Sequential Media Differentiation of iPSCs into Haemogenic Endothelial Cells

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Solution Overview

Problem

Current adoptive T cell therapies are limited by a lack of suitable patient and tumor-specific T cells, necessitating the development of methods for producing therapeutically sufficient and functional antigen-specific T cells for effective immunotherapy.

Innovation Solution

A process for differentiating induced pluripotent stem cells (iPSCs) into haemogenic endothelial cells (HECs) using sequential mesoderm and haemogenic endothelium induction media, which allows for the production of T cells without the need for purification or isolation, and in the absence of feeder cells or serum, enabling the generation of clinical-grade blood cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional methods are used to produce T cells for immunotherapy, then the process requires purification or isolation steps and uses feeder cells or serum, but this limits the availability and scalability of antigen-specific T cells

Engineering Contradiction:

Improveproduction yield of HECsVSAvoidcomplexity of differentiation process

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The differentiation process is divided into distinct sequential stages with specific media formulations for each stage: mesoderm induction stage, haemogenic endothelium induction stage, and T cell differentiation stage. Each stage uses optimized growth factors and conditions to guide cells through specific developmental transitions, enabling systematic production of HECs without purification steps

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Defined culture media containing specific growth factors (BMP4, VEGF, bFGF, SCF, CHIR-99021, Activin A) serve as intermediaries to mediate the differentiation process. These media formulations replace the need for feeder cells or serum by providing precise biochemical signals that guide cell fate decisions through each differentiation stage

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If rapid and reproducible differentiation is achieved, then high yields of HECs are produced, but the process requires precise control of multiple signalling pathways

Engineering Contradiction:

Improvereproducibility of differentiationVSAvoidnumber of signalling pathways to control

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The differentiation protocol systematically modulates key signaling pathway parameters by adding or removing specific growth factors at defined time points. The media formulations control SMAD1/5/9, VEGFR, cKIT, and GSK3β pathway activities through controlled addition of BMP4, VEGF, bFGF, SCF, and CHIR-99021, enabling reproducible differentiation while managing pathway complexity through parameter optimization

Inventive Principle:

Principle #35Parameter changes

3Productivity

If purification or isolation steps are eliminated, then the process is simplified and faster, but cell purity may be compromised

Engineering Contradiction:

Improvespeed of productionVSAvoidpurity of HECs

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The differentiation system is designed to self-organize into the desired cell type through intrinsic developmental programming guided by extrinsic signaling cues. The sequential media formulations exploit the natural differentiation hierarchy of stem cells, allowing HECs to self-differentiate without external purification or isolation interventions, maintaining both speed and purity

Inventive Principle:

Principle #25Self-service

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 provides a rapid, reproducible, and high-yield production of HECs, which can be differentiated into T cells, enhancing the availability of antigen-specific T cells for immunotherapy and expanding the patient population and range of tumor types that can be treated.

Implementation Method 1

The first mesoderm induction medium stimulates SMAD1, SMAD5 and SMAD9 mediated signalling pathways

Methodology Applied

Scientific EffectSMAD1, SMAD5 and SMAD9 mediated signalling pathways:

Implementation Method 2

inhibits glycogen synthase kinase 3β

Methodology Applied

Scientific EffectGlycogen synthase kinase 3β inhibition:

Implementation Method 3

The first HE induction medium (i) stimulates VEGFR mediated signalling pathways

Methodology Applied

Scientific EffectVEGFR mediated signalling pathways:

Implementation Method 4

The second HE induction medium (i) stimulates cKIT receptor (CD117; KIT receptor tyrosine kinase) mediated signalling pathways

Methodology Applied

Scientific EffectcKIT receptor (CD117; KIT receptor tyrosine kinase) mediated signalling pathways:

Data Source

PatentUS20240124841A1Methods of producing haemogenic endothelial cells from pluripotent stem cells

Publication Date: 2024.04.18 ADAPTIMMUNE

AI summary

This invention relates to the production of haemogenic endothelial cells (HECs). A population of induced pluripotent stem cells (iPSCs) is differentiated into mesoderm cells by culturing the IPSCs sequentially in first, second and third mesoderm induction media to induce differentiation into mesoderm cells. The mesoderm cells are then differentiated into HECs by culturing the mesoderm cells sequentially in first and second haemogenic endothelium (HE) induction media to induce differentiation into HECs. The HECs may be further differentiated into hematopoietic progenitor cells (HPCs) and progenitor T cells.