Supplementary MaterialsReporting Summary 41467_2020_16225_MOESM1_ESM. Parkinson?s disease (PD). Cells for transplantation can be obtained from fetal brain tissue or from stem cells. However, after transplantation, dopamine (DA) neurons are seen to be?a minor component of grafts, and it has remained difficult to determine the identity of other cell types. Here, we report analysis by single-cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts p75NTR from human embryonic stem cells (hESCs) and fetal tissue after functional maturation in a pre-clinical rat PD model. We show that neurons and Tradipitant astrocytes are major components in both fetal and stem cell-derived grafts. Additionally, we identify a cell type closely resembling a class of recently identified perivascular-like cells in stem cell-derived grafts. Thus, this study uncovers previously unknown cellular diversity in a clinically relevant cell replacement PD model. and were prominently expressed in the yellow fetal cell-dominated cluster as well as in the blue and green hESC-dominated clusters consistent with the expression of these genes under DA neurogenesis in mouse and humans9,10 (Fig.?1g, Supplementary Fig.?2c). Regulators of neurogenesis ((refs. 9,10) (Fig.?1h, Supplementary Fig.?2d). Importantly, transcription factors such as that are critical in VM development and DA neurogenesis, as well as genes that are predictive of successful graft Tradipitant outcome (and (Fig.?1iCk, Supplementary Fig.?2eCg). Of note, cells expressing markers for more mature DA neurons could be distinguished in one part of the orange cluster, thus indicating Tradipitant neuronal diversity Tradipitant among more mature fetal cells (Supplementary Fig.?2g). Markers of pluripotency (and and and were expressed in cells in the neuron cluster indicating, as expected, a high proportion of DA neurons (Fig.?2e, Supplementary Fig.?4f). Further analyses of publicly available datasets reporting both bulk and scRNA sequencing provided additional strong support for the assignment into astrocytes, oligodendrocytes, and neurons14C17 (Supplementary Fig.?5a). Of note, despite transplantation of the cells at a Tradipitant highly proliferative stage, only a very small number of cells (1.3%) showed cell cycle scores indicative of some cycling cells after 6 months in vivo, and these cells all belonged to the oligodendrocyte and astrocyte clusters (Supplementary Fig.?3b). Open in a separate window Fig. 2 scRNA-seq analysis and histological validation of grafted cells into the striatum.a t-SNE showing clustering of 746 cells grafted to striatum (683 cells of hESC origin, grafted rats and transgene) was used to isolate grafted hESC-derived cells by FACS. To validate and extend the findings, hESCs patterned by the same protocol as initially used (Figs.?1 and ?and2)2) were grafted to the midbrain of nude rats (homotopic graft placement) and analyzed by scRNA-seq after 9 months of in vivo maturation (Fig.?4aCd). Importantly, to ensure that the GFP reporter and method of cell isolation did not influence or bias the results, cells in the new experiment were either sequenced directly after dissociation or after FACS isolation (outlined in Fig.?4a). In addition, the 10X Genomics Platform was used to allow for higher throughput. After QC and filtering to exclude rat cells (see Supplementary Fig.?8aCc), a total of 7875 cells were retained for analysis. The resulting UMAP embedding and graph-based clustering showed that, as with the hESC-derived cells grafted to the striatum (Fig.?2), VM-patterned hESCs transplanted to the midbrain gave rise to three main clusters which, based on marker expression, were clearly classified as astrocytes, neurons, and VLMCs (Fig.?4eCi, Supplementary Data?4). A small number of cells expressing astrocyte markers expressed cycling genes and clustered separately (Fig.?4e). Comparable results were derived regardless of whether cells had been isolated and sequenced directly or isolated by FACS before sequencing (Fig.?4j). Open in a separate window Fig. 4 scRNA-seq analysis and histological validation of grafted cells into the midbrain.a Schematic overview of experimental design. VM-patterned hESCs grafted to midbrain of 6-OHDA rats and analyzed at 9 months (n?=?6). These rats were used as follows: scRNA-seq and several other DA neuron markers including (Supplementary Fig.?10f). These markers were broadly distributed within the cluster and individual.