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A Novel Protocol to Generate Dopaminergic Neurons for Research and Therapy

This ‘spotting-based’ culture and differentiation protocol lets you reliably produce midbrain dopamine progenitors and neurons.

Human pluripotent stem cells (hPSCs) have a vast amount of potential in both research and therapy, but they certainly aren’t the easiest things to work with. To differentiate them with any degree of consistency you need to have a robust and reliable protocol. And when the intention is to use those cells in a therapeutic setting, that protocol needs to be scalable and compatible with good manufacturing practice (GMP) standards. 

New work, led by Dr Jisun Kim and a team from Harvard Medical School, and published in Nature protocols, has resulted in a novel protocol that initially cultures and differentiates hPSCs in a ‘spot’ (a small, confined area) rather than the conventional 2D monolayer.1 The 28-day protocol consistently yields an enriched population of midbrain dopamine progenitors (mDAPs) and midbrain dopamine neurons (mDANs), ideal for both research and therapy.

Their protocol has already been successful in research around Parkinson’s disease (PD), where one of the pathological hallmarks is loss of in a loss of midbrain dopaminergic (mDA) cells in the substantia nigra pars compacta, which makes cell replacement therapy an attractive prospect. Just recently, researchers used this spotting protocol in a preclinical mouse model of PD, where transplanted mDAPs restored motor function.2 Even more excitingly, this protocol was used in the first autologous cell replacement therapy in a patient with sporadic PD without the need for immunosuppressants; the patient’s PD symptoms stabilized or improved 18–24 months after progenitor cells were implanted.3

Making mDAs

Thanks to a sizable body of historical research, we know that critical factors, like Sonic Hedgehog (SHH), WNT1 LMX1A, FOXA2, and various growth factors coordinate not only the embryological development of mDANs in vivo but the differentiation of hPSCs into mDANs in vitro as well.4,5 This understanding led to several differentiation protocols and showed just how important small molecules are for maintaining a dopaminergic lineage following differentiation; transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) signals, for example,  prevent spontaneous differentiation into other lineages like meso-endodermal and nonneuronal cell types.

Getting the differentiation mix of growth factors and signaling molecules just right requires a great deal of trial and error and must be optimized for different types of hPSCs. That means there’s no ‘one-size-fits-all’ approach. For example, the exact concentration of the Wnt agonist, CHIR, varies between studies: one group of researchers found 3 μM CHIR to be optimal for mDA neuronal induction,6 a different group found <1 μM also produced mDA neurons,7 while yet another group showed that >1.0 μM resulted in hindbrain neurons.8Neurotrophic factors like GDNF, BDNF, and FGF8 also play a critical role in mDA neuronal differentiation and also need to be optimized alongside the other components.

Finally, there are physical parameters to consider as physical cues will affect cell fate and differentiation.9 The most important physical factor is of course the culturing method. While 3D culture does have a lot of use in research – organoids, in particular, have seen steady popularity in the media – the vast majority of culture happens in a 2D monoculture. The main problem with the 2D monolayer culture is that it’s inefficient, i.e., it incurs a substantial loss of cells due to detachment, heterogeneous populations across the diameter of the culture dish, and often results in inconsistent outcomes. 

Spotting cells for better outcomes

In looking at the 2D monolayer systems it seemed that space is a major issue for successful culture. Professor Kwang-Soo Kim from the Harvard team said this culture method was akin to “…having people living in a concentrated area of town or city instead of spreading the population over a larger area.” 

So, what if you limited the physical space the cells were allowed to grow in but maintained a comparably high density? That’s precisely what the team from Harvard Medical School did here. They cultured 1 x 104 hPSCs in six 10 μl spots on a single 6 cm dish. Over 28 days the cells undergo three stages: the induction of floor plate (FP), neural progenitor (NP), and mDAP/mDAN stages.

Following FP and NP stages, the cells were replated on day 15 as a monolayer culture to avoid an excessive build-up of cells and allowed to continue differentiating. The FP and NP stages included the addition of a combination of signaling chemicals, like LDN, FGF8, and CHIR, while neurotrophic factors like BDNF and GDNF are added later in the final mDAP/mDAN stages. 

But getting those growth conditions right was no easy task. The Harvard team said they experienced frequent failures at the start. “We initially attempted to identify the optimal initial cell density/concentration. Then, we found that cells’ growth and differentiation properties were mediocre when seeded below a certain concentration, resulting in poor yield and unhealthy dopaminergic cells. At above a certain concentration, cells grow exponentially and detach.

Eventually, they overcame the laborious optimization process, and even went to test “…the universality of this novel spotting technique using multiple human iPSC and embryonic stem cell (ESC) lines,” said the team. They eventually went on to characterize cell phenotypes via immunocytochemistry with key mDA markers at days 0 and 28, and via immunofluorescence co-staining of TH+, ALDH1A1+, and GIRK2+ (APC-006, Alomone Labs).

The results were striking. Compared to the 2D monolayer culture method, the team was able to reliably generate mDA cells with significantly less cell detachment-associated loss caused by unfavorable culture conditions, such as culture acidification. Close to 90% of the monolayer experiments failed to provide meaningful data at day 28 due to major cell loss. The mDA cells generated by spotting also looked to be healthier than those generated in the traditional 2D monolayer culture.

Their results clearly showed how cells in the 2D monolayer quickly became overcrowded despite starting at a similar density to those in the spotting experiments. This overcrowding led to a cycle of detachment and regrowth that not only made maturation difficult to achieve but led to heterogeneous cell populations, depletion of the culture’s nutrients, acidification of the medium, and subsequent cell death. 

Cells grown under the spotting method on the other hand showed homogenous populations that showed steady growth and differentiation, complete with the development of neurite-like structures between cells. The spotting cell cultures also had a more stable pH and overall chemical environment. Following the 28 days, these cells can also be cryopreserved and thawed without loss in viability or functionality.

Putting the protocol to use

As we mentioned at the start, the spotting protocol has already been used to generate hPSC-derived mDA cells in preclinical work. The protocol has also been used at scale, in a GMP facility, in the first autologous cell transplant in a patient with PD. So, it’s safe to say it has incredible potential may be a bit of an understatement. But the creators of the protocol see it doing more, specifically, they have speculated that it may be modified to produce different neuronal lineages, such as GABAergic, glutamatergic, and serotonergic neurons.

Speaking with Professor Kim, he told us, “Since this technique works equally well for any human pluripotent stem cells, it should be useful for any type of cell therapy, disease modeling studies, drug screening, and toxicology-related studies using human iPSCs and ESCs. Thus, the availability of pre-printed spotted plates would contribute to generalizing the spotting technique.

When it comes to scaling this protocol is that the culture plates need to be lined manually. Professor Kim points out, “If manufacturers could produce already appropriately lined plates, it will make the process greatly efficient and save efforts and time” – which sounds like a worthy challenge for vendors out there.  

The complete protocol, with detailed steps, reagents, and data can be found over at Nature Protocols and we strongly urge you to go take a look. 

Reagents

Here’s a selection of the reagents used in the protocol:

References

  1. Kim, J. et al. Spotting-based differentiation of functional dopaminergic progenitors from human pluripotent stem cells. Nat. Protoc. 1–25 (2022) doi:10.1038/s41596-021-00673-4.
  2. Song, B. et al. Human autologous iPSC-derived dopaminergic progenitors restore motor function in Parkinson’s disease models. J. Clin. Invest. 130, 904–920 (2020).
  3. Schweitzer, J. S. et al. Personalized iPSC-Derived Dopamine Progenitor Cells for Parkinson’s Disease. N. Engl. J. Med. 382, 1926–1932 (2020).
  4. Arenas, E., Denham, M. & Villaescusa, J. C. How to make a midbrain dopaminergic neuron. Dev. 142, 1918–1936 (2015).
  5. Tao, Y. & Zhang, S. C. Neural Subtype Specification from Human Pluripotent Stem Cells. Cell Stem Cell. 19, 573–586 (2016).
  6. 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).
  7. 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).
  8. Xi, J. et al. Specification of midbrain dopamine neurons from primate pluripotent stem cells. Stem Cells30, 1655–1663 (2012).
  9. Madl, C. M., Heilshorn, S. C. & Blau, H. M. Bioengineering strategies to accelerate stem cell therapeutics. Nature 557, 335–342 (2018).

Photo by Coni Wang