Resetting Human Pluripotent Stem Cells with Defined Inhibitor Media
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Solution Overview
Problem
Current methods have not successfully generated stable, reproducible, and homogeneous human pluripotent stem cells in a 'naïve' or 'ground state' with the transcriptional and epigenetic characteristics of mouse embryonic stem cells, relying on growth factors and lacking evidence for rewiring of transcriptional control circuitry.
Innovation Solution
A method involving the use of reprogramming factors and inhibitors to reset human stem cells to a naïve state, utilizing a first medium with MEK, tankyrase, and STAT3 activators, and a second medium with MEK, PKC, and GSK3 inhibitors to sustain this state, inducing expression of key transcription factors like ESRRB.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If human PSCs are cultured in conventional conditions with growth factors, then they maintain pluripotency, but they do not achieve stable ground state with rewired transcriptional control circuitry
Solution Approach 1:
The patent applies parameter changes by systematically modifying culture conditions including inhibition of FGF signaling, TGFβ signaling, and GSK3 activity, combined with specific small molecule inhibitors to transition human PSCs from conventional pluripotent state to ground state, achieving stable transcriptional reprogramming without complex formulations
Solution Approach 2:
The patent extracts and removes the dependency on growth factors and serum replacement factors from the culture system by using defined small molecule inhibitors that block specific signaling pathways, thereby simplifying the culture formulation while maintaining ground state stability
2Adaptability or versatility
If human PSCs are converted to ground state using complex culture formulations, then they show altered characteristics, but they remain dependent on FGF, TGFβ and serum replacement factors
Solution Approach 1:
The patent extracts the dependency on growth factors by using small molecule inhibitors to block signaling pathways, thereby achieving ground state characteristics without requiring FGF, TGFβ, or serum replacement factors in the culture medium
Solution Approach 2:
The patent enables human PSCs to self-maintain ground state characteristics through endogenous transcriptional circuitry reprogramming, making them independent of external growth factor support and capable of self-renewal in defined inhibitor-containing medium
3Productivity
If human PSCs are dissociated for passage, then they can be expanded, but cloning efficiency is low
Solution Approach 1:
The patent applies parameter changes by modifying the cellular state through signaling pathway inhibition, which alters cell adhesion and proliferation characteristics to improve both expansion capability and cloning efficiency simultaneously in dissociated cell passages
Data Source
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AI summary
The invention provides methods and materials for resetting or sustaining a human stem cell in a "naïve" or "ground" state, based on the use of media including combinations of inhibitors. An example naïve culture medium comprises a PKC inhibitor, a MEK inhibitor. Also provided are methods of obtaining or propagating such cells, cells obtained using these methods, and novel culture media, which can be used in these methods.