Additionally, no AQP4+ astrocytes or 5-hydroxytryptamine (5-HT+) serotonergic neurons are detected in the final cell product, which has been reported in some of the in vivo grafts [25, 30]

Additionally, no AQP4+ astrocytes or 5-hydroxytryptamine (5-HT+) serotonergic neurons are detected in the final cell product, which has been reported in some of the in vivo grafts [25, 30]. during the current study are available from the corresponding author on reasonable request. Abstract Background Midbrain dopaminergic (DA) progenitors derived from human pluripotent stem cells are considered to be a promising treatment for Parkinsons disease (PD). However, the differentiation process produces undesired cell types, which influence the in vivo evaluation of DA cells. In this paper, we analyze the cell fate choice during differentiation and provide valuable information on cell preparation. Methods Human embryonic stem cells were differentiated into DA progenitors. We applied single-cell RNA sequencing (scRNA-seq) of the differentiation cells at different time points and investigated the gene expression profiles. Based on the differentially expressed genes between DA and non-DA cells, we investigated the impact of (DA enriched) overexpression on DA differentiation and the enrichment effect of CD99 (non-DA enriched) sorting. Results Transcriptome analyses revealed the DA differentiation trajectory as well as non-DA populations and three key lineage branch points. Using genetic gain- and loss-of-function approaches, we found that overexpression of and the midbrain marker and DA markers cells, weakly expressing and and gradually decreased from C1 to C3 (Fig.?1c and Additional file2: Fig. S1a, b), and this is usually also consistent with recently published article [30]. Open in a separate window Fig. 1 Single-cell RNA sequencing of DA differentiation in vitroSchematic depicting DA differentiation from hESCs at different differentiation stages. b We defined 11 clusters in DA differentiation. c Heat map showing expression levels of the differentially expressed genes of each cluster Trajectory mapping reveals three branch points in DA differentiation UMAP projections of cells from day 5 to 25 revealed dynamic changes in cell populations over time (Fig.?2a). On day 5, hESCs first give rise to RGCs, accompanied by downregulation of pluripotent genes and (Fig.?1c). We found that RGCs were the prominent clusters at early stages. Day 5 and day 11 cells contained 95% and 70% of RGCs, respectively (Fig.?2a, b). Early DA progenitors (C6) appeared on day 17, while young DA neurons (C8) emerged on day 25. In contrast, the two non-DA populations were detected on day 11 (C4, 20% of cells) and day 17 (C5, 42% of cells), respectively. Notably, we also identified a CPECs population (C7) specific to day 25. Moreover, two rare cell types ( ?5% of cells at any time point), including base plate cell population (C9) and an unknown population (C10), were detected in the differentiation process. Open in a separate window Fig. 2 Differentiation trajectory from hESCs to DA. a UMAP projections of cells sampled from day 5 to 25. b Cell numbers at each time point. c Pseudo-time trajectories calculated from UMAP d Expression of selected marker genes along pseudo-time. e Expression of marker genes projected onto UMAP We next investigate the lineage trajectories from hESCs to DA progenitors using Monocle 3. Pseudo-time sequence analysis revealed Rabbit Polyclonal to BRF1 that this DA lineage (C1: C2: C3: C6: C8) diverts from hESCs into RGCs populations (C1, C2, C3) on day 5 and turns into the early DA progenitors (C6) on day 17, which further mature into young DA neurons (C8) (Fig.?2c). The trajectory mapping Defactinib hydrochloride revealed three branching points from pluripotent cells. The first branch gives rise Defactinib hydrochloride to an population (C2: C4) on day 11, indicating part of the RGCs differentiate to a fate of non-DA lineage. The second branch generates from RGCs into another population (C3: C5) on day 17. The third branch gives rise to CPECs (C6: Defactinib hydrochloride C7) on day 25. Expression of known markers during DA differentiation displaying expected temporal-specific transcriptional profiles (Fig.?2d, e). Specifically, the expression of pluripotency gene (is usually highly expressed from day 5 and sustained high expression to day 25. The.