Somatic Cell Reprogramming With Cytoplasm-Free GV Extract

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Solution Overview

Problem

Existing methods for somatic cell reprogramming to pluripotent stem cells are slow, yield is low, and involve undesirable DNA replication and cell division, limiting their effectiveness.

Innovation Solution

Using germinal vesicle (GV) extracts from meiotic oocytes without oocyte cytoplasm for somatic cell nuclear reprogramming, optionally with additional factors to enhance efficiency, and resealing somatic cell membranes post-incubation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If Yamanaka factors are overexpressed in MEFs to generate iPSCs, then pluripotent cells can be derived, but the process is slow (1-3 weeks) and yield is small

Engineering Contradiction:

Improvereprogramming efficiencyVSAvoidreprogramming time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent extracts the essential reprogramming factors from oocyte cytoplasm and concentrates them into GV extract, removing unnecessary components. This extraction allows the reprogramming process to be accelerated while maintaining efficiency, resolving the contradiction between reliability and time loss by isolating only the critical reprogramming agents.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the concentration and composition parameters of reprogramming factors by using GV extract at optimized concentrations (e.g., 1-10 μg/μL). This parameter optimization enables rapid reprogramming within 24 hours while maintaining high efficiency, directly addressing the time-loss issue without sacrificing reprogramming reliability.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If metaphase eggs are used for nuclear transfer, then reprogramming can occur, but DNA replication and cell division continue during the process

Engineering Contradiction:

Improvereprogramming completenessVSAvoidundesirable DNA replication and cell division

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent extracts reprogramming factors specifically from the germinal vesicle (GV) of meiotic oocytes, separating these factors from the rest of the oocyte cytoplasm. This extraction eliminates DNA replication and cell division activities while retaining the essential reprogramming components, thus removing harmful factors while preserving reprogramming completeness.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The GV extract serves as an intermediary that transfers reprogramming factors to somatic cells without requiring the presence of living oocytes. This intermediary approach allows reprogramming to occur in vitro without triggering DNA replication or cell division, resolving the contradiction between reprogramming completeness and harmful cellular activities.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If oocyte cytoplasm is used in reprogramming, then reprogramming factors are present, but undesirable factors may interfere with downstream applications

Engineering Contradiction:

Improvereprogramming capabilityVSAvoidundesirable oocyte cytoplasm factors

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent performs a selective extraction of reprogramming factors from the germinal vesicle, separating them from the surrounding oocyte cytoplasm. This extraction process isolates beneficial reprogramming factors while leaving behind undesirable cytoplasmic components, thus maintaining reprogramming capability while eliminating harmful factors that could interfere with downstream applications.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent segments the oocyte into its germinal vesicle component and cytoplasmic component, utilizing only the GV for factor extraction. This segmentation allows the isolation of pure reprogramming factors without contamination from cytoplasmic factors, resolving the contradiction between maintaining reprogramming capability and removing harmful substances.

Inventive Principle:

Principle #1Segmentation

4Reliability

If additional factors such as protease inhibitors are added during incubation, then reprogramming efficiency may increase, but process complexity increases

Engineering Contradiction:

Improvereprogramming efficiencyVSAvoidincubation process complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The GV extract is prepared and optimized to contain all necessary reprogramming factors in appropriate concentrations and formulations. This self-service approach eliminates the need for additional supplements like protease inhibitors during the incubation process, maintaining high reprogramming efficiency while simplifying the overall protocol and reducing process complexity.

Inventive Principle:

Principle #25Self-service

Data Source

PatentUS20250333693A1Cell type conversion

Publication Date: 2025.10.30 CAMBRIDGE ENTERPRISE LTD

AI summary

This disclosure relates to a method of somatic cell nuclear reprogramming to alter the cell type comprising preparing a GV extract, permeabilising somatic cells, incubating the somatic cells with the GV extract to alter the cell type, and rescaling somatic cell membranes, wherein said GV extract does not comprise oocyte cytoplasm. The disclosure also relates to a GV extract comprising reprogramming factors.