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Seq-ing Markers of Midbrain Dopamine Neurons

2017; Elsevier BV; Volume: 20; Issue: 1 Linguagem: Inglês

10.1016/j.stem.2016.12.014

1934-5909

Teresia Osborn, Penelope J. Hallett,

Neuroscience and Neural Engineering

Transplantation of human pluripotent stem cell-derived dopaminergic neurons is a promising approach to treating Parkinson's disease. In this issue of Cell Stem Cell, Kee et al., 2017Kee N. Volakakis N. Kirkeby A. Dahl L. Storvall H. Nolbrant S. Lahti L. Björklund A.K. Gillberg L. Joodmardi E. et al.Cell Stem Cell. 2017; 20 (this issue): 29-40Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar and Kirkeby et al., 2017Kirkeby A. Nolbrant S. Tiklova K. Heuer A. Kee N. Cardoso T. Ottosson D.R. Lelos M.J. Rifes P. Dunnett S.B. et al.Cell Stem Cell. 2017; 20 (this issue): 135-148Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar identify specific markers of midbrain dopaminergic progenitors to improve their derivation and predict dopamine neuron content after engraftment. Transplantation of human pluripotent stem cell-derived dopaminergic neurons is a promising approach to treating Parkinson's disease. In this issue of Cell Stem Cell, Kee et al., 2017Kee N. Volakakis N. Kirkeby A. Dahl L. Storvall H. Nolbrant S. Lahti L. Björklund A.K. Gillberg L. Joodmardi E. et al.Cell Stem Cell. 2017; 20 (this issue): 29-40Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar and Kirkeby et al., 2017Kirkeby A. Nolbrant S. Tiklova K. Heuer A. Kee N. Cardoso T. Ottosson D.R. Lelos M.J. Rifes P. Dunnett S.B. et al.Cell Stem Cell. 2017; 20 (this issue): 135-148Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar identify specific markers of midbrain dopaminergic progenitors to improve their derivation and predict dopamine neuron content after engraftment. Cell replacement using midbrain dopamine (mDA) neurons for Parkinson's disease (PD) provides a unique opportunity to address the root cause of the debilitating motor symptoms of this disease, the selective loss of mDA neurons. mDA neurons transplanted into the parkinsonian brain grow and connect to their appropriate target and provide cellular and synaptic functional repair. Transplantation with fetal ventral mDA neurons has proven successful and durable in the clinic (Barker et al., 2013Barker R.A. Barrett J. Mason S.L. Björklund A. Lancet Neurol. 2013; 12: 84-91Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, Mendez et al., 2005Mendez I. Sanchez-Pernaute R. Cooper O. Viñuela A. Ferrari D. Björklund L. Dagher A. Isacson O. Brain. 2005; 128: 1498-1510Crossref PubMed Scopus (368) Google Scholar) but presents several complicating factors. An alternative to fetal grafts is transplantation of substantia nigra dopamine neurons differentiated from human pluripotent stem cells (hPSCs). Scalable production of such cells is rapidly evolving, to the point where clinical transplantation of hPSC-derived mDA neurons is imminent. Developmental specification of hPSCs into mDA neurons is relatively well understood and transplanted PSC-derived mDA neurons are functionally similar to fetal ventral midbrain grafts (Grealish et al., 2014Grealish S. Diguet E. Kirkeby A. Mattsson B. Heuer A. Bramoulle Y. Van Camp N. Perrier A.L. Hantraye P. Björklund A. Parmar M. Cell Stem Cell. 2014; 15: 653-665Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, Hallett et al., 2015Hallett P.J. Deleidi M. Astradsson A. Smith G.A. Cooper O. Osborn T.M. Sundberg M. Moore M.A. Perez-Torres E. Brownell A.L. et al.Cell Stem Cell. 2015; 16: 269-274Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) in animal models of PD. Although several protocols have been established to obtain mDA progenitor and/or post-mitotic mDA neurons for transplantation (Cooper et al., 2010Cooper O. Hargus G. Deleidi M. Blak A. Osborn T. Marlow E. Lee K. Levy A. Perez-Torres E. Yow A. Isacson O. Mol. Cell. Neurosci. 2010; 45: 258-266Crossref PubMed Scopus (169) Google Scholar, Kirkeby et al., 2017Kirkeby A. Nolbrant S. Tiklova K. Heuer A. Kee N. Cardoso T. Ottosson D.R. Lelos M.J. Rifes P. Dunnett S.B. et al.Cell Stem Cell. 2017; 20 (this issue): 135-148Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, Kriks et al., 2011Kriks S. Shim J.W. Piao J. Ganat Y.M. Wakeman D.R. Xie Z. Carrillo-Reid L. Auyeung G. Antonacci C. Buch A. et al.Nature. 2011; 480: 547-551Crossref PubMed Scopus (1319) Google Scholar, Sundberg et al., 2013Sundberg M. Bogetofte H. Lawson T. Jansson J. Smith G. Astradsson A. Moore M. Osborn T. Cooper O. Spealman R. et al.Stem Cells. 2013; 31: 1548-1562Crossref PubMed Scopus (182) Google Scholar), several markers commonly used for in vitro quantification of mDA progenitors (e.g., Lmx1a and FoxA2) are not predictive of DA neuron content following engraftment (Kirkeby et al., 2017Kirkeby A. Nolbrant S. Tiklova K. Heuer A. Kee N. Cardoso T. Ottosson D.R. Lelos M.J. Rifes P. Dunnett S.B. et al.Cell Stem Cell. 2017; 20 (this issue): 135-148Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Identification of additional markers to maximize mDA content in hPSC-derived populations intended for transplantation is therefore desirable. Now in Cell Stem Cell, two studies from the groups of Thomas Perlmann (Kee et al., 2017Kee N. Volakakis N. Kirkeby A. Dahl L. Storvall H. Nolbrant S. Lahti L. Björklund A.K. Gillberg L. Joodmardi E. et al.Cell Stem Cell. 2017; 20 (this issue): 29-40Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and Malin Parmar (Kirkeby et al., 2017Kirkeby A. Nolbrant S. Tiklova K. Heuer A. Kee N. Cardoso T. Ottosson D.R. Lelos M.J. Rifes P. Dunnett S.B. et al.Cell Stem Cell. 2017; 20 (this issue): 135-148Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) provide a detailed understanding of mDA neuron development at single-cell resolution and assess how in vitro cell preparations can predict in vivo graft outcome. Together, these studies enable further refinement and standardization of mDA neuron differentiation protocols for application in the clinic. To identify better predictive markers of developing mDA neurons, Kee et al., 2017Kee N. Volakakis N. Kirkeby A. Dahl L. Storvall H. Nolbrant S. Lahti L. Björklund A.K. Gillberg L. Joodmardi E. et al.Cell Stem Cell. 2017; 20 (this issue): 29-40Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar performed single-cell RNA-seq analysis on Lmx1a-expressing and -non-expressing cells harvested from ventral mesencephalic and diencephalic regions of Lmx1a reporter mouse embryos. They identified two separate Lmx1a sublineages (Axis-1 and -2) that both expressed many of the commonly used markers for quantification of mDA content during hPSC differentiation (e.g., Lmx1a/b, FoxA1/A2, Otx2, Msx1, and Nurr1). However, several classic DA markers such as Corin, En1, Msx2, and Pitx3 were only found in Axis-1 whereas Axis-2 appeared to be a non-dopaminergic sublineage. Immunohistochemistry confirmed a caudal (Axis-1) domain destined for mDA development and a rostral (Axis-2) domain with a unique gene expression pattern (e.g., Barhl1/2, Dbx1, Wnt8b, Nkx2-1/4, and Pitx2) destined to generate glutamatergic subthalamic nucleus (STN) neurons. Further, the authors show by tamoxifen-inducible deletion that Lmx1a and b are required for development of both STN and mDA neurons. Analysis of human fetal tissue using qPCR and immunohistochemistry confirmed segregated gene expression of BARHL1/2, WNT8B, NKX2-1/4, and DBX1 in Axis-2, expression of EN1/2, WNT1, and CPNY1 in Axis-1, and expression of FoxA2 in both populations. The authors then modified rostrocaudal patterning in their differentiation protocol to control Axis-1 versus Axis-2 sublineage determination. Lmx1/Pitx2+ Axis-2 cells were generated by exposure to low concentrations (at or below 0.4μM –0.6μM) of the GSK3B inhibitor CHIR99021 (CHIR), and forced expression of the rostral domain marker Barhl1 further pushed the cells toward an STN fate. Importantly, Lmx1/Pitx2+ cell content could be reduced by increasing the concentrations of CHIR to levels used in other published differentiation protocols (Kriks et al., 2011Kriks S. Shim J.W. Piao J. Ganat Y.M. Wakeman D.R. Xie Z. Carrillo-Reid L. Auyeung G. Antonacci C. Buch A. et al.Nature. 2011; 480: 547-551Crossref PubMed Scopus (1319) Google Scholar, Sundberg et al., 2013Sundberg M. Bogetofte H. Lawson T. Jansson J. Smith G. Astradsson A. Moore M. Osborn T. Cooper O. Spealman R. et al.Stem Cells. 2013; 31: 1548-1562Crossref PubMed Scopus (182) Google Scholar). In a complementary study, Kirkeby et al., 2017Kirkeby A. Nolbrant S. Tiklova K. Heuer A. Kee N. Cardoso T. Ottosson D.R. Lelos M.J. Rifes P. Dunnett S.B. et al.Cell Stem Cell. 2017; 20 (this issue): 135-148Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar observed considerable variation in the size and survival of grafts following xenotransplantation of hPSC-derived mDA neuron preparations into a rat model of PD. The authors classified 30 batches of grafts 6–24 weeks after transplantation as containing either high or low mDA neuron content, and they assessed expression of common mDA neuron markers in the cell preparations that the corresponding grafts were prepared from. Intriguingly, they found that expression of several markers commonly used to assess mDA progenitors and neurons (LMX1A, tyrosine hydroxylase (TH), FOXA2, OTX2, CORIN, and AADC) in the pre-transplantation preparations did not correlate with in vivo mDA yields after engraftment. By elegantly combining RNA-seq analysis of those same cell preparations paired with the mDA yield, mDA density, and size of the original grafts, the authors identified markers representing caudal ventral midbrain (VM) in the cell batches, including EN1, WNT1, SPRY1, CNPY1, FGF8, and PAX8, which correlated positively with in vivo outcome. Similarly to the findings of Kee et al., Kirkeby and colleagues also found that STN lineage markers were enriched in grafts with low DA neuron content. Moreover, those grafts contained Barhl1+ STN neurons in vivo and the corresponding in vitro cell preparations were relatively enriched for STN progenitors. To further direct hPSC differentiation toward the caudal VM domain, Kirkeby et al. showed that specific application of FGF8b at later stages (d9–d16) of the mDA progenitor culture increased the yield of mDA neurons, whereas earlier application upregulated forebrain/diencephalic markers. Lastly, after adapting their refined differentiation protocol to use clinical-grade (or highest available grade) growth factors and chemicals, Kirkeby et al. demonstrated increased yields of DA progenitors with high expression of caudal VM markers. Those progenitors provided robust functional outcomes following xenotransplantation into 6-OHDA lesioned rats, as defined by both a reduction of amphetamine-induced rotational asymmetry and an increase in the use of the contralateral forepaw. To move forward with clinical transplantation of cells, whether progenitor cells or post-mitotic neurons (or a mix thereof), it is crucial to have a defined set of markers that correlate with in vivo cell survival and function to ensure quality control between batches. Robust and reliable in vitro analysis of cell preparations prior to transplantation allows precise determination of the content of cells to be transplanted and proper prediction of the resulting dopaminergic cell load in the graft. Positive and negative cutoffs for marker expression need to be determined for any cell source and differentiation protocol to be used in the clinic, although those criteria may vary depending on the maturity stage of the cells to be transplanted. For transplantation of post-mitotic dopaminergic neurons, TH can be used to determine mDA neuron content in vitro (Cooper et al., 2010Cooper O. Hargus G. Deleidi M. Blak A. Osborn T. Marlow E. Lee K. Levy A. Perez-Torres E. Yow A. Isacson O. Mol. Cell. Neurosci. 2010; 45: 258-266Crossref PubMed Scopus (169) Google Scholar, Doi et al., 2014Doi D. Samata B. Katsukawa M. Kikuchi T. Morizane A. Ono Y. Sekiguchi K. Nakagawa M. Parmar M. Takahashi J. Stem Cell Reports. 2014; 2: 337-350Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, Hallett et al., 2015Hallett P.J. Deleidi M. Astradsson A. Smith G.A. Cooper O. Osborn T.M. Sundberg M. Moore M.A. Perez-Torres E. Brownell A.L. et al.Cell Stem Cell. 2015; 16: 269-274Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, Kriks et al., 2011Kriks S. Shim J.W. Piao J. Ganat Y.M. Wakeman D.R. Xie Z. Carrillo-Reid L. Auyeung G. Antonacci C. Buch A. et al.Nature. 2011; 480: 547-551Crossref PubMed Scopus (1319) Google Scholar, Sundberg et al., 2013Sundberg M. Bogetofte H. Lawson T. Jansson J. Smith G. Astradsson A. Moore M. Osborn T. Cooper O. Spealman R. et al.Stem Cells. 2013; 31: 1548-1562Crossref PubMed Scopus (182) Google Scholar) in combination with expression of FoxA2, Lmx1a/b, En1, Otx2, Pitx3, and Nurr1. However, the papers by Kee et al. and Kirkeby et al. illustrate that successful transplantation of mDA progenitors requires additional markers, since the commonly used combination of Lmx1a and FoxA2 are also expressed in rostral progenitor cells that develop into glutamatergic STN neurons. Together, the transcription factors identified in Kee et al. and the refined differentiation protocol reported by Kirkeby et al. can improve yield of hPSC-derived mDA progenitor cells and decrease contamination by STN progenitor cells. These are important steps in ensuring consistency and reproducibility of mDA progenitor cell preparations, a critical step as cell transplantation therapies for PD move closer to clinical reality. Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's DiseaseKirkeby et al.Cell Stem CellOctober 27, 2016In BriefKirkeby et al. show that identification and application of a set of predictive markers can help refine differentiation protocols and improve transplant outcome in a preclinical model for hESC-based treatment of Parkinson's disease. Full-Text PDF Open AccessSingle-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal LineagesKee et al.Cell Stem CellOctober 27, 2016In BriefKee et al. use single-cell RNA-seq to reconstruct Lmx1a+ differentiation in silico, revealing an unexpectedly close relationship between mesDA and STN neuronal lineages during differentiation. Application of markers that distinguish the two can help optimize mesDA differentiation of hESCs with a view to improving therapeutic translation. Full-Text PDF Open Archive