Källberg, M. et al. Template-based protein structure modeling using the RaptorX web server. Nat. Protoc. 7, 1511–1522 (2012). Kirkeby, A. et al. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep. 1, 703–714 (2012). Amit, M. et al. Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nat. Protoc. 6, 572–9 (2011). Ribeiro, E. et al. The Structure and Regulation of Human Muscle α-Actinin. Cell 159, 1447–1460 (2014). KDM5B elution volumes were studied using a Superdex 200 5/150 Increase column (GE Health Care) using an HPLC system (Agilent 1100). KDM5B in concentrations in the range 0.05–2 mg/ml were applied using a buffer of 50 mM HEPES 300 mM NaCl pH 7.7 and 1 mM DTT and a flow rate of 0.25 mL/min. Samples were kept at 5 °C until the application to the column. The column was calibrated using standards from the LMW and HMW kits (Sigma A6103). SAXS

Yamane, K. et al. PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation. Mol. Cell 25, 801–12 (2007). Homology modelling was performed using the RaptorX server 37. Prediction of coiled coil regions from the amino acid sequence was done with the COILS server 38. EM data collection, 3D particle reconstruction and analysisPouton, C. W. & Haynes, J. M. Embryonic stem cells as a source of models for drug discovery. Nat. Rev. Drug Discov. 6, 605–16 (2007). Luger, K., Rechsteiner, T. J. & Richmond, T. J. Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol. Biol. 119, 1–16 (1999). Winter Village Train Ride is a modification of LEGO set 40573-1 "Christmas Tree" and Lego Set 40262-1 "Christmas Train Ride". It makes a great centerpiece to any LEGO Winter Village. Jerzy Dorosz, Line Hyltoft Kristensen, Nanda G. Aduri, Osman Mirza, Rikke Lousen, Saskia Bucciarelli, Ved Mehta, Selene Sellés-Baiget & Michael Gajhede Day 25 mDA neurons differentiated in parallel on 2D Matrigel coated surfaces or in 3D biomaterial platforms, one batch for each platform, were harvested and dissociated to small ~50–100 μm clusters using 0.5 mM EDTA and pipetting. 250,000 cells were implanted into the striatum of isoflurane anesthetized 150–200 g adult female Fischer 344 rats (at stereotaxic coordinates AP: +1.0, ML: −2.5, DV −5.0). Four animals were assigned per group. 10 mg/kg Cyclosporine was injected intraperitoneally daily starting 24 h before surgeries and until the animals were euthanized. 6 weeks after cell implantations, animals were transcardially perfused with 4% PFA. Brains were harvested and incubated in 4% PFA overnight, and transferred into a 30% (w/v) sucrose solution the following day.

However, standard 2D culture systems generally face challenges for producing high quality and yields of cells. At a minimum, approximately 100,000 mDA neurons would need to engraft and survive within the striatum for effective disease treatment 5. With purities of ~15–30% hPSC-derived mDA neurons 1, 6, 7, and only 1–5% of implanted cells surviving as TH+ neurons post-implantation in pre-clinical models 1, 2, 3, generating sufficient numbers of cells to treat the estimated 1 million PD patients in the US alone would be challenging. Even producing the ~10 9 cells typically needed for an in vitro pharmacology, toxicology, or genetic screen is daunting 8, 9. Furthermore, current mDA neuron derivation systems entail the use of animal- and human-derived culture components that limit reproducibility and risk pathogen transfer 10, 11. To achieve higher capacity cell production, a longstanding approach in cell bioprocess engineering is to “scale up” to three-dimensional (3D) platforms rather than “scale out” to additional 2D surface area. The former offers several potential advantages: a more biomimetic 3D environment for cell culture, the potential for higher cell densities per unit culture volume, and ease of harvesting cells for implantation. Suspension or microcarrier culture offers the potential for scale up; however, human pluripotent stem cells in such cultures can aggregate into large clumps whose interiors undergo necrosis or non-specific differentiation 12, 13. Unfortunately, agitation, the most common approach to avoid such aggregation, can result in hydrodynamic shear stress that adversely affects cell growth and differentiation 12, 14.

Delivery & returns

Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480, 547–551 (2011). We also analyzed expression of non-dopaminergic neuronal markers, 5HT for serotonergic and GABA for GABAergic, in mDA neuronal cultures generated in 2D or 3D cultures, and did not find a significant difference ( Figure S6). Finally, to demonstrate the general applicability of this platform to generate mDA neurons, we differentiated 3 additional hPSC cell lines – H9 hESCs, WIBR3 hESCs (NIH registry number NIHhESC-1-0079), and 8FLVY6C2 hiPSCs, a cell line derived from healthy fibroblasts 30 – which all showed robust TH and TUJ1 expression at Day 25 of differentiation ( Figure S7). mDA neurons generated in 3D are more electrophysiologically active than cells generated in 2D culture Schmitz, S. U. et al. Jarid1b targets genes regulating development and is involved in neural differentiation. EMBO J. 30, 4586–4600 (2011). A Christmas Story, Scrooged, and National Lampoon's Christmas Vacation, then Monty Python movie marathon for Boxing Day.

The price for the kit is a bit on the expensive side, but the ability to wire as one sees fit is a slight bonus.

Our difference

In addition to early stage markers, expression of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine production that is crucial for mDA neuronal function, was significantly higher at day 25 on 3D (36% of cells) than on 2D (20%), suggesting rapid differentiation in 3D. Furthermore, >90% of the TH positive neurons in 3D were also FOXA2 positive ( Figure S3c), indicating a floorplate origin. Finally, by D40 of differentiation, TH+ neuronal differentiation plateaued at similar, high levels on both platforms (47% on 3D and 49% on 2D for H1 hESC derived mDA neurons), demonstrating equally strong potential for generating TH+ cells at levels comparable to previous reports 1, 4. Saha, K. et al. Substrate modulus directs neural stem cell behavior. Biophys. J. 95, 4426–4438 (2008). Biostructural Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, 2100, Copenhagen, Denmark Krawetz, R. et al. Large-scale expansion of pluripotent human embryonic stem cells in stirred-suspension bioreactors. Tissue Eng. Part C. Methods 16, 573–82 (2010). Arenas, E., Denham, M. & Villaescusa, J. C. How to make a midbrain dopaminergic neuron. Development 142, 1918–1936 (2015).