Embryo implantation is dependent on the human endometrium transforming into the decidua of pregnancy, a process of intensive tissue remodelling. Disruptions of this process can lead to reproductive disorders, such as recurrent implantation failure, happening in about 5% of all in vitro fertilisation (IVF) patients and recurrent pregnancy loss, experienced by approximately 2.5% of women trying to conceive.

The University of Warwick research group of Professor Jan Brosens, including Dr Emma Lucas and Dr Tom Rawlings, recently published a preprint using human endometrial assembloids to gain insights into the underlying mechanisms of these kinds of reproductive disorders.

You can find the full preprint here

Development of a human endometrial assembloid model

Schematic for establishing endometrial assembloid _ dolomite bioi

Schematic for establishing endometrial assembloids. Source: Adapted from Rawlings et al. 2021

The endometrial model established by the research group consisted of gland organoids and primary stromal cells. The gland organoids were established by seeding endometrial epithelial progenitor cells in MatrigelTM with expansion medium. The medium includes growth factors and signal transduction pathway modulators, helping to maintain long-term cultures and to keep cells genetically stable. Assembloids were created by propagating purified endometrial stromal cells in monolayer culture and combining them in a 1:1 ratio with the epithelial progenitor cell organoids. The mixture was further seeded in a hydrogel matrix, consisting of a collagen mixture present in midluteal endometrium and cultured in an expansion medium.

Single cell sequencing to investigate cellular complexity of decidualizing assembloids

The authors’ aimed to understand if epithelial cells and endometrial stromal cells adopt different cellular states upon decidualization of endometrial assembloids. To test for this hypothesis, they performed single cell RNA-Sequencing (scRNA-Seq) with the Nadia Instrument, comparing undifferentiated assembloids in an expansion medium and decidualized assembloids in a minimal differentiation medium.

They were able to identify 11 distinct cell clusters, of which 5 matched unambiguous and 1 ambiguous epithelial cell types as well as 5 stromal cell populations.

endometrial assembloid paper _ UMAP visualizing epithelial and stromal subsets.

UMAP visualizing epithelial and stromal subsets. Source: Rawlings et al. 2021

Within those populations only one epithelial subgroup, the ciliated cells, was present in both undifferentiated and decidualized assembloids. Interestingly, the 2 cell clusters identified after decidualization of endometrial assembloids, expressed genes used in clinical tests to aid the timing of embryo transfer in IVF patients.

The stromal fraction of the undifferentiated assembloids consisted of actively dividing cell populations expressing proliferative phase marker genes.

A population of ambiguous cells, termed ‘transitional population’, harboured within the decidualized endometrial assembloids expresses both epithelial and stromal genes. This subset expressed genes that are enriched in Gene ontology (GO) terms such as ‘wound healing’, ‘regulation of stem cell proliferation’, ‘blood coagulation’ and ‘blood vessel development’ indicating a role in tissue repair and regeneration.

Decidualization of assembloids for 4 days led to the emergence of pre-decidual, decidual, and senescent decidual cells. Pre-decidual and decidual cells mapped to the early- and mid-luteal phase of the cycle whereas senescent cell genes peaked in the late-luteal phase, i.e. prior to menstrual breakdown.

Both the pre-decidual and the senescent cells are enriched in GO terms such as ‘Wound healing’, ‘Response to hypoxia’, and ‘Inflammatory response’, suggesting that both clusters comprise stressed cells. However, only senescent cells expressed genes enriched in terms such as ‘Embryo implantation’ ‘Cellular senescence’, ‘Aging’, and ‘Leukocyte activation’.

In summary, scRNA-Seq with Nadia showed distinct epithelial and stromal subpopulations mapping to specific phases of the menstrual cycle. Actively dividing cells can mostly be found in proliferating assembloids, whereas senescent epithelial and decidual subpopulations emerge upon differentiation. The authors were able to show that decidualized assembloids closely resemble midluteal endometrium and that endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

In case you are interested in the method applied we have summarized them in short below:

Materials and Methods for single cell sequencing of human endometrial assembloids with the Nadia

Sample preparation:

1. Dissociation of assembloids with 0.5 mg/ml Collagenase I for 10 minutes in a 37°C water bath with regular vigorous shaking.
2. Sample wash with additive free DMEM/F12 phenol free medium
3. Incubation with 5×TrypLE Select diluted in additive free DMEM/F12 phenol free medium for 5 minutes in a 37°C water bath
4. Cell clumps were disrupted by manual pipetting
5. Suspension in 0.1% BSA in PBS and passage through a 35 µm cell sieve

ScRNA-Seq on Nadia

1. Droplet generation was performed using the Nadia Instrument (Dolomite Bio)
2. Reagents used as described in the scRNA-Seq on Nadia v1.8 protocol (Dolomite Bio)
3. Pooled beads were processed as described previously (Lucas et al., 2020)
1. NextSeq 500 with high output 75 cycle cartridge (Illumina)

Bioinformatics analysis

1. Data processing was performed using Drop-Seq_tools-2.3.0 as described previously (Lucas et al., 2020)
2. Cells with at least 200 genes detected were included
3. Genes were included if detected in at least 3 cells
4. Cells with more than 5000 genes and more than 5 % mitochondrial gene transcripts were excluded
5. The Seurat v3 standard workflow (Stuart et al., 2019) was used to integrate datasets from biological replicates
6. Gene ontology (GO) analysis was performed on differentially expressed genes from specified ‘FindMarkers’ comparisons in Seurat v3 using the Gene Ontology Consortium database (Ashburner et al., 2000, The Gene Ontology, 2019, Mi et al., 2013)
7. Dot plots of significantly enriched GO terms (FDR adjusted P < 0.05) were generated in RStudio (version 1.2.5042)


Further reading

Read our digests’ of other papers involving single cell experiments performed using the Nadia Platform