Genetically edited human placental organoids cast new light on the role of ACE2

Abstract

ACE2 expression is altered in pregnancy disorders and ACE2 gene variants are associated with several major pregnancy complications including small-for-gestational-age, fetal growth restriction and preeclampsia. This study utilised gene-editing to generate both ACE2 knockout and ACE2 rs2074192 placental organoids, facilitating mechanistic studies into the role of ACE2 in placental development, and the effect of fetal carriage of ACE2 rs2074192 CC, CT and TT genotypes. Parameters of cell and organoid growth were measured, together with qPCR, Western Blotting, and ELISA assessments, in all groups from both organoid models. Here, we report that ACE2 knockout results in delayed placental cell growth and increased cell death. ACE2 knockout organoids had lower ACE protein expression, reduced organoid diameters and asymmetrical growth. Placental organoids with the ACE2 rs2074192 TT genotype had significantly higher expression of ACE2 mRNA and ACE2 protein with elevated ACE2:ACE expression ratio and no change in ACE protein. Despite increased expression of ACE2 protein, ACE2 enzyme activity was significantly decreased in ACE2 rs2074192 TT placental organoids. TT organoids also had reduced diameters and asymmetrical growth. Our research provides a new molecular understanding of the role of ACE2 in placental development, with potential implications for pregnancy in the carriage of the ACE2 rs2074192 gene variant.

Fig. 1

The ACE- and ACE2-mediated opposing arms of the renin-angiotensin system.

Fig. 2. Method describing gene-editing to generate different TSC groups.

Early gestation placenta tissue was collected from n = 3 patients. For each tissue sample, trophoblast stem cells (TSCs) were isolated and cultured prior to separating into two groups: (1) ACE2 KO and (2) rs2074192. Within group (1), cells were transfected to achieve genotypes of two copies of the ACE2 gene (ACE2^+/+), one copy (ACE2^+/-) or zero copies (ACE2^-/-). Within group (2), cells were transfected to achieve genotypes of two dominant alleles (CC), one dominant and one recessive allele (CT) or two recessive alleles (rs2074192 SNP; TT). Once successfully transfected, cells were used to create organoids. All experiments performed in technical triplicate.

Fig. 3. Expression of ACE2 and ACE in ACE2 KO organoids, and ACE2 KO cell growth.

The abundance of A ACE2 mRNA, B ACE2 protein in cell media and C ACE2 protein in the cell lysate of ACE2^+/+, ACE2^+/- and ACE2^-/- (ACE2 KO) organoids. D Percentage of dead cells in the 24 h post seeding. E In situ localisation of ACE2 in ACE2^+/+, ACE2^+/- and ACE2 KO organoids. F Merged xCELLigence line trajectories for proliferation and G proliferation rate (represented by the slope of xCELLigence trajectory, 1/h) of ACE2^+/+, ACE2^+/- and ACE2 KO trophoblast stem cells (TSCs). H Merged xCELLigence line trajectories for migration and I migration rate of ACE2^+/+, ACE2^+/- and ACE2 KO TSCs. J In situ localisation of ACE in ACE2^+/+, ACE2^+/- and ACE2 KO organoids. K Level of ACE protein in cell lysate and L ACE2/ACE (ACE2:ACE) ratio (from cell lysate levels) of ACE2^+/+, ACE2^+/- and ACE2 KO organoids. Representative immunofluorescence images of organoid sections after 20 days of culture. Scale bars: 100 μm. Nuclei are stained with DAPI. Data are presented as a 10–90 percentile interleaved box-and-whisker plot. Statistics: linear mixed model with random intercept accounting for individual patient correlation. White bars denote ACE2^+/+ group, grey bars denote ACE2^+/- group, and red bars denote the ACE2 KO group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns indicates non-significance (n = 9).

Fig. 4. ACE2 KO organoid growth and trophoblast cell differentiation.

Representative image (Brightfield) of symmetrical organoid growth A ACE2^+/+, B ACE2^+/− and C asymmetrical organoid growth (ACE2 KO). D The average diameter (µm) of ACE^+/+, ACE^+/− and ACE2 KO organoids. E The ratio of diameter 1 (horizontal) to diameter 2 (vertical) of ACE^+/+, ACE^+/− and ACE2 KO organoids as a measure of symmetrical organoid growth. A value of 1.0 indicates perfect symmetry. In situ localisation of PSG1 and PEG10 in F early culture (day 6) and G late culture (day 28) ACE^+/+, ACE^+/− and ACE2 KO organoids. Scale bars: 100 µm. PSG1 staining signifies syncytiotrophoblast cells, and PEG10 staining signifies villus cytotrophoblast cells. Nuclei are stained with DAPI. Data are presented as a 10–90 percentile interleaved box-and-whisker plot. Statistics: linear mixed model with random intercept accounting for individual patient correlation. White bars denote the ACE^+/+ group, grey bars denote the ACE^+/− group, and red bars denote the ACE2 KO group. ns indicates non-significance, *p < 0.05, ****p < 0.0001 (n = 9).

Fig. 5. Successful induction of the ACE2 rs2074192 SNP.

Successful gene-editing at position 6: the reference C allele (A) and the alternate T allele (B).

Fig. 6. Expression and activity of ACE2 and ACE in CC, CT and TT organoids.

The abundance of A ACE2 mRNA, B ACE2 protein in cell media and C ACE2 protein in cell lysate; D Level of ACE protein in cell lysate, E ACE2/ACE ratio (from cell lysate levels); enzyme activity of ACE2 in F cell media and G cell lysate, and the ratio between ACE2 activity and ACE2 levels in H cell media and I cell lysate of CC, CT and TT organoids. Data are presented as a 10–90 percentile interleaved box-and-whisker plot. Statistics: linear mixed model with random intercept accounting for individual patient correlation. White bars denote the CC group, beige bars denote the CT group, and brown bars denote the TT group. ns indicates non-significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 9).

Fig. 7. Asymmetrical growth of TT organoids.

A The average diameter (µm) of CC, CT and TT organoids. B The ratio of diameter 1 (horizontal) to diameter 2 (vertical) of CC, CT, and TT organoids as a measure of symmetrical organoid growth. A value of 1.0 indicates perfect symmetry. Data are presented as a 10–90 percentile interleaved box-and-whisker plot. Statistics: linear mixed model with random intercept accounting for individual patient correlation. White bars denote the CC group, beige bars denote the CT group, and brown bars denote the TT group. ns indicates non-significance, *p < 0.05, **p < 0.01 (n = 9).