Fig 1.
Simultaneous detection of four pluripotency markers by flow cytometry.
(A) Flow cytometric analysis of permeablized H9 hESCs. Cells were exposed to antibodies against TRA1-60, SSEA4, NANOG and OCT4. IgG antibody was used as negative control. Top: Cytometry profiles of undifferentiated H9 cells. Bottom: Cytometry histograms of H9 cells 9 days after removal of bFGF. (B) Flow cytometric analysis of undifferentiated H9 cells for the co-stained for TRA1-60 and OCT4. (C) Quantification of positive cells for TRA1-60, SSEA4, NANOG and OCT4 in comparison to IgG control for both undifferentiated and differentiated H9 cells.
Fig 2.
Outline of human quantitative embryoid body (hqEB) system strategies.
(A) Preparation of spheroids from TRA1-60+ magnetically purified hESCs. Low passage H9 cells were dissociated to single cells by Accutase treatment, then TRA1-60+ cells were magnetically purified. These were then plated at known density into Aggrewell plates to from spheroids of uniform size. (B) Alternative plating and differentiation approaches. Three methods were assessed: (1) Spheroids were deposited individually into cell culture plate wells, and imaged every 3 days. (2). Pools of EBs are plated into 6 well dishes under non-adherent conditions, imaged, quantitated for size. (3) Spheroids are attached onto Matrigel coated plates and allowed to proliferate and spread under adherent conditions.
Fig 3.
Representative images of hESC spheroids prepared from TRA1-60+ purified cells.
Spheroids containing uniform numbers of cells were prepared in Aggrewell plates using TRA1-60+ cells. (A) Aggrewell plate containing spheroids aggregated for 3 days (1.25X magnification). (B) Aggrewell plate containing spheroids aggregated for 3 days (4X magnification). (C) Aggrewell plate containing spheroids aggregated for 3 days (10 X magnification). (D) Spheroids imaged immediately after aggregation.
Fig 4.
Consequences of teratogen exposure on individually cultured EBs.
(A) H9 hESCs were dissociated with aAccutase, formed into spheroids, and plated into individual wells under non-adherent conditions (day 0). Media was changed to EB differentiation medium and individual EBs were imaged microscopically every 3 days for a total of 12 days of differentiation. Individual EBs were treated with vehicle only (DMSO), Caffeine, Penicillin-G, and Valproic acid and images were acquired after 0, 3, 6, 9, and 12 days of differentiation. (B) EBs were quantitated by measuring the apparent size (μm2) of individual embryoid bodies after treatment with DMSO, Caffeine, Penicillin-G or Valproic acid. Results shown indicate the means and standard deviations by quantitatively imaging a cohort of 3 independently cultured EBs each tested with each of the 3 compounds. (*P<0.05; **P<0.01; ***P<0.001 as compared to DMSO). (C) Shape quantification expressed by the circularity coefficient of individual EBs treated with DMSO, Caffeine, Penicillin-G or Valproic acid. Results shown indicate the means and standard deviations obtained by quantitatively imaging a cohort of 3 independently cultured EBs each tested with each of the 3 compounds.
Fig 5.
Monitoring EB growth in pooled populations of EBs grown under non-adherent conditions.
(A) Representative images of EBs grown in the presence of DMSO (vehicle only), Caffeine, Penicillin-G and Valproic acid. (B) Size quantification of EB populations treated with DMSO, Caffeine, Penicillin-G or Valproic acid. Results are the means +/- standard deviation. (n = 17 to 69 independent EBs after 3, 6, and 9 days of culture in the presence of teratogens (*P<0.05; **P<0.01; ***P<0.001 as compared to DMSO). (C) Analysis of mRNA expression of differentiation markers from EBs treated with DMSO, Caffeine or Penicillin-G over the course of 12 days of EB differentiation. RT-PCR performed to detect mesodermal (KDR, C-ACTIN and BRACHYURY) and ectodermal mRNAs (NETO2, NCAM, NES, BIII-TUB and NEFH). GAPDH is used as loading control. (D) EB spreading assay performed on attached EBs cultured on Matrigel (0 hours) and treated with DMSO, Caffeine, Penicillin-G or Valproic acid for 72 hours in differentiation medium.