Analysis of Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells as a Means to Assess Pluripotency

Abstract

Human induced pluripotent stem cells (hiPSCs) have core properties of unlimited self-renewal and differentiation potential and have emerged as exciting cell sources for applications in regenerative medicine, drug discovery, understanding of development, and disease etiology. Key among numerous criteria to assess pluripotency includes the in vivo teratoma assay that has been widely proposed as a standard functional assay to demonstrate the pluripotency of hiPSCs. Yet, the lack of reliability across methodologies, lack of definitive clinical significance, and associated expenses bring into question use of the teratoma assay as the “gold standard” for determining pluripotency. We propose use of the in vitro embryoid body (EB) assay as an important alternative to the teratoma assay. This paper summarizes the methodologies for creating EBs from hiPSCs and the subsequent analyses to assess pluripotency and proposes its use as a cost-effective, controlled, and reproducible approach that can easily be adopted to determine pluripotency of generated hiPSCs.

Inhibition of glycogen synthase kinase-3 (GSK3) promotes the neural differentiation of full-term amniotic fluid-derived stem cells towards neural progenitor cells

“…Moreover, these cells acquired immunoreactivity for the neuron-specific marker class III ␤-tubulin (Tuj1), which indicated the post-mitotic neurons. Additionally, they also acquired immunoreactivity for GFAP, which is expressed in glia cells of the CNS, such as astrocytes and ependymal cells (Domowicz et al, 2012;Sheridan et al, 2012). Interestingly, the differentiated hAFS cells acquired immunoreactivity for the neural precursor cell markers NESTIN, post-mitotic neuron marker class III ␤-tubulin and glial marker GFAP at the same time.…”

Comparative analysis of ABCG2+ and ABCG2-retinoblastoma cells in three-dimensional culture

“…We examined our ABCG2+ and ABCG2-enriched Rb cells in order to determine their degree of developmental potency. We used a novel in vitro approach by adapting what would normally have been embryoid body assay [22,23] used for pluripotent iPSCs and fashioned it into an in vitro tumor assay. We chose this method to examine Rb cells in threedimension but without confounding host factors of animal xenografts or human tumors.…”

Dataset in support of the generation of Niemann-Pick disease Type C1 patient-specific iPS cell lines carrying the novel NPC1 mutation c.1180T>C or the prevalent c.3182T>C mutation – Analysis of pluripotency and neuronal differentiation

“…Teratoma formation assay in immune deprived mice was not performed any more, following the 3Rs principle, to replace, refine or reduce, the use of animals, mandated by the German Protection of Animal Act. In regards of the teratoma formation assay in stem cell research the applicability of the in vitro embryoid body formation assay is discussed by Sheridan and colleagues [4].…”