BDL-E5 Fluorescent Probe for Pluripotent Stem Cell Detection

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Solution Overview

Problem

Current methods for detecting and isolating pluripotent stem cells, particularly at early reprogramming stages, are inefficient and lack specificity, relying on cumbersome genetic engineering techniques and non-specific markers that are costly and not widely applicable.

Innovation Solution

Development of a fluorescent compound, BDL-E5, which specifically binds to pluripotent stem cells and cells undergoing reprogramming, allowing for early detection and enrichment through fluorescence-activated cell sorting without the need for genetic modification or extensive washing steps.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If fluorescent dye conjugated antibodies or fluorescent substrates are used to detect pluripotent stem cells, then detection is possible, but the markers are not specific to bona fide pluripotent stem cells and are detectable in adult stem cells

Engineering Contradiction:

Improvedetection specificityVSAvoidmarker applicability

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The invention changes the detection parameter from protein-based markers (antibodies, substrates) to small molecule fluorescent probes that detect endogenous metabolites. This parameter change enables specific detection of pluripotent stem cells through their unique metabolic profile without cross-reactivity with adult stem cells

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention substitutes the mechanical/chemical binding mechanism of antibody-antigen interaction with a small molecule fluorescent probe that detects endogenous metabolites. This substitution eliminates the need for large protein markers and provides more specific detection of pluripotent stem cells

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Measurement precision

If gene reporters such as fluorescent proteins driven under OCT4 or NANOG are used, then specific detection of pluripotent stem cells is achieved, but the constructs need to be inserted by viral or gene editing methods which are cumbersome and potentially disruptive

Engineering Contradiction:

Improvedetection specificityVSAvoidgenetic engineering complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The invention extracts the detection function from the cell's genetic machinery by using exogenously added small molecule fluorescent probes that detect endogenous metabolites. This eliminates the need for viral transduction or gene editing to insert fluorescent protein reporters

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention introduces an intermediary small molecule fluorescent probe that binds to endogenous metabolites in pluripotent stem cells. This intermediary approach allows detection without direct genetic modification of the target cells

Inventive Principle:

Principle #24Intermediary (Mediator)

3Measurement precision

If conventional fluorescent markers are used, then detection of well-developed colonies is possible, but early reprogramming cells cannot be detected or sorted

Engineering Contradiction:

Improvedetection timingVSAvoidreprogramming efficiency

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The invention enables preliminary detection of reprogramming cells at early stages before colony formation. The small molecule fluorescent probes detect metabolic changes that occur early in reprogramming, allowing identification and sorting of cells before they develop into visible colonies

Inventive Principle:

Principle #10Preliminary action

4Measurement precision

If fluorescent probes for pluripotent stem cells are manufactured, then detection is enabled, but the manufacturing cost is relatively expensive which hinders clinical and commercial development

Engineering Contradiction:

Improvedetection capabilityVSAvoidmanufacturing cost

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

The invention uses small molecule fluorescent probes that are chemically synthesized and relatively inexpensive to manufacture compared to antibody-based probes or genetically encoded fluorescent proteins. These small molecules can be produced through standard chemical synthesis routes, reducing manufacturing costs

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

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

BDL-E5 enables efficient and specific identification and isolation of pluripotent stem cells at early stages of reprogramming, improving the efficiency and cost-effectiveness of stem cell reprogramming protocols by providing a high signal-to-background fluorescence ratio.

Implementation Method 1

fluorescent compound of formula (I)... a fluorescent compound, BDL-E5, which specifically binds to pluripotent stem cells and cells undergoing reprogramming, allowing for early detection and enrichment through fluorescence-activated cell sorting

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS11634437B2Fluorescent compounds specific for pluripotent stem cells and reprogramming-ready cells and methods of using the same

Publication Date: 2023.04.25 AGENCY FOR SCI TECH & RES

AI summary

The present invention provides fluorescent compounds of formula (I) as disclosed herein or pharmaceutically acceptable salts thereof. Further provided are uses of said fluorescent compounds in methods of determining, in a sample, the presence and/or amount of pluripotent stem cells or cells undergoing reprogramming to become induced pluripotent stem cells.