Development of a quantitative metric to assess the role of hESC microenvironment on hESC fate. Quantitative fluorescence microscopy was used to obtain images. (A) hESCs were stained for Hoechst (Ai) and Oct-4 (Aii). A mask was drawn around each nucleus (Aiii). The fluorescence intensity corresponding to the species of interest, in this case Oct-4, within this mask is obtained. A spatial map is generated using the centroid of the nucleus and the fluorescence intensity measurement indicated by the color bar (Aiv). Scale bar is 50 μm. (B) An algorithm is used to determine the number of neighbors within a 300 μm radius. This information is superimposed on the spatial location map to create heat maps (Bi). Blue indicates low number of cells and red indicates high density. The spatial location is superimposed on the Oct-4 fluorescence for each cell to obtain an Oct-4 map (Bii). Scale bar is 408 μm and the color threshold to determine positive populations is as indicated. (C) The cells are classified into bins based on the localized cell density, which is the number of neighbors within a 300 μm radius. The number of cells in each bin is shown here. (D) Oct-4 histograms are plotted for the cells in bin 1 and bin 7 which contain 0–100 cells (low) and 500–600 cells (high) as the localized cell density. (E) The percentage of Oct-4+ cells is plotted as a function of the localized cell density.

Endogenous FGF and TGF-β signaling regulates hESC self-renewal and exogenous FGF-2 suppresses Smad1 signaling in Oct-4+ hESCs. HESC cells were cultured in XFT media. Cells were plated on Matrigel-coated plates in medium without any exogenous cytokines. After 11 h, the medium was replaced with fresh medium or medium containing FGF (FI) or TGF-β inhibitors (TI). After a further 37 h, the cells were fixed and analyzed. (A) The percentage of Oct-4+ cells under the different conditions after 48 h is shown. (B) The percentage of Oct-4+ cells in each condition is plotted as a function of the localized cell density, that is the local microenvironment. (C) The mean single-cell pSmad1 levels in the Oct-4+ subpopulation are plotted. Data are normalized to the mean single-cell pSmad1 value in the XV condition. Mean single-cell pSmad1 levels are plotted as a function of the number of Oct-4+ cells (D), number of Oct-4− cells (E) and the fraction of Oct-4+ cells (F) within a 300 μm radius. (G) Total cellular content (cytoplasmic and nuclear) of pSmad1 and total Smad1 (TSmad1) after 90-min simulation by XV media alone (XV) or XV media supplemented with 80 ng/ml of FGF-2 (F), 25 ng/ml of BMP2 (B) or 80 ng/ml of FGF-2 and 25 ng/ml of BMP2 (B+F). (H) Cytoplasmic to nuclear ratio of pSmad1 and TSmad1 after stimulation. Error bars in (B) represents the standard error of the mean of five wells in a single representative trial that has been repeated seven times.

Niche-size-mediated control of self-renewal acts through the BMP/GDF3 pathway and by controlling pSmad1 activation. (A) The signaling antagonism between hESCs and ExE described earlier is recapitulated by the artificial signaling gradients established by micropatterning. Cells in D=200 μm and D=800 μm colonies were pulsed for 90 min with unconditioned media (XV), media conditioned by D=200 μm cells (200-CM), D=800 μm cells (800-CM), 25 ng/ml BMP2, hESC-CM and ExE-CM. As expected, 200-CM increased pSmad1 levels of larger colonies, whereas 800-CM decreased pSmad1 levels in smaller colonies. (B) Oct-4 expression in D=200 μm can be maintained by the addition of 100 nM of siRNA against BMP2 (siBMP2) or Smad1 (siSmad1), the addition of 300 ng/ml of Noggin or 10 μM Y-27632 Rho-kinase associated (ROCK) inhibitor. Larger colonies D>400 μm differentiate in 100 nM of siRNA against GDF3 (siGDF3). However, Y-27632 and 100 nM siSmad1 have little effect on larger colonies. (C) pSmad1 levels decrease in the presence of siBMP2, noggin, siSmad1 and Y-27632 in small colonies, whereas pSmad1 levels increase in larger colonies in the presence of siGDF3. Interestingly, no effect of pSmad1 is observed when siSmad1 and Y-27632 are added to larger colonies. (D) Protein loss of TSmad1 by siSmad1 was detected by quantified fluorescence microscopy while ELISA assays were used to confirm loss of BMP2 protein by siBMP2 in 200 μm colonies and loss of GDF3 protein by siGDF3 in larger colonies.

The composition of the hESC microenvironment modulates Smad1signaling. (A) Images of hESCs at low and high cell density within a single well showing inverse correlations between the number of Oct-4+ cells in the hESC microenvironment and pSmad1. Cells are stained for Hoechst, Oct-4, pSmad1. Scale bar is 50 μm. (B) Quantification of the effect of local cell density can be visualized macroscopically by superimposing the single cell local cell density metric (Bii) and pSmad1 levels (Biii) onto spatial maps. Blue indicates low values and red indicates high values. Scale bar is 408 μm. (C) PSmad1 levels in the HNF3β+and Oct-4+ subpopulations (Ci). PSmad1 levels of the Oct-4+ subpopulation as a function of the localized Oct-4+ cell density (Cii). PSmad1 levels of the Oct-4+ subpopulation as a function of the localized HNF3β density (Ciii). PSmad1 levels in the Oct-4+ subpopulation with fixed HNF3β localized cell density and increasing Oct-4+ localized cell density. Data are normalized to the pSmad1 levels of the lowest localized cell density (Civ). (D) PSmad1 levels in hESCs after being pulsed for 90 min with unconditioned media (UCM), media conditioned by either H9 or I6 hESCs (hESC-CM), media conditioned by H9- or I6-derived extra-embryonic endoderm (ExE-CM), or 25 ng/ml of BMP2. (E) ELISA data for GDF3 and BMP2 present in hESC-CM and ExE-CM.

Micropatterning can be used to manipulate the hESC microenvironment and control hESC fate. To demonstrate that hESC microenvironment directly controls hESC fate, H9 hESCs were plated on patterned tissue-culture substrates with Matrigel using microcontact printing. Colony diameters (D) ranged from 200–800 μm and distance between colonies, pitch (P), was fixed at 500 or 1000 μm. (A) Quantitative fluorescent microscopy of patterned H9 cultures in XV media after withdrawal of all growth factors for 48 h and stained for Hoechst, Oct-4 and pSmad1. Scale bar is 200 μm. (B) Analysis of micropatterned colonies after 48 h. The percentage of Oct-4 cells as a function of colony size (Bi). PSmad1 in the Oct-4+ population as a function of colony size (Bii). Gene expression for Oct-4, Sox2, Nanog normalized to β-actin as a function of colony size (Biii). (C) To elucidate the molecular mechanism for the changes in pSmad1 levels, gene expression was quantified as a function of colony size for Smad1 agonist, BMP2, and antagonists, Follistatin, GDF3 and LeftyB and I-Smads, Smad6 and Smad7. Gene expression after normalization to β-actin and relative to the 200 μm data set (Ci). Ratios of the transcripts of Smad1 antagonists, Follistatin, GDF3 and LeftyB, to the transcripts of Smad1 agonist, BMP2, as a function of colony size (Cii). GDF3 gene expression as a function of colony size normalized to the percentage of Oct-4+ cells present (Ciii). For (Ci), statistical comparisons were made between gene expression for each cytokine at 400 and 800 μm colonies against the expression at 200 μm colonies. For (Cii), statistical comparisons were made between the ratio of the levels of transcripts of the antagonist to BMP2 between the 400 and 800 μm colonies against the ratio present in the 200 μm.

Two niche determinants—composition and size—regulate hESC self-renewal by local modulation of pSmad1 agonists and antagonists. (A) pSmad1 levels in hESCs are a balance of agonist(s) secreted by differentiated progeny including HNF3β+ extra-embryonic endoderm and antagonists secreted by hESCs (Oct-4+). The levels of pSmad1 of a single hESC will be dependent on the localized cell density of the Oct-4+ and HNF3β+ neighbors around it. (B) By manipulating colony size through micropatterning, the levels of Smad1 antagonists, GDF3 and LeftyB, increase while BMP2 remains constant resulting in pSmad1 levels that decrease with colony size. This decrease in pSmad1 levels can be corelated with the increased pluripotency seen with increasing colony size.