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Novel method for isolation and cultivation of ground state cells from heterogenous human pluripotent stem cell cultures

Reference Number TO 03-00400

Challenge

Pluripotent stem cells (PSCs) show different stages of pluripotency during development. Human ESC and iPSC lines generated routinely are generally heterogeneous and are mainly in the primed state, containing naive cell clusters (Figure). Primed PSCs show already a certain bias regarding lineage-specific differentiation. Naïve PSCs, by contrast, can differentiate into the three embryonic germ layers without bias. They may hold a great potential for therapeutic applications in regenerative medicine including gene therapy applications. Owing to their heterogeneity and low single-cell clonogenicity, stable long-term cultivation of human PSCs is still challenging. Only few culture media are described so far, that claim reproducible conversion and stable, long-term maintenance of ground state human PSCs, each coming with certain limitations. Accordingly, novel media and culturing methods are highly sought-after.

GFP-tagged ground state hESCs in a culture of hESCs
GFP-tagged ground state hESCs in a culture of hESCs

Technology

A novel medium and methods for isolation and stable long-term culturing of ground state human PSCs is provided. In contrast to media and methods described so far, this culturing method is highly reproducible. The cells can be passaged stably (over 100 times), and they form typical naïve-like, dome-shaped cell colonies. Importantly, there is evidence that the cells are close to the human inner cell mass (ICM), but not to the morula. The cells do not differentiate spontaneously, but have the unbiased potential to be differentiated to the three germ layers and can be also differentiated to the primordial germ cells. The medium is for xeno-free and feeder-free conditions, without bFGF/TGFbeta/Activin A and has only very few key components. It was developed by identification and isolation of ground state human PSCs, tagged with a LTR7/HERVH reporter, since elevated HERV transcription is a hallmark of ground-state human ESCs. Unlike naïve-like cells described so far, the cells isolated by this method are not artificially modified or reprogrammed, since these cells are naturally present in a low abundance within cultures of both human ESCs and induced pluripotent stem cells (hiPSCs). A small-molecule screen and subsequent optimization of the medium identified the key compositions enabling the long-term preservation of naïve-like features. Since the reporter can also be used transiently, this new method and medium allows to derive standardized, naïve-like hPSCs of robust differential potential including stable long-time cultivation without permanent genomic modification.

Commercial Opportunity

Available for licensing or co-development

 

 

 

 

Patent Situation

EP and US patent applications with priority date from October 7, 2014

EP3204490

US2017/0313978

Further Reading

"Isolation and cultivation of naive-like human pluripotent stem cells based on HERVH expression": Nature Protocol (2016); doi: 10.1038/nprot.2016.016.

"Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells": Nature (2014); doi:10.1038/nature13804.

"Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates": Nat Genet. 2009, 41(6), 753-61.