Dataset Information

Molecular barcodes and single cell transcriptomics to map graft diversity and lineage of human DA neurons in a rat xenograft model of Parkinson’s Disease

ABSTRACT: Human pluripotent stem cells (PSCs) differentiated into dopamine (DA) neurons via a ventral floor plate intermediate provide an unlimited cell source for cell replacement therapy for Parkinson’s disease (PD). However, histology and scRNA-seq profiling of resulting grafts in in vivo models have revealed that seemingly homogenous cell preparations give rise to heterogenous grafts not only made up of dopamine neurons. Here, we used single nuclei RNA-seq combined with molecular barcode tracing delivered to the PSC-derived DA progenitors both in vitro and in vivo to show that three cell types (neurons, astrocytes and vascular leptomeningeal cells, VLMCs) make up the cellular composition of the grafts and that these cell types all share a common progenitor. Additionally, barcoding proliferative cells in vivo 1 month after transplantation, showed that a fraction of the cells retained their multipotent potential also after transplantation. Single-cell chromatin accessibility and RNA expression paired maps identified seemingly subtle chromatin accessibility differences among progenitors supporting their homogeneity, and together with the tracing data shows that the PSC-derived floor plate cells used for transplantation are tri-potent, giving rise to dopamine neurons, astrocytes and VLMCs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE200610 | GEO | 2023/05/01

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However the effective use of hPSCs for cell therapy has lagged far behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons derived from human PSCs generally display poor in vivo performance. There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive in vitro molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD animal models in three host species. Long-term engraftment in 6-OHDA-lesioned mouse and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models tested indicate considerable promise for the development of cell based therapies in PD. Differentiated hESC with three conditions (LSB, LSB/S/F8, LSB/S/F8/CHIR) were subjected to RNA extraction in specific timepoint (day 0, 1, 3, 5, 7, 11, 13, 25) and hybridization on Illumina microarrays. Each sample has 3 or 4 biological repeats. Based on previous study* of dual SMAD inhibition neural induction, we developed new midbrain dopamine neuron protocol. It depends on time specific treatment of below factors (LSB/S/F8/CHIR): L (LDN193189 (BMP inhibitor) , day 0-11), SB (SB431542 (TGF-b signal inhibitor), day 0-5), S (SHH + Purmorphamine (Smo agonist), day 1-7), F8 (FGF8, day 1-7) and CHIR (CHIR99021 (GSK3b inhibitor), day 3-13) LSB and LSB/S/F8 are limited control conditions of dual SMAD only (LSB) or traditional patterning with Sonic and FGF (LSB/S/F8) *Chambers,S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275-280 (2009).

Dataset Information

Molecular barcodes and single cell transcriptomics to map graft diversity and lineage of human DA neurons in a rat xenograft model of Parkinson’s Disease

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets