Recommendation 2 .1 .1 .a: For studies using cell lines where pluripotency has been established as described above and reported in peer-reviewed publications, it may not be necessary to repeat multi-lineage differentiation assays . Minimally, however, the undifferentiated status of the cells should be monitored by quantitative marker analysis (see Table A4 .1).

Recommendation 2 .1 .1 .b: For large scale studies describing the derivation of extensive panels of new pluripotent cell lines by well-established techniques, where in depth characterization of all lines may not be possible, a subset of the lines should be confirmed to be pluripotent by differentiation assays . For the remaining lines, the undifferentiated status of the cells should be monitored by quantitative marker analysis; these lines should then be designated putative pluripotent lines.

Recommendation 2 .1 .1 .c: Where novel reprogramming techniques, cell culture methodologies, or other non-established protocols are used, confirmation of the undifferentiated status and developmental potential should be comprehensive . These include evaluation of larger panels of markers of the undifferentiated state and the capacity for differentiation into progenitors of the three embryonic germ layers, and of more differentiated cells, by multiparametric analysis.

Recommendation 2 .1 .2: Xenograft (teratoma) assays are not required to indicate pluripotency.

Although xenografting of pluripotent stem cells into immunocompromised animals provides a strong test of pluripotency and has been widely used in the past, concerns for animal welfare and increasing regulation in different jurisdictions make this assay undesirable if equivalent information can be derived from in vitro assays. The xenograft assay can provide evidence of the ability of differentiated cells to undergo histogenesis to yield complex tissues. However, several studies have confirmed that adequate evidence for pluripotency can be obtained from in vitro differentiation (Allison et al., 2018; Bock et al., 2011). Organoid or 3D assays in vitro may yield information on capacity for morphogenesis and histogenesis. Thus, xenograft assays are not recommended as a routine method for assessing pluripotency, although they may be a useful adjunct for the assessment of potential malignancy. If xenograft assays are used, criteria for assessment of teratomas and teratocarcinomas are described in Appendix 4.

The Undifferentiated State

Recommendation 2 .2 .1: The expression of recognized cell surface markers and transcripts can be used to assess and monitor the undifferentiated status of a cell line . However, the expression of such markers does not demonstrate pluripotency.

None of the markers present on undifferentiated cells are uniquely expressed in these cells, although they are often incorrectly used to identify pluripotent stem cells. Equally, there are many examples of cells that have little or no capacity for differentiation, notably nullipotent embryonal carcinoma cells from germ cell tumors, but that express many of these markers of undifferentiated cells, including OCT4 (POU5F1) and NANOG (see Table A4.1, for a list of markers). Therefore, these markers should not be called pluripotency markers as pluripotency cannot be defined by marker expression from undifferentiated cells. Nevertheless, such markers and gene expression profiles are useful to indicate undifferentiated status and as surrogate measures for retention of identity in cell types whose pluripotency has been well-established by prior differentiation assays, e.g., conventional hESCs or hiPSCs. They may also be used to screen putative pluripotent cells created by well-established methods for producing pluripotent cells. The absence of certain key markers, e.g., OCT4, can be strong indicators of loss of pluripotency, but these data cannot be considered definitive.

Developmental State

Recommendation 2 .3 .1: Evidence that a stem cell culture represents a particular developmental state should be based on relatedness to a stage and region of embryo development as assessed by specific gene expression and as far as possible by global profiling . Developmental state should be corroborated by demonstration of appropriate lineage-specific differentiation, including potential lineage biases . Profiling comparisons need to clarify the relationship to other reported stem cell states, and information should be provided on differentiation or interconversion between states . The culture conditions for generating and propagating the specific stem cell state should be reported in full, together with information on stability (or transience), homogeneity/heterogeneity and clonogenicity.

Stem cells in culture are expected to correspond to staging points along the developmental trajectory in the embryo between zygote and late gastrulation. Such correspondence means high global similarity in transcriptome and epigenome together with relatedness in features such as cell morphology, metabolic parameters, and differentiation competence.

Epiblast cells in the preimplantation human embryo and corresponding pluripotent stem cell lines are designated naïve. Naïve pluripotency is considered a discrete state. It is succeeded by an intermediate or formative stage of pluripotency that has shed naïve characteristics but not gained lineage specification. The formative phase appears to last several days in humans, and it is probable that a series of formative sub-states may be captured in culture. Formative pluripotency is a continuum with regionally specified and fated epiblast at the time of gastrulation, termed primed. Studies with mouse epiblast stem cells (EpiSCs) demonstrate that primed pluripotent stem cells can reside in different sub-states, related to different regions of gastrulation stage epiblast.

While cells in culture cannot be completely identical to cells in vivo, it should be considered that adaptations to the culture environment may induce features and identities that do not exist in the embryo. Stem cell properties that are discordant with those known for cells in the embryo should be declared. These aberrant pluripotent states may be of interest for certain applications, but caution is required in interpretations related to normal development.

Recommendation 2 .3 .1 .a: Heterogeneity within the culture should be quantitatively addressed at the single cell level, by flow cytometry, high content screening, live cell imaging, and/or single cell -omics . Composition of the cultures should be assessed at multiple time points . Ideally, sub-populations should be characterized for inter- conversion and for clonogenic potential.

Pluripotent stem cell cultures are often mixed in composition. Such heterogeneity may arise in several ways including: continuous differentiation of a fraction of cells; maintenance of different pluripotent states in the same culture conditions; hierarchical pluripotency progression with more primitive naïve or formative stage cells giving rise to later stages; interconversions between pluripotent sub-states reflecting inherent plasticity but not necessarily relevant to developmental events; and spread of genetic or epigenetic changes that influence cell identity.