0 50 100 150 200 250 300 Nuclei number/field 0 20 40 60 80 Percent multinucleation 0 1 2 3 4 5 6 MediumBr-cAMP Percent TUNEL positive Fresh cells Frozen.

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Nuclei number/field Percent multinucleation MediumBr-cAMP Percent TUNEL positive Fresh cells Frozen cells Fig 1s The effect of cryopreservation on the number of viable CT in culture. Viability is based on the number of cells that adhere in 5 days of culture with and without Br-cAMP, the number that undergo multinucleation (differentiation) in 5 days in medium alone and with Br-cAMP and the number of cells undergoing apoptosis after 5 days with and without Br-cAMP. The open histograms are the performance of fresh Cells, directly after isolation and before cryopreservation, and the filled histograms are those cryopreserved For 1 year and then thawed. Procedures are detailed in the Methods. The histograms are the mean ± SD of data from 4 replicate wells per group. Results are from a single experiment.

Multinucleated nuclei (Percent) MediumEGF Br-cAMP - zVAD + zVAD Fig 2s Syncytium formation is greater with Br-cAMP stimulation than with EGF and neither is inhibited by zVAD-fmk. EGF was applied at 10 ng/ml and Br-cAMP at 10  g/ml for 6 days, with the medium changed every other day at 6% oxygen. The percent multinucleation was determined as described in the methods. Shown are triplicate experiments carried out at different times with cells isolated from a single placenta. Depicted are the means +/- SD.

Figure 3s:Effect of caspase inhibitors on TNF  –stimulated villous CT apoptosis. Inhibitors were added after a period of 4 hrs for plating cells, then 15 min later, TNF  was added. zVAD and zIETD were added at 20  M and qVD at 10  M. The cells were incubated for 18 hrs and TUNEL was carried out as described in the Methods. Depicted are the means and SD of three experiments with cells isolated from different placentas. Different letters by the bars (a,b,c) indicate statistically different groups (ANOVA analysis, p<0.05) MediumTNFTNF+ zVAD TNF+ zIETD TNF+ qVD Apoptosis (percent TUNEL positive) a b c a a

* ** IETD concentration (uM) Percent Fig 4s: Relationship of apoptosis (lower curve, diamonds) and cell fusion (upper curve, squares) to zIETD-fmk concentration. Depicted is the mean and SD of three separate experiments each having triple replicates. * marks significant difference (Students T test, relative to 0 concentration zIETD-fmk) with p< 0.05 and ** marks a p<0.01. Fig 4s

Medium Br-cAMP EGF Placental CT preparation hCG  secretion (mIU/ml) Br-cAMP/ Medium EGF/ Medium Secretion hCG  normalized to Medium Normalizing differing hCG  secretion from different trophoblast preparations by dividing secretion by the medium control. Panel A: hCG  secretion from three different preparations (1-3) treated by medium alone (open bars), Br-cAMP (grey bars) and EGF (black bars). Panel B: Normalizing secretion from each trophoblast preparation by dividing by secretion with medium alone in that preparation (the dashed line at a ratio of 1.0). * marks a significant reduction (p<0.05) compared to medium (Students paired T test). Fig 5s *

Fig 6s HeLaTrophoblasts 1hr2hr4hr1d2d4d Time with Br-cAMP Trophoblasts Med TNF  Actin floating 16 B Actin Caspase-8 Time in hrs with TNF  MWt C A GAPDH Caspase-8 Caspase-8 cleavage in CT cultured from 30 min to 16 hrs with TNFa (Panel A) and for up to four days with Br-cAMP (Panel C). Panel B shows the positive control (HeLa cells treated with cyclohexamide and TNFa for 4 hrs). The upper portion of each panel shows caspase-8 and the lower portion shows expression loading controls as noted, both run at the same time on a two color LiCOR imaging detector (see Methods for details). Arrows mark the 56 kDa proform and the p43/41 kDa, the p26 kDa and the p18 cleaved products. The blots in panel A includes cytotrophoblasts well advanced in apoptosis (floating cells at 16 hrs). All other cells were adherent. Depicted are one of two nearly identical data sets.