3 minute lightning oral presentation (and poster) ESA-SRB-APEG-NZSE 2022

Term side-population trophoblasts can be differentiated to mature trophoblast populations and form organoids (#97)

Cherry Sun 1 , Katie M Groom 2 , Charlotte Oyston 1 , Larry W Chamley 1 , Joanna L James 1
  1. Obstetrics & Gynaecology, University of Auckland, Auckland, New Zealand
  2. Liggins Institute, University of Auckland, Auckland, New Zealand

Trophoblast stem cells (TSC) give rise to all mature trophoblast lineages of the human placenta (cytotrophoblast, syncytiotrophoblast (STB) extravillous trophoblast (EVT)). The side-population technique allowed the isolation of TSCs directly from first-trimester placentae1. A similar trophoblast side-population can be isolated from term placentae, and are reduced 10-fold in fetal growth restriction. Our prior work established that term side-population trophoblasts require different culture conditions to first-trimester TSCs, in line with morphological/transcriptomic placental differences across gestation. Here, we aimed to use these refined conditions to determine whether term side-population trophoblasts exhibit similar functional capacity as their first-trimester counterparts.  

Term side-population trophoblasts1 were seeded at 6000 cells/well in a 96-well plate coated with Laminin-521. Differentiation to STB/EVT was undertaken by modifying prior TSC differentiation protocols2. Undifferentiated controls were maintained in TSC-Medium containing 25ng/mL decorin and 50ng/mL IL-8. Organoid formation was undertaken in Matrigel domes3, with halved dome volume for term side-population trophoblasts, and decorin and IL-8 medium supplementation. All experiments were run in triplicate. Data are mean±SEM.

Term side-population trophoblasts could be differentiated into; a) STB  as shown by up-regulation of Syncytin-1 (66.09%±9.428 in STB-Medium; 23.37%±2.307 in undifferentiated controls, p<0.05) and hCG, (25.28%±11.09 in STB-Medium, no expression in undifferentiated controls, ), or b) EVT as shown by up-regulation of HLA-G (29.05%±11.42 of cells in EVT-Medium; no expression in undifferentiated controls, p=0.0637). First-trimester side-population trophoblasts formed organoids (90.53±29.93μm diameter at 28 days), demonstrating self-renewing capacity. Term side-population trophoblasts formed smaller organoids (38.57±5.568μm diameter at day 28). Organoids could not form from pure FACS-sorted first-trimester cytotrophoblasts that had side-population trophoblasts excluded, demonstrating that side-population trophoblasts are required for organoid formation.

These data indicate term side-population trophoblasts exhibit the differentiation potential expected of a TSC population. Formation of the first term trophoblast organoids opens up their potential use as disease models.

  1. Gamage, T. K., Perry, J. J., Fan, V., Groom, K., Chamley, L. W., & James, J. L. (2020). Side-population trophoblasts exhibit the differentiation potential of a trophoblast stem cell population, persist to term, and are reduced in fetal growth restriction. Stem Cell Reviews and Reports, 16(4), 764-775.
  2. Okae, H., Toh, H., Sato, T., Hiura, H., Takahashi, S., Shirane, K., ... & Arima, T. (2018). Derivation of human trophoblast stem cells. Cell stem cell, 22(1), 50-63.
  3. Haider, S., Meinhardt, G., Saleh, L., Kunihs, V., Gamperl, M., Kaindl, U., ... & Knöfler, M. (2018). Self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta. Stem cell reports, 11(2), 537-551.