Naïve Pluripotent Stem Cell Conversion Using Let-7 miRNA and BMP
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Solution Overview
Problem
Current methods for producing naive pluripotent stem cells from primed cells are inefficient and require lengthy subculturing procedures, often involving gene introduction and low molecular compounds like valproic acid, which pose risks of genomic mutation.
Innovation Solution
A method involving culturing primed pluripotent stem cells in a specific medium, followed by introduction of miRNA from the let-7 family or their similar sequences, or their activity inhibitors, along with bone morphogenetic protein, to promote conversion to naive pluripotent stem cells without the need for HDAC inhibitors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chemical resetting methods using low molecular compounds like valproic acid are used, then the risk of genomic mutation is reduced compared to gene introduction methods, but the production efficiency remains poor and requires long periods for subculturing procedures
Solution Approach 1:
The invention changes the chemical parameters of the culture medium by incorporating specific compounds (SB-431542 as TGF-β inhibitor, CHIR99021 as Wnt pathway activator, and bFGF) to optimize the conversion process. This parameter optimization enables efficient naive cell production while maintaining safety, resolving the contradiction between reliability and productivity.
Solution Approach 2:
The invention uses specific chemical compounds as intermediaries to mediate the conversion from primed to naive state. These compounds (SB-431542, CHIR99021, bFGF) act as mediators that facilitate the transition without requiring gene introduction, thus maintaining safety while improving production efficiency through optimized chemical intervention.
2Productivity
If gene introduction methods are used to convert primed state to naive state, then conversion can be achieved, but the risk of genomic mutation increases
Solution Approach 1:
The invention replaces the mechanical/genetic system (gene introduction) with a chemical system (culture medium optimization using specific compounds). This substitution eliminates the risk of genomic mutation associated with gene introduction while maintaining the ability to achieve conversion from primed to naive state through chemical mediation.
Solution Approach 2:
The invention uses chemical compounds as intermediaries to mediate the conversion process, replacing direct genetic manipulation. The compounds SB-431542, CHIR99021, and bFGF serve as safe intermediaries that achieve the desired conversion without integrating into the genome, thus resolving the contradiction between conversion capability and genomic safety.
3Reliability
If conventional chemical resetting methods are used, then gene introduction is avoided, but the production time is extended and efficiency is poor
Solution Approach 1:
The invention optimizes the temporal parameters of the culture process by using specific compound concentrations and timing (SB-431542, CHIR99021, bFGF combinations). This parameter optimization accelerates the conversion process, reducing the time loss associated with subculturing while maintaining the safety advantage of avoiding gene introduction.
Solution Approach 2:
The invention maintains continuous useful action by optimizing the culture conditions to sustain efficient conversion throughout the process. The combination of SB-431542, CHIR99021, and bFGF creates a continuous favorable environment for naive cell production, minimizing idle time and reducing the need for extensive subculturing procedures.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method significantly enhances the efficiency of producing naive pluripotent stem cells, achieving up to 80% conversion in a relatively short time, while avoiding genetic manipulation and reducing the risk of genomic mutations.
Implementation Method 1
introduction of miRNA selected from among miRNA belonging to the let-7 miRNA family and their similar sequences into the cell to be cultured in step (1) or (2), or expressing the miRNA in the cell
Implementation Method 2
introducing bone morphogenetic protein into the cell to be cultured in step (1) or (2) and expressing the bone morphogenetic protein in the cell, or adding the bone morphogenetic protein to the medium of step (1) or (2)
Implementation Method 3
Methods have also been developed recently for converting primed pluripotent stem cells from the primed state to the naive state by addition of specified low molecular compounds (see NPLs 2 and 3, for example). Methods involving addition of low molecular compounds are known as 'chemical resetting methods'
Data Source
AI summary
Naïve pluripotent stem cells are produced at high efficiency. A method for producing naïve pluripotent stem cells, wherein the method includes the following steps: (1) a step that cultures primed pluripotent stem cells in primed pluripotent stem cell culture medium; and (2) a step that cultures the cells obtained in step (1) in naïve pluripotent stem cell culture medium, including at least one of the following steps: (i) a step that introduces miRNA selected from miRNA belonging to the let-7 miRNA family and similar sequences thereto into the cells cultured in steps (1) or (2) or causes said miRNA to be expressed in said cells; or, a step that introduces an activity inhibitor of a let-7 miRNA family target protein into the cells cultured in steps (1) or (2) or causes said activity inhibitor to be expressed in said cells; and (ii) a step that introduces a bone morphogenetic protein into the cells cultured in steps (1) or (2), causes said bone morphogenetic protein to be expressed in said cells, or adds said bone morphogenetic protein to the medium of (1) or (2); or a step that adds a bone morphogenetic protein activator to the medium of steps (1) or (2).