A Chemically Defined Feeder-free System for the Establishment and Maintenance of the Human Naive Pluripotent State

Abstract

SummaryThe distinct states of pluripotency in the pre- and post-implantation embryo can be captured in vitro as naive and primed pluripotent stem cell cultures, respectively. The study and application of the naive state remains hampered, particularly in humans, partially due to current culture protocols relying on extraneous undefined factors such as feeders. Here we performed a small-molecule screen to identify compounds that facilitate chemically defined establishment and maintenance of human feeder-independent naive embryonic (FINE) stem cells. The expression profile in genic and repetitive elements of FINE cells resembles the 8-cell-to-morula stage in vivo, and only differs from feeder-dependent naive cells in genes involved in cell-cell/cell-matrix interactions. FINE cells offer several technical advantages, such as increased amenability to transfection and a longer period of genomic stability, compared with feeder-dependent cells. Thus, FINE cells will serve as an accessible and useful system for scientific and translational applications of naïve pluripotent stem cells.

Large-scale analysis of imprinting in naive human pluripotent stem cells reveals recurrent aberrations and a potential link to FGF signaling

“…RNA-seq samples of hPSCs are often obtained from cells grown on mouse embryonic fibroblasts as feeders. This is especially true for naive cell cultures, which in most protocols rely on feeders ( Szczerbinska et al., 2019 ). The presence of RNA fragments that originate from mouse cells were shown to be a source for the potential detection of false-positive SNVs if they are mistakenly aligned to the human genome ( Avior et al., 2021 ; Stirparo et al., 2021 ).…”

“…To this end, we compared conversion protocols which had at least five naive samples as representatives. We also combined samples cultured in two different medium conditions (4i/L/A + FINE; Szczerbinska et al., 2019 ; Theunissen et al., 2016 ) that do not contain GSK3i, which was shown to reduce methylation in imprinted loci in mice ( Popkie et al., 2010 ). This comparison showed a significant association between naive conversion protocol and LOI extents and divided the protocols into three distinct groups, which we denote as having high, medium, and low LOI levels ( Figure 4 A).…”

Temporal activation of LRH‐1 and RAR‐γ in human pluripotent stem cells induces a functional naïve‐like state

“…The main motivation for development of an efficient protocol to establish human naïve pluripotency is the production of cells that have a high growth rate, are resistant to single‐cell dissociation, and show the capability to differentiate into three germ layers while maintaining genome integrity. Various approaches have been tried, including forced expression of naïve‐related transcription factors (Li et al , ; Buecker et al , ; Hanna et al , ; Takashima et al , ; Chen et al , ), manipulation of different signaling pathways with small molecules (Chan et al , ; Ware et al , ; Duggal et al , ; Qin et al , ), or targeting of numerous protein kinases such as PKC, p38, JNK, BRAF, SRC, CDK, and ROCK (Gafni et al , ; Theunissen et al , ; Guo et al , , ; Zimmerlin et al , ; Szczerbinska et al , ) to induce the naïve pluripotent state in human cells. These different culture conditions induce different levels of naivety in PSCs.…”

Pluripotent stem cells for the study of early human embryology

“…In contrast, activated LTR7Y elements are specifically observed in pre‐implantation blastocysts (Goke et al., 2015). Recently, the promoter activity of LTR7Y element was used as a specific reporter for naïve PSCs (Szczerbinska et al., 2019). The expression of LTR7 elements is induced by the binding of transcription factors that are highly expressed in naïve human PSCs, such as OCT3/4, NANOG, KLF4, and TFCP2L1 (Fort et al., 2014; Lu et al., 2014; Wang et al., 2014).…”

“…Although genetic reporters allow us to perform cost‐effective and large‐scale screening (Szczerbinska et al., 2019; Theunissen et al., 2014), genetic manipulation is required. In contrast, cell surface markers are useful for isolating live cells by flow cytometry when combined with antibodies and fluorescent dyes.…”