Abstract
Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.
Highlights
The isolation of embryonic stem cells from developing mouse blastocysts, their maintenance in culture, and genetic manipulation has provided a fundamentally important research tool for experimental biology [1]
As embryoid bodies (EBs) growth and disaggregation beyond day 7 of differentiation compromised the quality and viability of the cell culture, we focused our investigation on timepoints up to and including day 7
We examined the expression of these same markers in EBs and confirmed the red cell expansion which occurs in parallel with increased expression of erythroid-specific genes (Fig 1B) [28, 29]
Summary
The isolation of embryonic stem cells from developing mouse blastocysts, their maintenance in culture, and genetic manipulation has provided a fundamentally important research tool for experimental biology [1]. In vitro differentiation of stem cells offers unparalleled access to developmental pathways including well defined multipotent cells, precursors and mature cell types representing a wide range of organ systems [2]. Developing robust protocols using mESCs to obtain specific cell types at scale would further allow the use of these cells for detailed molecular analysis and large scale, high throughput screens. For maximum value, it is crucial that mESC-derived cells and tissues faithfully represent the corresponding primary cell populations. Primitive haematopoiesis originates in the blood islands of the yolk sac (embryonic day E7.25–8.5). This is transiently accompanied by definitive haematopoiesis arising from Erythroid Myeloid
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