Proteome reprogramming of endometrial epithelial cells by human trophectodermal small extracellular vesicles reveals key insights into embryo implantation

Poh, Qi Hui; Rai, Alin; Carmichael, Irena; Salamonsen, Lois A.; Greening, David W.

doi:10.1002/pmic.202000210

Cited by 26 publications

(84 citation statements)

References 186 publications

(337 reference statements)

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“…To gain insight into the proteome composition of EV cargo and whether cell growth conditions in 2D or 3D impacted protein expression, quantitative mass spectrometry was employed ( Poh et al, 2021 ; Zhu et al, 2021 ). We report for EVs derived from cells cultured in 2D format (2D EVs), 1,266 proteins and from 3D condition (3D EVs), 1,035 proteins ( n = 5, independent replicates).…”

Section: Resultsmentioning

confidence: 99%

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies

Kusuma

Zhu

et al. 2022

Front. Cell Dev. Biol.

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Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

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Section: Resultsmentioning

confidence: 99%

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies

Kusuma

Zhu

et al. 2022

Front. Cell Dev. Biol.

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“…We next performed proteome profiling of sEVs and their parental cells using nLC-MS/MS and data-dependent acquisition ( Rai et al, 2021c ; Kompa et al, 2021 ). We found that the proteomes of parental cells versus their derived sEVs (fraction 6–8, 1.06–1.11 g/ml) were distinct ( Supplementary Figure S2 ), with sEVs compared to cells significantly enriched in sEV markers (e.g., CD9/63/81, Alix/PDCD6IP, TSG101) ( Greening et al, 2017 ; Rai et al, 2021d ; Mathieu et al, 2021 ), which includes CD63 as well as proposed universal marker of sEVs/exosomes, SDCBP ( Kugeratski et al, 2021 ) ( Figure 1D , Supplementary Table S1 ) ( Rai et al, 2021c ; Poh et al, 2021 ). Further, we show sEVs display lower abundance for non-EV (intracellular/organelle) proteins (e.g., nucleus; HNRNPC, mitochondria; CYSC, endoplasmic reticulum; CALR) ( Poh et al, 2021 ) ( Figure 1D , Supplementary Table S1 ).…”

Section: Resultsmentioning

confidence: 99%

“…We found that the proteomes of parental cells versus their derived sEVs (fraction 6–8, 1.06–1.11 g/ml) were distinct ( Supplementary Figure S2 ), with sEVs compared to cells significantly enriched in sEV markers (e.g., CD9/63/81, Alix/PDCD6IP, TSG101) ( Greening et al, 2017 ; Rai et al, 2021d ; Mathieu et al, 2021 ), which includes CD63 as well as proposed universal marker of sEVs/exosomes, SDCBP ( Kugeratski et al, 2021 ) ( Figure 1D , Supplementary Table S1 ) ( Rai et al, 2021c ; Poh et al, 2021 ). Further, we show sEVs display lower abundance for non-EV (intracellular/organelle) proteins (e.g., nucleus; HNRNPC, mitochondria; CYSC, endoplasmic reticulum; CALR) ( Poh et al, 2021 ) ( Figure 1D , Supplementary Table S1 ). Using these multiple sEV inclusion proteins to verify the presence of sEVs, is in direct agreement with MISEV guidelines ( Thery et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

Endometrial small extracellular vesicles regulate human trophectodermal cell invasion by reprogramming the phosphoproteome landscape

Fatmous

Rai

Poh

et al. 2022

Front. Cell Dev. Biol.

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A series of cyclical events within the uterus are crucial for pregnancy establishment. These include endometrial regeneration following menses, under the influence of estrogen (proliferative phase), then endometrial differentiation driven by estrogen/progesterone (secretory phase), to provide a microenvironment enabling attachment of embryo (as a hatched blastocyst) to the endometrial epithelium. This is followed by invasion of trophectodermal cells (the outer layer of the blastocyst) into the endometrium tissue to facilitate intrauterine development. Small extracellular vesicles (sEVs) released by endometrial epithelial cells during the secretory phase have been shown to facilitate trophoblast invasion; however, the molecular mechanisms that underline this process remain poorly understood. Here, we show that density gradient purified sEVs (1.06–1.11 g/ml, Alix+ and TSG101+, ∼180 nm) from human endometrial epithelial cells (hormonally primed with estrogen and progesterone vs. estrogen alone) are readily internalized by a human trophectodermal stem cell line and promote their invasion into Matrigel matrix. Mass spectrometry-based proteome analysis revealed that sEVs reprogrammed trophectoderm cell proteome and their cell surface proteome (surfaceome) to support this invasive phenotype through upregulation of pro-invasive regulators associated with focal adhesions (NRP1, PTPRK, ROCK2, TEK), embryo implantation (FBLN1, NIBAN2, BSG), and kinase receptors (EPHB4/B2, ERBB2, STRAP). Kinase substrate prediction highlighted a central role of MAPK3 as an upstream kinase regulating target cell proteome reprogramming. Phosphoproteome analysis pinpointed upregulation of MAPK3 T204/T202 phosphosites in hTSCs following sEV delivery, and that their pharmacological inhibition significantly abrogated invasion. This study provides novel molecular insights into endometrial sEVs orchestrating trophoblast invasion, highlighting the microenvironmental regulation of hTSCs during embryo implantation.

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“…Trophectoderm also secretes soluble biomolecules into uterine fluid [28,29]. Furthermore extracellular vesicles (EV) are released into uterine fluid from both maternal and trophectodermal surfaces [30][31][32]. EV membranes protect their 'cargo' from enzymatic degradation and deliver these biomolecules, which include proteins, lipids and miRNA, to their specific target cells, thus changing their phenotype to promote implantation [33].…”

Section: Uterine Microenvironmentmentioning

confidence: 99%

Infertility and the Endometrium

Salamonsen¹,

Dimitriadis²

2022

Clin. Exp. Obstet. Gynecol.

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Background: A couple's infertility can originate from the male and/or the female. In women, the uterus provides the site where the maternal-fetal interface is established and maintained. Final blastocyst development occurs within the uterine cavity, then the blastocyst must attach to and implant into the endometrium (the inner uterine surface), via its outermost trophectodermal cells. Beneath the epithelium, these differentiate into syncytial trophoblast and invasive trophoblast -the latter progress through the endometrium to invade the spiral arteries converting them to the flaccid blood sacs of the placenta. Therefore, the endometrium plays a critical role in establishment of pregnancy. Objectives: To critically examine current knowledge of endometrial preparation for blastocyst implantation and placental development at the cellular and molecular level and to evaluate measures to improve implantation success. Mechanism: Literature searching by leading experts in the field. Findings: A wealth of new knowledge resulting from 'omics' technologies and new functional models has greatly enhanced our knowledge, but this information is yet to be translated into enhanced outcomes. Conclusions: The endometrium remains the 'black box' of infertility. Extensive trials do not support current adjuvant therapies as being better than placebo while effectively timed testing for endometrial preparedness for implantation is still urgently needed.

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Proteome reprogramming of endometrial epithelial cells by human trophectodermal small extracellular vesicles reveals key insights into embryo implantation

Cited by 26 publications

References 186 publications

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies

Endometrial small extracellular vesicles regulate human trophectodermal cell invasion by reprogramming the phosphoproteome landscape

Infertility and the Endometrium

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