Resolving developing neuronal lineages in the ventral midbrain

The numerous cells and cell types of our bodies originate from a fertilised egg. Through the study of the developing embryo, we have come to understand much about how different cells in different tissues arise from this single cell, and how they are related to one another within an expansive family tree. Knowledge of these developmental instructions has birthed the field of regenerative stem cell biology, where efforts aim to engineer therapeutic cell types for clinical use. However, time and again we find that the details are more complicated than first thought. The road to the clinic will require extensive understanding of precisely what cell types we want (or do not want), and precisely what cellular instructions will get us there.

Neurodegenerative disorders such as Parkinson's Disease (PD) are becoming a major and increasing health burden for developing societies. In PD patients, loss of dopamine releasing neurons in the ventral midbrain (vMB) leads to a decline of movement control. While prognosis has improved for PD patients in recent years, transplantation of stem cell derived dopaminergic neurons, to replace those that degenerate, remains an attractive prospect. In this light two questions emerge: i) how may stem cells be instructed towards the dopaminergic lineage? and ii) what criteria precisely identify these dopaminergic cells?

Developmental biology research has uncovered morphogen permutations that specify the dopamine lineage. Thus, stem cell differentiation protocols targeting PD incorporate these extrinsic signaling molecules to instruct stem cells to choose a dopaminergic identity. However, this approach is not completely efficient, often yielding neurons from a mix of different lineages. In paper I we show that forced expression of intrinsic transcriptional determinants can also supply these commitment instructions. Following a lineage specific logic, the transcription factor Lmx1a selects for dopaminergic neurons, while Nkx2.2, Phox2b, and Olig2 select for serotonergic neurons, visceral motor neurons and somatic motor neurons respectively, however only in permissive morphogen contexts. Moreover, in each scenario untargeted neuronal lineages are depleted, and in some cases combinations of intrinsic transcriptional determinants can override even a conflicting extrinsic environment.

Enrichment alone is not enough for clinical grade transplantable material, in particular if contaminating neuron types are detrimental. In addition to enriching for neurons that we want, we must also identify and eliminate neurons that we do not want. In paper II, we employ single-cell RNA-sequencing technologies to characterise the Lmx1a-expressing lineage in the mouse vMB. We show that previous stalwarts of dopaminergic identity, such as Lmx1a/b, FoxA1/2, Shh, and Wnts, are in fact all shared with rostrally adjacent glutamatergic lineages. Computational analysis uncovered additional markers specific to these cells, that we further validate in vitro and in vivo in both mouse and human systems. Finally, in paper III we show that appreciation of this new rostro-caudal division can guide fine-tuning of morphogen application, leading to improvements in existing stem cell protocols targeted to PD.

In summary, we have probed neuron lineage commitment in the vMB, finding that Lmx1a can instruct stem cells down the dopaminergic lineage, but only when care is also taken to specify the appropriate rostro-caudal level. Interestingly, regenerative medicine efforts outside the PD field are tackling conceptually identical challenges. Using developmental biology as a guide, efforts aim to derive cells of additional neuron types, including muscle, skin, neural crest and other organs, with high purity and efficacy. Given that bone marrow transplants, a routine hematopoietic stem cell therapy, required several decades from inception to clinic, one can hope that with patience and diligence, more stem cell therapies will, in time, become accessible standard medical procedures.

List of scientific papers

I. Lia Panman, Elisabet Andersson, Zhanna Alekseenko, Eva Hedlund, Nigel Kee, Jamie Mong, Christopher W. Uhde, Qiaolin Deng, Rickard Sandberg, Lawrence W. Stanton, Johan Ericson, and Thomas Perlmann. Transcription factor-induced lineage selection of stem-cell-derived neuralprogenitor cells. Cell Stem Cell. (2011) vol. 8 (6) pp. 663-751.
https://doi.org/10.1016/j.stem.2011.04.001

II. Nigel Kee, Nikolaos Volakakis, Agnete Kirkeby, Lina Dahl, Helena Storvall, Sara Nolbrant, Laura Lahti, Åsa K. Björklund, Linda Gillberg, Eliza Joodmardi, Rickard Sandberg, Malin Parmar, and Thomas Perlmann. Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages. Cell Stem Cell. 20, 1–12 January 5, 2017.
https://doi.org/10.1016/j.stem.2016.10.003

III. Agnete Kirkeby, Sara Nolbrant, Katarina Tiklova, Andreas Heuer, Nigel Kee, Tiago Cardoso, Daniella Rylander Ottosson, Mariah J. Lelos, Pedro Rifes, Stephen B. Dunnett, Shane Grealish, Thomas Perlmann and Malin Parmar. Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson’s Disease. Cell Stem Cell. 20, 1–14 January 5, 2017.
https://doi.org/10.1016/j.stem.2016.09.004