Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Summary

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Abstract

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGFβ/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Introduction

hPSCs, comprising embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are pluripotent meaning that they can give rise to various cell lineages in vitro ^1-2. Numerous differentiated cell types have been derived from hESCs to date, supporting the notion that these cells present valuable tools for scientific research and cell-based screening approaches, and hold promise for cell-based therapeutics.

Neuronal differentiation protocols for hESCs are usually complex and time-consuming as they are often based on first inducing overall neural fate, followed by manual isolation of neural progenitors, and subsequent terminal differentiation into a given neuron type of interest^3-6. In some regard, this complexity is not surprising and inevitable given that such state-of-the-art directed differentiation protocols tend to stepwise mimic early human development, which is in itself extremely complex. On the other hand, it may in part also reflect our lack of understanding of the underlying molecular processes, resulting in differentiation protocols that are still far from being fully optimized.

With regards to the conversion of undifferentiated hPSCs into neuroectoderm, Pera et al. found that suppression of BMP / SMAD1/5/8 signaling enhances neural induction of hESCs⁷. Moreover, inactivation of SMAD2/3 signaling has been shown to promote neuroectoderm formation⁸. Accordingly, combined inactivation of these two pathways tends to lead to more efficient neural induction of hPSCs^4,9. More recently, we have shown that a third signaling pathway, FGF/ERK, serves as a strong repressor of the earliest steps of neuroectodermal commitment in hESCs. Conversely, simultaneous repression of all these three pathways lead to near-homogeneous conversion of hESCs into neuroectoderm within just four days¹⁰. Subsequently, highly efficient terminal differentiation into functional neurons was observed within eight days. The neurons obtained were likely to be sensory neurons of the peripheral nervous system, which may in part explain their rapid derivation¹⁰. Defects in sensory neuron maintenance is regarded a cause of certain human disorders such as familial dysautonomia¹¹. For modeling such diseases based on hiPSC technology¹², for further functional characterization, as well as for applied purposes not requiring any particular type of neurons, this differentiation procedure may present a useful basis.

However, the differentiation strategy we originally employed involved formation of embryonic bodies (EBs) from clumps of hESC colonies¹⁰, and this resulted in quite a degree of heterogeneity regarding EB sizes, and also appeared to compromise neuron formation efficiency in some cell lines. Moreover, due to the heterogeneity in EB sizes, the ability to systematically test effects of additional growth factors or small molecules during and after neuron formation appeared to be somewhat confounded.

In this report, we adapted our previous procedure to a medium throughput-compatible, forced aggregation-based technique to generate EBs in a highly size-controlled manner, as also shown in other contexts¹³. Subsequently, the EBs generated in V-shaped wells of 96-well plates were transferred to U-shaped ultra-low attachment 96-well plates, to initiate neuroectoderm formation in suspension. Four days later, the EBs could be plated out giving rise to terminally differentiated neurons at high efficiency and homogeneity. Alternatively, day-4 EBs were dissociated into single cells and plated out as such, which resulted in low-density monolayers of human neurons. Coherent results were thus far obtained with two independently derived hESC lines, HuES6 and NCL3.

As a prerequisite, successful EB formation was strictly dependent on the use of a synthetic polymer, polyvinyl alcohol (PVA), together with ROCK inhibitor Y-27632 known to promote hESC survival^13-14. As a result, homogeneity of the EBs formed in 96-V-plates was substantially enhanced compared to formation of EBs as mass cultures based on random splitting techniques. This system thus provides a significantly improved platform for neuroectoderm formation in suspension culture, followed by highly controlled neuron formation under adherent conditions.

Protocol

1. Preparation of Matrigel-coated Plates and Media

1. Preparation of Matrigel-coated plates Matrigel has been found to be a preferred substrate for the attachment of differentiating EBs and also promotes the efficient outgrowth of neurons from these¹⁰.
   1. Thaw 10 ml of Matrigel overnight on ice.
   2. Next day, dilute the jelly-like Matrigel with two volumes of ice-cold DMEM/F-12 and prepare aliquots of 1 ml in prechilled 15 ml conical tubes which may be stored frozen at -20 °C.
   3. Decide upon a plate format for neuronal differentiation. For instance, as a starting point, we recommend to perform an initial round of differentiation in a 12-well format. Pre-cool four 12-well plates at 4 °C. Dissolve one aliquot of frozen Matrigel by adding 9 ml of pre-chilled DMEM/F-12 followed by pipetting up and down until all the prediluted Matrigel has thawed and dissolved.
   4. Then, transfer the contents into a new 50 ml tube containing 15 ml of DMEM/F-12 on ice (final Matrigel dilution: 1:75). Then, add 0.5 ml of diluted Matrigel to each well of the pre-cooled 12-well plates. Wrap plates with Parafilm and let stand at RT overnight.
   5. Next day, transfer plates to 4 °C. Coated plates can be stored for several weeks before use.
2. Media

EB formation medium (~15 ml needed to perform differentiation in one 96-well plate - see Table 1 and scheme in Figure 2):
DMEM/F-12 with 0.4% (PVA)
1x N2
1x B27
0.05% BSA
20 ng/ml FGF2
10 μM Y-27632
1x PenStrepGln

Differentiation medium (~30 ml needed to perform differentiation in one 96-well plate, followed by neuron formation in one Matrigel-coated 12-well plate, see Table 1):
DMEM/F-12
1x N2
1x B27
0.05% BSA
0.5 μM PD0325901
15 μM SB431542
0.5 μM Dorsomorphin
1x PenStrepGln

2. Forced EB Formation

1. Grow hPSCs under your preferred feeder-free conditions. We use MEF-conditioned medium on Matrigel-coated 6-well plates¹⁵. However, other culture systems such as homemade or commercial defined media should also work. At the time of starting a differentiation experiment, hPSCs should be sub-confluent and actively growing.
2. Mechanically remove spontaneously differentiated colonies using a sterile plastic pipette tip under a stereo microscope.
3. Wash remaining undifferentiated colonies using PBS, and digest to single cells using Accutase containing 1X Y-27632. Pellet single cells by centrifugation at 200 x g for 2 min, resuspend in a small volume of EB formation medium and determine cell titre using a hematocytometer.
4. Add an aliquot of cells to the remaining EB formation medium to result in a titre between 40,000 and 80,000 cells per ml.
5. Using a multichannel pipette, transfer 100 μl per well to a 96V-bottom plate, resulting in 4,000 to 8,000 cells per well. Spin 96-well plate in a swing-out plate centrifuge at 400 x g for 1 min to collect cells at the bottom of the wells, and transfer to cell culture incubator. EBs will form overnight, as shown in Figure 1.

3. Neuroectoderm Induction

1. Next day (day 0), collect EBs from 96V plate under a stereo microscope and transfer in a small volume into a 3.5 cm bacterial dish containing 2 ml of differentiation medium. Gently agitate the dish for several seconds to wash the EBs.
2. Add 100 μl of differentiation medium per well of an ultra-low-attachment U-bottom 96-well plate. Under a stereomicroscope, transfer EBs in small volumes from the washing dish into the 96U-wells (one EB per well). Place 96U plate into cell culture incubator for 4 days to allow neuroectoderm formation.

4. Neuron Formation

1. On day 4, prepare a washing dish as in step 3.1 and additionally replace DMEM/F-12 in a pre-warmed Matrigel-coated 12-well plate by differentiation medium (1 ml per well).
2. Plating of EBs
   1. Collect EBs from 96U plate, wash these as above, and carefully seed them one by one into wells of the Matrigel-coated 12-well plate (about 8 EBs per well): EBs should be equally distributed within the wells to allow undisturbed outgrowth of neurons over the next days (see "day 5" image in Figure 2).
   2. Before transfer of the 12-well plate back into the incubator, allow EBs to loosely attach for around 10 min at RT. Then, carefully transfer 12-well plate to cell culture incubator. Neurons will grow out from the plated EBs in a radial manner within the next 4 days, as shown in Figure 2 ("day 8" image on the right).

5. Neuron Formation as Monolayers

1. As an alternative to the steps of point 4), at day 4 of the procedure, collect EBs into a 3.5 cm bacterial dish containing 2 ml of differentiation medium, then transfer EBs in a small volume to a PBS-containing dish, and then into another dish containing Accutase.
2. Let digest to single cells, centrifuge at 200 x g for 2 min, resuspend in a small volume of differentiation medium and determine cell titre using a hematocytometer. Adjust titre to 1-2X10⁶ cells per ml using differentiation medium (without Y-27632).
3. Plate cells out in pre-warmed Matrigel-coated 12-well plate (1 ml per well), and transfer to cell culture incubator. Neuron formation as monolayers was observed between days 7 to 8 (Figure 3C). It should be noted, however, that this monolayer variant of the procedure is not fully optimized with regards to cell survival and most suitable substrate.

Results

In our previous report, in line with many others, EBs from hPSCs could be generated simply by replating aggregates upon routine splitting of hPSCs into low-attachment dishes¹⁰. This usually results in a wide distribution of resulting EB sizes (Figure 1C, top). Another problem resulting from this is that EBs maintained in suspension tend to aggregate with one another, leading to an even higher heterogeneity of EB sizes. To circumvent these problems, we therefore attempted to generate EBs ...

Discussion

Most differentiation protocols involving EB formation from hPSCs, including our previously published procedure¹⁰, are based on manual or enzyme-assisted harvesting of hESCs as aggregates, which inevitably leads to heterogeneity in EB sizes. This fact leads to variability in differentiation efficiency with some cell lines, thereby rendering systematic analyses difficult, in particular at a medium throughput scale. Furthermore, manual dissection is labor-intensive which by itself compromises scalability.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Matrigel HC	Becton Dickinson	354263	See text for details on handling
DMEM/F-12	Life Technologies	21331-020
Poly(vinyl alcohol)	Sigma	363170	Dissolve at 0.4% in DMEM/F-12 using an ultrasonic waterbath / avoid overheating / can be kept at 4 °C for several weeks
N2 Supplement	Life Technologies	17502-048	100X, store as frozen aliquots
B27 Supplement	Life Technologies	12587-010	50X, store as frozen aliquots
Bovine Serum Albumin	Sigma	A1595	10% = 500X, store as frozen aliquots
L-Glutamine with Penicillin / Streptomycin	PAA	P11-013	Or equivalent
FGF2	Peprotech	100-18B	Dissolve at 10 μg/ml in 0.1% BSA / PBS = 1000X, store as frozen aliquots
ROCK inhibitor Y-27632	abcamBiochemicals	Asc-129	Dissolve at 10 mM in DMSO = 1000X, store as frozen aliquots
PBS	PAA	H15-002	Or equivalent
Accutase	PAA	L11-007
96-well V-bottom plates	Nunc	277143
PD0325901	Axon Medchem	Axon 1408	Dissolve at 0.5mM in DMSO = 1000X, store as frozen aliquots
SB431542	abcamBiochemicals	Asc-163	dissolve at 15 mM in DMSO = 1000X, store as frozen aliquots
Dorsomorphin	Santa Cruz	sc-200689	dissolve at 0.5 mM in DMSO = 1000X, store as frozen aliquots
96-well U-bottom ultra-low attachment plates	Corning	7007
beta-III Tubulin antibody (mouse)	Sigma	T8660	use at 1:1000
beta-III Tubulin antibody (rabbit)	Covance	PRB-435P	use at 1:2000
BRN3A (POU4F1) antibody	Santa Cruz	sc-8429	use at 1:500
Table 1. Specific reagents and equipment.