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Scalable in vitro production of defined mouse erythroblasts

['Helena S. Francis', 'Mrc Molecular Haematology Unit', 'Mrc Weatherall Institute Of Molecular Medicine', 'University Of Oxford', 'Oxford', 'United Kingdom', 'Caroline L. Harold', 'Robert A. Beagrie', 'Andrew J. King', 'Matthew E. Gosden']

Date: 2022-02 

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.

Funding: The study was not specifically funded. The main contributing authors are funded as designated below. UKRI | Medical Research Council (MRC): MR/T014067/1; Wellcome Trust:Helena Francis 109097/Z/15/Z; Sir Henry Wellcome Fellowship:Robert Beagrie 209181/Z/17/Z The first author Helena Francis was on a Wellcome Trust Studentship at the University of Oxford (109097/Z/15/Z). The co-author Rob Beagrie is a Sir Henry Wellcome Fellow (209181/Z/17/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Francis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we exploit the spontaneous differentiation of erythroid cells within EBs as a readily-accessible and scalable erythroid cell population. By selecting erythroid cells expressing the transferrin receptor (CD71), we can isolate and characterize a large population of mouse erythroid cells. We show that this population is homogeneous, faithfully represents normal primitive erythropoiesis and accurately mimics the resulting phenotypic effects of genetic manipulations seen in primary cells obtained from mice harbouring the same genetic perturbations, potentially avoiding the need to establish full mouse models to assess the effects of such manipulations. Finally, we present a protocol for miniaturization of the differentiation protocol, which offers the potential to perform high-throughput studies on tens to hundreds of genetic models in a single experiment.

The precise origins and cellular outputs of mESC-EB cultures have been of main interest to the field of developmental biology for years [ 4 , 6 , 9 , 10 ]. In haematopoiesis, initial studies used colony re-plating assays of EB-derived cells and detailed the emergence of erythroid progenitors of primitive and definitive nature from day four onwards [ 8 ]. This was subsequently shown to closely reflect erythropoiesis in mouse development in vivo [ 3 , 11 ]. Within a similar time-frame, haemoglobinized erythroid cells also begin to arise in cultured EBs [ 8 ]. Whilst in early studies haemoglobinization was observed in only ~1% of EBs, optimisation of culture conditions has now increased erythrocyte-containing EB production to almost 100% [ 12 , 13 ]. Isoelectric focusing [ 12 ], RNase protection [ 14 ], and RT-PCR data [ 8 ] from earlier studies identified embryonic globins, suggesting the presence of erythrocytes that most likely recapitulate some aspects of primitive erythropoiesis. Later studies showed that under specific conditions, the cultures may yield both primitive and definitive AGM-like progenitor cells [ 10 ]. More recent immunophenotypic characterization and re-plating of progenitors revealed that the primitive and definitive outputs detected in mESC-EB culture resemble primitive and EMP outputs in mouse embryos [ 4 ]. It is not yet clear if truly definitive long-term repopulating hematopoietic stem cells are represented in these cultures [ 10 , 15 , 16 ]. As demonstrated over almost two decades of work in this system, the co-emergence of progenitors of mixed origins hinders the use of the culture system as a source of lineage-specific haematopoietic cells for detailed molecular studies and obviate the need for further characterisation of the cells produced. If better defined, the erythroid cell production in the mESC-EB system would expand the in vitro system from a developmental biology platform to a mammalian genetics and molecular screening platform.

During development, mouse haematopoiesis occurs in three distinct waves. 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 Progenitors (EMP; E8.25–9.5) and eventually replaced by long-term definitive haematopoiesis emerging from the aorta-gonad-mesonephros (AGM) at around E10.5 [ 3 – 5 ]. Lineage specification, differentiation and maturation of haematopoietic cells at each stage of development has been extensively investigated using mESCs [ 2 , 3 , 6 ]. Such studies have informed researchers of normal blood formation and also elucidated some of the mechanisms underlying inherited and acquired blood diseases [ 3 , 5 , 7 ]. To investigate haematopoietic differentiation, mESCs are cultured in the absence of leukaemia inhibitory factor (LIF), which results in the formation of a 3D organoid or embryoid body (EB). This promotes differentiation that faithfully represents early mouse development; resulting EBs therefore contain a mixture of all three germ layers [ 8 ]. The use of additional cytokines, alternative plating strategies, and/or re-plating can be used to support particular differentiation pathways of interest [ 6 ].

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. Nevertheless, for maximum value, it is crucial that mESC-derived cells and tissues faithfully represent the corresponding primary cell populations.

ATAC-seq, ChIP-seq, and Capture-C data from EB day 7 CD71+ reported in this article have been deposited in the Gene Expression Omnibus (GEO) database under the following accession number: GSE184435. ATAC-seq and ChIP-seq data used for mESC, E10.5 blood, and APH Ter119+ spleen are already published as referenced in the manuscript and are accessible following these accession numbers: GSE108434, GSE27921, GSE97871.

Single-cell RT-PCR: single-cell expression analysis necessitated isolation of 185 single erythroid cells by indexed FACS into 96-well plates. See S1 Methods for details. Briefly, cells were lysed, RNA reverse transcribed and material used for 43 TaqMan assays ( S3 and S4 Tables). When performing reverse transcription and pre-amplification on sorted single cells, one aliquot of RNA standard was included (prepared and used as detailed in supplemental data).

To compare ATAC-seq datasets genome-wide, triplicate data for each tissue of interest were peak-called with the Model-based Analysis of ChIP-seq tool (MACS2) [ 23 ] using default parameters. PCA was then performed using the DiffBind package in R [ 24 ]. H3K4me3 and CTCF ChIP-seq data files were aligned and processed as described. Peaks were then called using the Lanceotron peak caller [ 25 ], using default settings. Peaks with scores ≥0.5 called in at least two replicates were extracted, and bedtools intersect was used to compare overlapping peaks between different biological stages.

ATAC-seq was performed on 70x10 3 cells from target populations as previously described [ 19 , 20 ]. ChIP-seq was performed as described [ 21 ] on aliquots of 5x10 6 CD71+ cells derived from two separate differentiation experiments per antibody. Chromatin fragmentation was performed for 8 minutes as optimized for the use of Bioruptor Pico sonication device. Data from both methods were analysed with an in-house pipeline as described [ 20 , 22 ]. In each case, for visualization, alignment files from two or three biological replicates were normalized to reads per kilobase per million mapped reads (RPKM) and averaged.

CD71+(high) cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer’s instructions. Briefly, cells from disaggregated EBs were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 μl per 10 7 cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 μl per 10 7 cells, according to manufacturer’s instructions). Bead-labelled cells were retained by LS columns. See S1 Methods for details.

24h prior to differentiation, mESCs were induced by passaging into IMDM based media supplemented with LIF. To start the differentiation culture (d0), cells growing in IMDM were trypsinized and plated in differentiation media in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x10 4 cells in 10 cm dishes or 100–1200 cells per well of a 96-well plate for up to seven days without further intervention except for daily gentle shaking of the dishes to disrupt potential EB attachment to the bottom of the dish. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. See S1 Methods for details.

E14TG2a.IV mESCs were maintained by standard methods [ 17 , 18 ]. To produce genetically modified mESC models, variations of CRISPR/Cas9 strategies were used in conjunction with homology-directed repair (HDR) when needed (YFP-tagged α-globin and D3839 models). mESCs were co-transfected with guide RNA and HDR vectors using TransIT-LT1 reagent (Mirus; according to manufacturer’s instructions) for YFP-tagged α-globin and Neon electroporation system (Invitrogen, according to manufacturer’s instructions) for DelR1 and D3839 mutants. Details on transfection conditions and sequences for guide RNA ( S1 Table ) and HDR vectors are in S1 Methods .

Results

A pure erythroid population can be isolated from a 7-day embryoid body culture The generation of EBs from mESCs is well-established [6]. To enrich for haematopoietic lineages, cells are plated at low density in differentiation media using bacterial dishes and harvested between days 2 and 7. Allowing EBs to differentiate undisturbed results in high levels of spontaneous erythroblast differentiation and haemoglobinization, observed as red EBs [13]. To optimize the number and purity of the obtained erythroid cells, we first established the kinetics of erythroid differentiation in the E14 mESC line. As EB 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. Previously published data show that globin expression increases in EBs as they differentiate in culture [8, 12, 14]. We confirmed this by RT-PCR at days 4–7 of differentiation (Fig 1A). Murine primary erythroid cells are usually staged by immunophenotyping using the erythroid markers CD71 and Ter119, as early erythroid progenitors are marked by high expression of CD71 (CD71+(high)) and subsequent populations first gain Ter119 and then lose CD71 [27]. 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]. By day 7, cells from disaggregated EBs were 40% distinctly CD71+(high), and about 30% also positive for Ter119. PPT PowerPoint slide

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TIFF original image Download: Fig 1. In vitro differentiated EBs as a source of erythroid cells. A) RT-PCR of mature mouse globin transcripts in mESCs and EBs between days 4–7 of differentiation, normalized to the 18S housekeeping gene. Levels are shown relative to the maximal detected expression for each gene. Bars represent mean values from three independent differentiations; error bars represent standard deviation of the mean. B) Cell counts for immunophenotypically-defined populations using antibodies for CD71 and Ter119 cell surface markers through days 4–7 of EB differentiation. Data shown are from a representative differentiation in 10 cm dish format. C) Fluorescence levels of an α-globin-YFP tag in mESCs and day 7 EBs. Top: a schematic of the tag shows the insertion site after the final exon of the gene with a 2A self-cleaving peptide sequence in yellow, the mVenus coding region in green, nuclear localization signal (NLS) repeats in blue, the STOP codon in red and the untranslated regions (UTRs) in pink. Bottom: brightfield images of mESCs and a single EB are overlaid with YFP fluorescence signal (left panels). Flow cytometry histograms for YFP fluorescence demonstrate the presence of an α-globin-positive population (green peak) in day 7 EBs (middle panels). Flow cytometry plots for the erythroid markers CD71 and Ter119 show the overlap of YFP+ population from the histogram with the CD71+ cell populations (YFP+ cells labelled green as in the histogram) in day 7 EBs (right panels). D) Protocol summary for the generation of EB-derived CD71+ erythrocytes in vitro. Example data for column-based CD71+ cell purification, starting with brightfield images of cultured mESCs (top panel), to whole EB (middle panel), to CD71-separated populations shown as cell pellet images for CD71+ (red pellet) and CD71- (clear pellet) fractions. A stained (modified Wright stain) cytospin preparation is shown for the purified CD71+ erythroid population (bottom panel) with an inset (black square) highlighting (black arrows) specifically mature primitive erythroid cells and their distinclive morphology; large nucleated hemoglobinised cells. E) Flow cytometry plots for CD71 and Ter119 markers are shown for populations at each step of the protocol. F) An overlay of CD71 histograms from all day 7 EB-derived populations (as indicated by colour) highlighting the varying intensities of CD71 expression at each step of the protocol. Note the highest CD71 intensity marking the CD71+ fraction retained by the LS column. Stained CD71- fraction (histogram and FACS plot) shows low CD71 expressing fraction unretained by the magnetic column. https://doi.org/10.1371/journal.pone.0261950.g001 To purify erythroid cells from day 7 EBs, we focused on the transferrin receptor surface protein CD71: a reliable marker of primitive and definitive erythroid cells [28, 29]. EB-derived cell populations almost all express CD71 with varying levels reflected by the intensity of CD71 staining by immunophenotyping and indicating the cycling nature of this population [30, 31]. The small portion of CD71+(high) cells derived from day 5 EBs have been shown to selectively mark primitive erythroid progenitors [31]. However, the more expanded and more mature erythroid output in differentiating EBs at day 7 needed characterisation. To that end, we monitored the most reliable erythroid marker, the globin expression, using a mESC line with one copy of the red cell specific α-globin gene heterozygously tagged with a yellow fluorescent protein (YFP). By flow cytometry, all YFP+ cells were also strongly stained for CD71 (Fig 1C) confirming that CD71+(high) is also a robust marker of erythroid cells derived from day 7 whole EB population. Next, we used a magnetic column-based selection method to isolate CD71+(high) erythroid cells from day 7 EBs (Fig 1D). Compared to FACS-based sorting, the use of a column-based selection results in low levels of cell death and allows for rapid selection of large numbers of cells. Cytospin staining confirmed that the isolated CD71+ cells are a mix of erythroid cell differentiation stages, in agreement with immunophenotyping data (Fig 1D). Moreover, the high stringency magnetic column selection resulted in high purity, as assessed by flow cytometry (>98% CD71+; Fig 1E) and was biased towards the desired population of CD71+(high); when closely inspected, the cell fraction retained by the column is of higher CD71+ intensity (Fig 1E, 1F) compared to the unretained population in the CD71- fraction (Fig 1F). The total cell yield from a typical single 10 cm dish of cultured EBs is 5-10x106 cells (see Methods); we obtain typically around 1–2 x106 CD71+ cells per plate. The cell numbers required for most molecular assays can be attained by scaling appropriately. In conclusion, we have developed a simple, scalable protocol for isolating a pure erythroid cell population from a single plating.

Genetically modified EB-derived erythroid cells recapitulate the phenotype of their in vivo derived counterparts When substituting in vivo models with in vitro cell systems, one must have confidence that the latter faithfully recapitulates the former [3, 37, 38]. We therefore compared the phenotypes of specific genetic manipulations in EB-derived cells with their counterparts in primary cells. We used the α-globin gene cluster as a well characterized model of mammalian gene expression. The α-globin genes are expressed and similarly regulated in both embryonic and adult red cells [34]. We initially showed that the pattern of α-globin-like gene expression in the EB-derived erythroid cells closely resembles that seen in normal in vivo primitive erythropoiesis (Fig 3A). To test whether key regulatory elements in this multi-gene locus acted similarly in EB-derived erythroid cells and primary in vivo mouse erythroid cells, we analysed the molecular phenotype of erythroid cells derived from genetically modified mESCs and their corresponding in vivo mouse models. Deletion of one of the major enhancers of the adult α-globin genes, R1, reduces the expression of alpha globin by 40% in definitive erythroid cells [20]; the same effect is seen in E10.5 primitive red cells with no associated effect on ζ-globin [34]. R1 was deleted from both alleles in ΔR1 mESCs and EB-derived erythroid cells were isolated and analysed for gene expression. ΔR1 EB-derived erythroid cells show downregulation of α-globin as observed in the equivalent mouse model both in E10.5 primitive and fetal liver definitive erythroid cells (Fig 4A). No effect on ζ-globin is observed in ΔR1 EB-derived red cells as in primary E10.5 erythroid cells (Fig 4A) [34]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. EB-derived ΔR1 erythroid cells recapitulate the molecular phenotype of their in vivo mouse-derived ΔR1 erythrocytes. A) Expression data for indicated genes based on mature transcripts from enhancer R1 knock out mESCs (ΔR1) day 7 EB-derived erythroid cells, normalized to the embryonic β-globin genes. Levels are shown relative to wildtype day 7 EB-derived erythroid cells (WT). Bars represent mean values from at least six independent differentiations; error bars represent standard deviation of the mean. Student’s t-test *P <0.001. B) Expression data for indicated genes based on mature transcripts from CTCF (HS38-39) knock out mESCs (D3839) day 7 EB-derived erythroid cells, normalized to the embryonic β-globin genes. Levels are shown relative to equivalent wildtype cells (WT). Bars represent mean values from at least six independent differentiations; error bars represent standard deviation of the mean. Student’s t-test *P <0.001. C) RPKM-normalized ATAC-seq and CTCF ChIP-seq tracks averaged for three replicates of wildtype and D3839 erythroid cells, both derived from day 7 EBs. D) Differential interactions (by NG-Capture-C) of α-globin regulatory regions and flanking genes between WT and D3839 d7 EB-derived erythroid cells. Capture-C data for the indicated viewpoints (black asterisks) in WT and D3839 erythroid cells are shown. Data representing at least 3 independent differentiation for two independently generated clones were used. Differential tracks show a subtraction (D3839-WT) of the mean number of normalized meaningful interactions per restriction fragment. https://doi.org/10.1371/journal.pone.0261950.g004 We also created an mESC line in which the CTCF boundary (HS38-39) was deleted in homozygosity (D3839) from the α-globin cluster as confirmed by ATAC-seq and CTCF ChIP-seq (Fig 4C) and compared the phenotype of the EB-derived erythroid D3839 cells to those from the corresponding mouse model [21]. Again, we found that the in vitro mESC-EB culture system largely recapitulated the in vivo phenotype. When compared to WT, D3839 EB-derived erythroid cells showed perturbation of gene expression similar to that reported in the D3839 mouse model [21]; the Mpg, Rhbdf1, and Snrnp25 genes, located 5′ of the deleted boundary, were upregulated (Fig 4B). Furthermore, an extension of the enhancer/promoter interaction domain to include promoters of the perturbed genes was also revealed by Capture-C in D3839 EB-derived erythroid cells (Fig 4D) as described in the D3839 mouse model [21]. The perturbed chromatin interaction profile is captured both from the α-globin R1 enhancer as well as the promoters of the affected genes (Rhbdf1 and Mpg); the subtraction tracks (D3839-WT) indicate a gain in significant chromatin interactions between Mpg, Rhbdf1 promoters and the α-globin cluster (Fig 4D). Recapitulating the complex phenotype of the HS38-39 boundary deletion supports the argument for the mESC-EB system as a faithful in vitro model for dissecting complex molecular mechanisms and address current outstanding questions such as the relationship between genome structure and function.

[END]

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