MIR302CHG RNA Detection for Residual Pluripotent Stem Cells
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Solution Overview
Problem
Existing methods for detecting residual undifferentiated pluripotent stem cells, such as iPS cells, in cell populations derived from nephron progenitor cells are inadequate due to issues like inefficient culture amplification and cross-reactivity with other cell types, making it difficult to ensure the safety of cell preparations for regenerative medicine.
Innovation Solution
The method involves measuring the level of RNA expressed from the MIR302CHG gene, specifically using splice variants ENST00000509938.1 and/or ENST00000505215.1, through quantitative PCR or 2-step ddPCR, with specific oligonucleotide primers and probes to detect and enrich undifferentiated pluripotent stem cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If iPS cell culture amplification method is used for detection, then iPS cells can be detected, but nephron progenitor cells are also highly efficiently cultured and amplified making selective detection impossible
Solution Approach 1:
The invention changes the detection parameter from general cell culture amplification to specific RNA expression level measurement of MIR302CHG. By measuring the RNA expression level rather than relying on cell culture amplification, the method achieves specific detection of iPS cells without being affected by the high amplification efficiency of nephron progenitor cells.
2Measurement precision
If LIN28A detection by PCR is used, then residual iPS cells can be detected, but LIN28A is expressed in nephron progenitor cells, pancreatic and liver organoids making detection unreliable
Solution Approach 1:
The invention applies local quality by targeting a specific RNA molecule (MIR302CHG) that has differential expression characteristics in specific cell types. The method uses cell-type-specific RNA expression patterns to achieve accurate detection, avoiding the cross-reactivity problem of LIN28A which is broadly expressed across multiple cell types including nephron progenitor cells, pancreatic and liver organoids.
3Quantity of substance
If general cell population analysis is performed, then cell preparation can be assessed, but it is very difficult to detect and evaluate a few residual iPS cells against a large amount of cell preparations
Solution Approach 1:
The invention replaces mechanical/cellular analysis methods with molecular RNA measurement. Instead of attempting to physically detect and evaluate individual residual iPS cells within a large cell population through mechanical means, the method substitutes this with RNA expression level measurement of MIR302CHG, which provides sensitive detection of residual iPS cells even when they are present in very small numbers within a large cell preparation.
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 approach allows for the reliable detection and enrichment of residual undifferentiated pluripotent stem cells, ensuring the safety of cell preparations for regenerative medicine applications, including nephron progenitor cells and other differentiated cell types.
Implementation Method 1
the step of determining amplifiability by quantitative PCR
Implementation Method 2
a step of determining amplifiability by 2-step ddPCR (Droplet digital PCR)
Data Source
AI summary
An object of the present invention is to provide a method and a reagent for detecting an undifferentiated pluripotent stem cell(s). The object is achieved by being able to detect a few residual undifferentiated pluripotent stem cells through measurement of the level of RNA expressed from the MIR302CHG gene.