Naive Pluripotent Stem Cell Conversion via Torin1 and Low Oxygen
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Solution Overview
Problem
Current methods have not successfully derived and maintained naive human pluripotent stem cells capable of generating a substantial amount of mature human cells in non-human animals, as they typically remain in a primed state of pluripotency, limiting their ability to contribute to all three germ layers in chimeric organisms.
Innovation Solution
Exposing human iPSCs or ESCs to Torin1, in the presence of MEK and GSK inhibitors, leukemia inhibitory factor, and insulin, under low glucose and reduced oxygen conditions, to convert them into naive pluripotent stem cells, which can then be implanted into non-human animals to form chimeras and generate human cells of all three germ layers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If human iPSCs or ESCs are cultured under conventional conditions, then they maintain a primed state of pluripotency, but they cannot generate a substantial amount of mature human cells in non-human animals
Solution Approach 1:
The patent applies parameter changes by modifying culture conditions including adding Torin1 (an mTOR inhibitor), using low glucose medium (5% glucose), reducing oxygen concentration (5% O2), and adding specific inhibitors (MEK inhibitor, GSK3 inhibitor) along with LIF and insulin. These parameter changes convert human PSCs from a primed state to a naive state, enabling them to generate mature human cells in non-human animals while maintaining pluripotency
2Manufacturing precision
If human PSCs are exposed to Torin1 for a short period, then they convert to naive pluripotent state, but requires specific culture conditions and time
Solution Approach 1:
The patent applies preliminary action by exposing human PSCs to Torin1 for a short period (1-24 hours) before the main culture phase. This preliminary exposure initiates the conversion to naive state, after which cells are continued in culture without Torin1 but with MEK inhibitor, GSK3 inhibitor, LIF, and insulin. This two-stage approach simplifies the overall process by separating the conversion step from the maintenance step
3Productivity
If human PSCs are implanted in blastocyst of non-human animal, then they can generate substantial amounts of human cells, but requires naive pluripotent state
Solution Approach 1:
The patent applies parameter changes by establishing specific culture conditions (Torin1 exposure, low glucose 5% medium, 5% oxygen, MEK inhibitor, GSK3 inhibitor, LIF, insulin) that convert and maintain human PSCs in a naive pluripotent state. This naive state is essential for the cells to be implanted in blastocysts and generate substantial amounts of human cells of all three germ layers with high reliability
Data Source
AI summary
Provided herein are compositions and methods for generation of naive human pluripotent stem cells. The method comprises incubation of iPSCs under 5% O2 in a medium comprising 5% glucose, an MEK inhibitor, a GSK3β inhibitor, human leukemia inhibitory factor (LIF), human insulin and Torin 1. The method does not need any other inhibitors or transgene expression. The naive human pluripotent cells can be used to generate a large amount of mature human cells from all three germ layers in host non-human animals.