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Single cell RNA sequencing of in vitro differentiation NMP protocols

ABSTRACT: Single cell RNA sequencing of in vitro differentiation NMP protocols

PROVIDER: PRJNA548192 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9264140.fastq.gz	Fastqsanger.gz
	SRR9264150.fastq.gz	Fastqsanger.gz
	SRR9264160.fastq.gz	Fastqsanger.gz
	SRR9264170.fastq.gz	Fastqsanger.gz
	SRR9264180.fastq.gz	Fastqsanger.gz

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Single cell RNA sequencing of in vitro differentiation NMP protocols

Project description:The mammalian embryos Caudal Lateral Epiblast (CLE) harbours bipotent progenitors, called Neural Mesodermal Progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here we performed a single cell analysis of different in vitro NMPs populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them to E8.25 CLE mouse embryos. In our analysis of this region our findings challenge the notion that NMPs can be defined by the exclusive coexpression of Sox2 and T at mRNA level. We analyse the in vitro NMP-like populations using a purpose-built Support Vector Machine (SVM) based on the embryo CLE and use it as a classification model to compare the in vivo and in vitro populations. Our results show that NMP differentiation from ESCs, leads to heterogeneous progenitor populations with few NMP-like cells, as defined by the SVM algorithm, whereas starting with EpiSCs, yields a high proportion of cells with the embryo NMP signature. We find that the population from which the Epi-NMPs are derived in culture contains a node-like population, which leads to suggest that this population probably maintains the expression of T in vitro and thereby a source of NMPs. In conclusion, differentiation of EpiSCs into NMPs reproduces events in vivo and suggests a sequence of events for the emergence of the NMPs population.

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Project description:Neuroblastoma is the most common extra cranial solid tumor derived from sympathoadrenal (SA) cells and characterized as either adrenergic or mesenchymal. Here, we compared four different protocols to differentiate induced pluripotent stem cells (iPSC) toward SA cells and intermediate cell states (neuromesodermal progenitors [NMP], trunk neural crest cells [tNCC]) as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two iPSC lines of different genders. This protocol also generated tumors with the highest level of the neuroblastoma-specific marker, PHOX2B. Transcriptomally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma.

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Normothermic machine perfusion (NMP) inhibits proinflammatory responses in the liver and promotes regeneration

Project description:Background and Aims: Liver transplantation is a successful treatment for patients with liver failure. However, organ shortage results in over 11% of patients loosing their chance of a transplant due to worsening of their liver disease. Ischemia/reperfusion injury (IRI) is the major cause leading to graft loss after liver transplantation. Therefore, novel methods of organ-preservation have been developed in recent years to minimize IRI. One such device is the OrganOx metra, a normothermic machine perfusion (NMP) device providing oxygen and nutrition to allow aerobic metabolism and minimizing IRI. OrganOx metra has presented with several advantages compared to conventional cold storage (CS). We aimed to confirm the impact of NMP on reducing IRI and to define the underling mechanisms. Methods: We compared 12 NMP with 27 CS-preserved livers by performing gene microarray, immunoprofiling of hepatic lymphocytes and immunochemistry staining of liver tissues for assessing necrosis, platelet deposition and neutrophil infiltration, and the status of steatosis after NMP or CS pre- and post-reperfusion. Results: Recipients receiving NMP grafts showed significantly lower peak AST levels than those receiving CS grafts. NMP altered gene-expression profiles of liver tissue from pro-inflammation to pro-healing and regeneration. NMP also reduced the number of IFN- and IL-17 producing T cells and enlarged CD4posCD25highCD127negFOXP3pos Treg pool. NMP liver tissues showed less necrosis and apoptosis in the parenchyma and fewer neutrophils and platelet infiltration compared to CS liver tissues. Conclusion: Reduced IRI in NMP recipients was the consequence of the combination of inhibiting inflammation and promoting graft regeneration.

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