBS                          25 ul
_____________________________________________________________________________
Add gently 1/8 volume at a time of solution A to solution B, while quickly pipetting several times or very brief intermittent vortexing for around 1 second. Repeat until solution A is completely added to solution B.
CRITICAL: Formation of the appropriate size of DNA-Ca²⁺-phosphate precipitate is critical to achieve adequate transfection efficiency.
Allow the precipitate to sit at room temperature for 20-30 minutes without any vortexing.
CRITICAL: Overmixing will result in large clusters of precipitate which is undesirable^.[4,10]
Add the complete mixture/precipitate (50ul) dropwise to the coverslip to be transfected (now in transfection medium). Shake gently to make sure the precipitate is evenly spread out over the whole well/coverslip.
Incubate in a cell culture incubator at 37 °C with 5% CO₂ for 1-3 hours.
Note: Longer incubation time increases the transfection efficiency with minimal effect on cell survival, because the precipitate is subsequently dissolved. After 20-60 minutes of incubation, if examined under the microscope, the precipitate should be homogeneous and resemble a cover of snow all over the field.^[4]
After 1-3 hours of incubation, dissolve the precipitate by incubating for 20 minutes in a 5% CO₂ incubator with transfection medium that has been pre-equilibrated in a 10% CO₂ incubator at 37°C. For the pre-equilibration, incubate the medium separately in a 10% CO₂ incubator for about 40-60 minutes, before adding to the wells.^[4]
CRITICAL: The 10% CO₂ will make the medium more acidic, and Ca²⁺- phosphate will dissolve in acidic solution. It is essential to dissolve the precipitate after incubation to reduce the cell toxicity. After microscopic examination, at the end of 20 minutes incubation, the precipitate should largely disappear.
CRITICAL: If neurons normally cultured in a 5% CO₂ incubator are exposed to 10% CO₂ for more than 15-20 minutes, cells will start dying. Thus, media should be pre-equilibrated and the plate to be returned to the 5% CO₂ incubator rather than the 10% CO₂ incubator.^[4]
Transfer the transfected coverslips back to their original wells containing the original neuronal culture medium (the original culture medium will minimize the cell toxicity, with 50% conditioned medium being the preferred choice over fresh medium).^[4]
Check the next day for protein expression. Protein expression can peak between days 3-6, depending on neuronal cell type, neuronal age and type of inserted DNA.
Note that the exogenous protein expression using this transfection method is very rapid. TdTomato expression in cultured neurons can be observed as early as 4 hours after the transfection, but in our experience cells that express e.g. TAU within a few hours after transfection usually show reduced viability/signs of stress within 1-2 days and die within 3-4 days, likely simply due to overexpression stress. We have not done side-to-side experiments, but feel that this is also true for supposing non-toxic fluorescent proteins, indicating that this kind of toxicity may simply be due to too much exogenous expression.

Critical considerations - Optimisation of transfection efficiency

pH of the transfection buffer

The pH of the transfection buffer is important for generating even and small precipitates. The pH of the HBS should be between 7.05 and 7.12. The pH of the solution may change with prolonged storage. Always use a HBS solution that has been properly stored. If the pH is low, no precipitate will form, and if too high, large aggregates tend to form. ^[3,4,6,10]
This sodium phosphate solution is prepared to accurately control the phosphate concentration in the 2× HBS solution. The phosphate concentration is critical for obtaining small and even calcium phosphate precipitation.^[3]

Transfection solution

Solution A should be added dropwise into solution B, with 1/8 volume of solution A each time and gentle, intermittent vortexing. Intensive and continuous vortexing may result in large and unevenly distributed particles. No further vortexing is necessary after mixing A and B.^[4]
Dropwise addition of the transfection mix plus HBS is critical for forming uniform precipitates. When doing this, the pipette tip should be about 2-3 cm above the liquid surface, so that the mixture is added in droplets. ^[3,4,10]

Incubation time (step 8)

Incubation time lower than 60 minutes might result in decreased transfection efficiency, but it can be extended up to 3 h to improve transfection efficiency. However, neuronal toxicity tends to increase after that point. We recommend to test several time points between 1 and 3 hours, even small changes in cell number or cell age can change outcomes significantly.

Plasmid profile

Inefficient expression due to the profile of the plasmid. A well-designed expression vector that includes a strong promoter is helpful to improve the transfection efficiency.^[4]

Culture media

Incubating in a 10% CO2 incubator will result in increased cell death. After adding transfection medium pre-equilibrated in a 10% CO2 incubator, place the plate in a 5% CO2 incubator. ^[4]
The culture media at this point may contains important trophic factors released by cells ("conditioned medium"), and therefore is kept for re-use after transfection. In our experience, re-using conditioned medium provides for better neuronal viability and reduces markers of stress, e.g. missorting of TAU into the somatodendritic compartment.

Age of cells and duration of expression:

Younger neurons (D8-12) are more efficiently transfected and show higher resistance to transfection-based toxicity. However, further experiments with the transfected neurons (eg.: immunostaining) are advised to be performed within 3-5 days following transfection, for TAU protein at least this appears to be the time when there is sufficient expression that can easily be detected and expected to influence the cellular phenotype, but not as much that expression alone is already toxic. While especially with this protocol it is easily possible to achieve transfection levels sufficient for detection after even half a day or so, these cells usually succumb to expression stress independent of the protein function (e.g. also the case for fluorescent proteins), which makes interpretation of the functional consequences of the transfected protein difficult.

TIMING

(excluding overnight incubation)

Step 1: 3-5 min
Step 2-4: 5-30 min (depending on number of wells to be transfected)

Step 5: 3 - 15 min (depending on number of wells to be transfected)

Step 6: 20-30 min

Step 7: 2 min

Step 8: 1-3 hours (incubation, longer times result in higher efficiency/number of cells transfected and expression/amount of protein expressed, but in particular if increased >3 hours also results in decreased cell viability)

Step 9: 25-30 min

Step 10: 3 min

Step 11: 10-15 min (without overnight incubation)

Neuronal gene delivery can be achieved using typical eukaryotic expression vectors^[7]. A number of genes and gene-combinations (i.e. co-transfection) have previously been carried out using Ca²⁺-phosphate transfection^[11]. Here we use MAPT/TAU knock-out human iPSC-derived neurons to introduce wildtype (wt) and mutant human TAU, co-transfected with a volume marker (tdTomato), 10 days after seeding. The WT and mutant TAU (used here: P301L-TAU) plasmids make use of the CMV promoter, and are both fused with a green fluorescent protein (here: Dendra2c, a particularly dim fluorescent protein). They are thus easily distinguishable from the cotransfected volume marker (here, tdTomato, red fluorescence). To summarize, using the procedure outlined below, we were able to transfect notoriously difficult to transfect human iPSC-derived neurons with decent efficiency using both TAU fused to Dendra2c, and tdTomato (Figure 1 and 2).

We also used single gene transfection (tdTomato only) to study the correlation between transfection efficiency and DNA concentration. We demonstrate that different DNA concentrations can be used to change the time course and quantities of protein expression^[6,12]. Low amount of DNA equals low efficiency, while after a certain threshold of DNA concentration the efficiency plateaued with gradual increase in cell death. We identified a range of 1.0-1.5 μg of DNA to be the optimal one for transfection efficiency in a well of a 24-well plate (Figure 3).

Last, we examined the duration of protein expression after transfection, by recording the expression levels under a epifluorescent microscope on each day up to day 10 after transfection. Daily monitoring of protein expression levels under the epifluorescent microscope concludes that protein expression (tdTomato) is visible even after a few (3-6) hours after transfection, reaching its peak between day 4 and day 6 after transfection. One week after transfection the protein expression levels begin to drop, correlating with increasing death of transfected cells (Figure 4).

Sun, M., Bernard, L.P., DiBona, V.L., Wu, Q., Zhang, H. Calcium Phosphate Transfection of Primary Hippocampal Neurons. J. Vis. Exp. (81).
Karra, D., Dahm, R. (2010) Transfection techniques for neuronal cells. J Neurosci 30(18): 6171–6177
Wang, S., Cho Y.K. (2016) An Optimized Calcium-Phosphate Transfection Method for Characterizing Genetically Encoded Tools in Primary Neurons. In: Kianianmomeni A. (eds) Optogenetics. Methods in Molecular Biology, vol 1408. Humana Press, New York, NY.
Jiang, M., Chen, G. (2006) High Ca²⁺-phosphate transfection efficiency in low-density neuronal cultures. Nat Protoc 1, 695–700
Jiang, M., Deng, L. & Chen, G. (2004) High Ca²⁺-phosphate transfection efficiency enables single neuron gene analysis. Gene Ther 11, 1303–1311.
Kwon, M., Firestein, B.L. (2013) DNA Transfection: Calcium Phosphate Method. In: Zhou R., Mei L. (eds) Neural Development. Methods in Molecular Biology (Methods and Protocols), vol 1018. Humana Press, Totowa, NJ.
Dudek, H., Ghosh, A. and Greenberg, M.E. (1998) Calcium Phosphate Transfection of DNA into Neurons in Primary Culture. Current Protocols in Neuroscience, 3: 3.11.1-3.11.6.
Graham, F.L. and van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 52, 456-467.
Dudek, H., Ghosh, A., Greenberg, M.E. (2001) Calcium phosphate transfection of DNA into neurons in primary culture. Curr Protoc Neurosci. Chapter 3:Unit 3.11. doi: 10.1002/0471142301.ns0311s03. PMID: 18428460.
Kumar, P., Nagarajan, A., Uchil, P.D. (2019) Transfection of Mammalian Cells with Calcium Phosphate-DNA Coprecipitates. Cold Spring Harb Protoc. 2019(10). doi: 10.1101/pdb.top096255. PMID: 31575800.
Eguchi, M., Yamaguchi, S. (2007) Double transfection into primary dissociated neurons. J Biosci Bioeng 103(5):497–499. doi: 10.1263/ jbb.103.497
Dahm, R., Zeitelhofer, M., Gotze, B., Kiebler, M.A., Macchi, P. (2008) Visualizing mRNA localization and local protein translation in neurons. Methods Cell Biol 85:293–327. doi: 10.1016/ S0091-679X(08)85013-3.
Craig, A.M. (1998) Transfecting cultured neurons. in Culturing Nerve Cells, 2nd edition. Banker, G.a.G., K., ed. MIT Press, Cambridge, MA.
Bachmann, S., Bell, M., Al Kabbani, M.A., Klimek, J., Zempel, H. (2021) Investigating isoform‐specific functions of Tau under healthy and pathological conditions using Tau‐deficient hiPSC‐derived cortical neurons. Alzheimer's & Dementia. 17. 10.1002/alz.058005.

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Optimized Calcium-Phosphate based co-transfection of TAU and tdTomato into human iPSC-derived neurons for the study of intracellular distribution of wildtype and mutant human TAU.

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Optimized Calcium-Phosphate based co-transfection of TAU and tdTomato into human iPSC-derived neurons for the study of intracellular distribution of wildtype and mutant human TAU.

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Introduction

Reagents

Equipment

Procedure

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Time Taken

Anticipated Results

References

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