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| Status |
Public on Jun 14, 2022 |
| Title |
Human midbrain dopaminergic neuronal differentiation markers predict cell therapy outcome in a Parkinson's disease model |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Human pluripotent stem cell (hPSC)-based replacement therapy holds great promise in treating Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we depicted the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including ventral midbrain, isthmus, and ventral hindbrain, resulting in heterogenous donor cell population. We reconstructed the differentiation trajectory of mDA lineage and identified CLSTN2 and PTPRO as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate highly enriched mDA neurons (up to 80%) following progenitor sorting and transplantation. Marker sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation, and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.
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| Overall design |
We collected cells for scRNA-seq at the end of five consecutive stages during the process of mDA neuron differentiation from hPSCs (day8-stage I; day 14-stage II; day 21-stage III; day 28-stage IV; day 35-stage V). We sought to define the cellular composition of these grafts by scRNA-seq. To remove the contamination of host (mouse) cells in the cell suspensions when dissecting graft tissue, we knocked a nuclear EGFP expressing cassette (EGFP- nucleoplasmin) into the AAVS1 locus of surface marker reporter hPSC lines (CLSTN2- tdT hPSCs or PTPTO-tdT hPSCs) to label all of the human cells . We transplanted surface marker-sorted or unsorted mDA progenitors derived from these hPSC lines into the striatum of PD mice. Four months after transplantation, we dissected grafts and prepared single-cell suspensions. Grafted human cells (EGFP+) were then isolated via FACS and subjected to scRNA-seq analysis.
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| Contributor(s) |
Xu P, He H, Gao Q, Wu Z, Zhou Y, Chen Y |
| Citation(s) |
35700056 |
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| Submission date |
May 25, 2022 |
| Last update date |
Jun 27, 2022 |
| Contact name |
Yuejun Chen |
| E-mail(s) |
yuejunchen@ion.ac.cn
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| Phone |
02154921897
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| Organization name |
Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences
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| Street address |
320 Yueyang Road
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| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (9)
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| GSM6194011 |
stage IV, day 28, scRNAseq |
| GSM6194012 |
stage V, day 35, scRNAseq |
| GSM6194013 |
transplanted cells derived from unsorted progenitors, batch 1, 4 months, scRNAseq |
| GSM6194014 |
transplanted cells derived from unsorted progenitors, batch 2, 4 months, scRNAseq |
| GSM6194015 |
transplanted cells derived from PTPRO sorted progenitors, 4 months, scRNAseq |
| GSM6194016 |
transplanted cells derived from CLSTN2 sorted progenitors, 4 months, scRNAseq |
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| Relations |
| BioProject |
PRJNA842351 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE204796_RAW.tar |
525.6 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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