Pluripotent Stem Cell Differentiation to Germ Cells

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Solution Overview

Problem

Current methods for inducing primordial germ cell-like cells (PGC-like cells) from pluripotent stem cells are inefficient and lack reproducibility, often resulting in random differentiation and failure to contribute to healthy offspring.

Innovation Solution

A method involving the induction of epiblast-like cells (EpiLCs) from pluripotent stem cells using activin A, BMP4, and other growth factors under serum- and feeder-free conditions, which allows for the precise differentiation into Blimp1- and stella-positive PGC-like cells capable of contributing to spermatogenesis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If random differentiation of ESCs as embryoid bodies under undefined conditions is used, then PGC-like cells can be obtained, but the efficiency is low and reproducibility is poor

Engineering Contradiction:

Improveefficiency of PGC-like cell inductionVSAvoidreproducibility of differentiation

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The invention first induces epiblast-like cells (EpiLCs) from pluripotent stem cells under defined conditions before inducing PGC-like cells. This preliminary step establishes a controlled intermediate state that improves both efficiency and reproducibility of the overall differentiation process

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention changes culture parameters from undefined conditions to defined conditions, specifically using serum-free medium with controlled growth factors (BMP4, activin A, bFGF) and cytokines. This parameter control enables reproducible induction of EpiLCs and subsequent PGC-like cells

Inventive Principle:

Principle #35Parameter changes

2Productivity

If serum-containing medium is used for culture, then cell growth is supported, but differentiation into PGC-like cells is inefficient

Engineering Contradiction:

Improvedifferentiation efficiency into PGC-like cellsVSAvoidculture condition definition

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The invention transitions from serum-containing medium to serum-free medium with defined growth factors. This parameter change enables efficient PGC-like cell differentiation while maintaining cell viability through controlled supplementation with BMP4, activin A, bFGF, and other cytokines

Inventive Principle:

Principle #35Parameter changes

3Reliability

If feeder cells are used for culture, then cell survival is improved, but differentiation control is reduced

Engineering Contradiction:

Improvecell survival rateVSAvoiddifferentiation control

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The invention extracts and removes feeder cells from the culture system, replacing them with defined growth factors and cytokines in serum-free medium. This extraction enables direct control over differentiation while maintaining cell survival through soluble factors rather than cell-cell interactions

Inventive Principle:

Principle #2Taking out (Extraction)

4Ease of operation

If undefined culture conditions are used, then spontaneous PGC formation occurs, but the process is random and inefficient

Engineering Contradiction:

Improvespontaneous differentiation capabilityVSAvoidPGC-like cell induction efficiency

Core Design Contradiction:

Ease of operationVSProductivity

Solution Approach 1:

The invention changes from undefined spontaneous conditions to defined conditions with specific growth factors (BMP4, activin A, bFGF) and cytokines. This defines the differentiation pathway while maintaining the spontaneous nature of the process, significantly improving efficiency

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention introduces a preliminary differentiation step to EpiLCs under defined conditions before PGC-like cell induction. This preliminary action creates an optimal intermediate state that enhances the efficiency of subsequent PGC formation

Inventive Principle:

Principle #10Preliminary action

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

The method achieves efficient and reproducible induction of functional PGC-like cells that can contribute to the generation of healthy offspring, providing a critical step in reconstructing germ cell development in vitro and advancing reproductive technology and medicine.

Implementation Method 1

culturing the pluripotent stem cell in the presence of activin A

Methodology Applied

Scientific EffectSignal transduction:

Implementation Method 2

culturing the EpiLC obtained in the step (i) in the presence of BMP4

Methodology Applied

Scientific EffectSignal transduction:

Data Source

PatentUS9938496B2Method of inducing differentiation from pluripotent stem cells to germ cells

Publication Date: 2018.04.10 KYOTO UNIV

AI summary

This invention provides a method of producing an epiblast-like cell (EpiLC) from a pluripotent stem cell, which comprises culturing the pluripotent stem cell in the presence of activin A; a method of producing a primordial germ cell-like (PGC-like) cell a pluripotent stem cell, which comprises culturing the EpiLC obtained by the method above in the presence of BMP4 and LIF. Also provided are a cell population containing PGC-like cells as obtained by the method, and reagent kits for the EpiLC- and PGC-like cell-induction from a pluripotent stem cell.