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The ability to differentiate into almost all tissue types is the hallmark of human pluripotent stem cells (hPSCs). However, as we study the biology of hPSCs for future clinical applications – whether they be under the influence of growth factors ( 1 – 4 ), pro-survival cocktails ( 5 , 6 ), genetic modification ( 7 –10), or other manipulations (1) – pluripotency testing remains a fundamental component of every research design. Assays generally used to test such “stemness” include genomic profiling for relative quantification of pluripotency genes, immunocytochemistry to detect pluripotency markers, embryoid body formation to test 3-germ-layer differentiation capability in vitro or in vivo , and teratoma formation to test 3-germ-layer differentiation capability in vivo (Table 1). Notwithstanding the in vitro assays, teratoma formation in vivo is considered the most stringent of pluripotency assays because it provides more reliable and comprehensive confirmation than does testing cells on a simplified, artificial petri dish. This in vivo assay, coupled with noninvasive, longitudinal imaging, have proven to be invaluable not only in the visualization of stem cell survival and migration post-delivery, but also have been crucial in studying the safety and viability of future stem cell applications (11–13).