Placenta Derived Adherent Cell (PDAC) Interaction and Response on Extracellular Matrix Isolated from Human Placenta
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Cell binding studies. After seeding for 3 hours, similar levels of PDAC attachment were observed on all pECMs (numbers 1–5); the levels of PDAC binding to pECMs were slightly less than that observed on purified collagen. Immunostaining of PDACs for fibronectin at this time revealed abundant intracellular staining, with no detectable extracellular fibronectin (data not shown). As shown in Figure 2, by hour 48 of culture, PDACs increased in number and adopted similar well-spread morphologies on purified collagen,pECM-2,and pECM-4. In contrast, PDACs did not thrive on pECM-1 or pECM-3.Not only were fewer cells observed, but also their morphologies were rounded and not well spread. Pluripotent progenitor cells on pECM-5 appeared more elongated and polarized than PDACs on other pECMs and collagen. Immunostaining for fibronectin at the 48-hour time point revealed an extensive network of extracellular fibronectin matrix fibrils on pECMs numbers 1–4.These fibronectin matrix fibrils were assembled by PDACs; the control pECM-only samples (ie, samples in which PDACs were not cultured on pECM) did not show evidence of fibronectin fibrils. It is also known from the literature that fibronectin deposition into the matrix is a celldependent process and does not occur spontaneously.9 In contrast to pECMs numbers 1–4, pECM-5 and collagen did not support fibronectin matrix assembly by PDACs and no extracellular fibrillar fibronectin was detected on these surfaces.
Cytokine array studies. The secretion of key cytokines/chemokines from PDACs resulting from the binding and proliferation to the pECM was investigated. Cytokine secretion on pECM was compared to that from PDACs incubated on tissue culture treated cell culture plates using a 25-multiplex cytokine array, which includes several interleukins and cytokines (Table 2). 
Of the 25 cytokines studied, increased secretion of 3 cytokines when on the pECM sheets (compared to tissue culture treated plates or TCP) was observed. These include IL-6, IL-8,and monocyte chemoattractant protein- 1 (MCP-1). Figure 3 (A-C), shows a time-dependent increase in cytokine secretion (IL-6, IL-8, and MCP-1) by PDACs on the 5 pECM constructs; data are normalized to cell numbers present on pECM sheets. 
Interestingly, pECM-5 was determined to be an anomaly in that there was no increase in MCP-1 secretion. As previously shown, pECM-5 did not support the expression of fibronectin, quite unlike pECM-1 through pECM-4.Taken together, these data suggest a possible change in cellular behavior or signaling when situated on this particular pECM. It is interesting to note that pECM-5 was the only matrix generated without the use of NaOH and had a biochemical composition that maintained the 2 key cell adhesion proteins—fibronectin and laminin.
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