Identifying Graft Composition in Parkinson's Disease Model Using Single-Cell Analysis
Summary
The achievement of cell replacement as a therapeutic modality for Parkinson's Disease (PD) has long been an aspiration in the field. While the procurement of transplantable cells can be derived from either fetal brain tissue or stem cells, previous studies have demonstrated that the dopamine (DA) neurons constituted a minor fraction of the grafted tissue. The identification of other cellular components in intracerebral grafts has proven to be a challenge. This study, utilizing a combination of single-cell RNA sequencing (scRNA-seq) and extensive histological evaluations, characterized the intracerebral grafts obtained from human embryonic stem cells (hESCs) and fetal tissue in a pre-clinical rat model of PD, following their functional maturation. The results revealed that neurons and astrocytes constituted the predominant cellular components in both fetal and stem cell-derived grafts. Additionally, a cell type strongly resembling the perivascular-like cells that have recently been identified was discovered in stem cell-derived grafts. In conclusion, this study brings to light previously unknown cellular diversity in the context of a clinically relevant cell replacement therapy for PD.
Research Criteria
Despite extensive efforts aimed at the amelioration of Parkinson's disease (PD) through cell replacement therapy, a satisfactory comprehension of the cellular composition of intracerebral grafts has remained elusive. This has been in part due to the limitations inherent to conventional histological methods, which are predicated upon pre-existing notions regarding the probable cell types present in the transplanted tissue. To circumvent this challenge, the authors of this study employed a combination of single-cell RNA sequencing (scRNA-seq) and histological analyses to meticulously characterize the cellular landscape of intracerebral grafts derived from human embryonic stem cells (hESCs) and fetal tissue after extended survival and functional maturation in a pre-clinical rat model of PD. Specifically, they focused on grafts that had been patterned to resemble the ventral midbrain (VM).
Fig.1 Experimental design. (Tiklová, 2020)
Sample Type
Single-cell sequencing was performed after human fetal tissue was transplanted into the rat brain for development.
Result—scRNA-Seq Identifies Composition of Fetal VM and hESC-Derived Neural Graft
The objective of this study was to examine the cellular composition of intracerebral transplants using scRNA-seq and histological analysis in a pre-clinical rat model of Parkinson's disease. The scRNA-seq data was analyzed through t-Distributed neighbor embedding and graph-based clustering, leading to the discovery of four major clusters. The top two clusters were primarily composed of VM-patterned hESCs, while the bottom two clusters were primarily composed of fetal cells. Despite both cell sources producing DA neurons with similar properties after transplantation, there were transcriptional differences between the two at the time of transplantation. Gene enrichment analysis using SAMseq showed prominent expression of neural progenitor/radial glial cell markers in both fetal and hESC-dominated clusters, reinforcing the role of these markers in DA neurogenesis in both mice and humans.
Fig.2 scRNA-seq analysis of progenitor cells before grafting in a pre-clinical cell therapy model of PD. (Tiklová, 2020)
Result—Graft Placement Does Not Affect the Cellular Composition of VM-Patterned hESC-Derived Grafts
The results of scRNA-seq analysis of human Embryonic Stem Cell (hESC)-derived transplanted cells, as well as hESCs grafted to the midbrain of nude rats and analyzed after 9 months of in vivo maturation, demonstrate that the transplanted cells give rise to neurons, astrocytes, and VLMCs but not oligodendrocytes. A UMAP embedding and graph-based clustering of the data retained for analysis after quality control and cell filtering showed the presence of three main clusters, classified as astrocytes, neurons, and VLMCs, with a small subset of cells expressing cycling genes and clustering separately. The results were consistent, regardless of the method of cell isolation and sequencing utilized.
Fig.3 scRNA-seq analysis and histological validation of grafted cells into the midbrain. (Tiklová, 2020)
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Reference
- Tiklová, K.; et al. Single cell transcriptomics identifies stem cell-derived graft composition in a model of Parkinson's disease. Nature Communications, 2020, 11(1): 2434.
