A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Summary

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Abstract

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.

In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.

The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

Introduction

During the last decades, the widely accepted concept of unidirectional progression towards cell commitment and differentiation was completely revised. It has been demonstrated that cell specification can be reversed, and a terminally differentiated cell can be pushed towards a less committed and higher permissive state, using different methods.

Among the several methods proposed, one of the most promising method involves the use of chemical compounds to induce cells into a so called chemically induced pluripotency. The small molecules used in this approach are able to interact and modify the epigenetic signature of an adult mature cell, avo....

Protocol

All studies were reviewed and approved by the Ethical Committee of the University of Milan. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH). Human cells isolation from healthy adult individuals was approved by the Ethical Committee of the Ospedale Maggiore Policlinico, Milano. All the methods in our study were carried out in accordance with the approved guidelines.

1. Skin fibro.......

Discussion

During the last decades, several studies focused on the development of strategies to revert a terminally differentiated cell towards a less committed and higher permissive state. The protocol here described allow the generation and long-term maintenance of pluripotent cells starting from adult mature terminally differentiated cells. The method combines two independent steps that involve the induction of a high permissive state which is achieved through chemical epigenetic erasing and its subsequent maintenance ensured us.......

Materials

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Sigma-Aldrich	M7522	Component of ESC medium
5-Azacytidine	Sigma-Aldrich	A2385	5-aza-CR, for fibroblast epigenetic erasing
Adenosine	Sigma-Aldrich	A4036	Component of nucleoside mix for ESC medium
Antibiotic Antimycotic Solution (100×)	Sigma-Aldrich	A5955	Component of fibroblast and ESC media
CFX96 Real-Time PCR	Bio-Rad Laboratories	NA	Thermal cycler for quantitative PCR
Cytidine	Sigma-Aldrich	C4654	Component of nucleoside mix for ESC medium
DMEM, high glucose, pyruvate	Thermo Fisher Scientific	41966052	For fibroblast isolation and culture medium
DMEM, low glucose, pyruvate	Thermo Fisher Scientific	31885023	For ESC medium
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	D5652	PBS; for biopsy and cell wash and for solution preparation
Dynabeads mRNA DIRECT Micro Purification Kit	Thermo Fisher Scientific	61021	mRNA estraction
ESGRO Recombinant Mouse LIF Protein	Sigma-Aldrich	ESG1106	Component of ESC medium
Fetal Bovine Serum, qualified, heat inactivated	Thermo Fisher Scientific	10500064	Component of fibroblast and ESC media
FGF-Basic (AA10-155) Recombinant Human Protein	Thermo Fisher Scientific	PHG0024	Component of ESC medium
Gelatin from porcine skin	Sigma-Aldrich	G1890	For dish coating
GeneAmp PCR System 2700	Applied Biosystems	NA	Thermal cycler for qualitative PCR
Global DNA Methylation ELISA Kit	CELL BIOLABS	STA-380	Methylation study
GoTaq G2 Flexi DNA Polymerase	Promega	M7801	Qualitative PCR
Guanosine	Sigma-Aldrich	G6264	Component of nucleoside mix for ESC medium
Ham's F-10 Nutrient Mix	Thermo Fisher Scientific	31550031	For ESC medium
KnockOut Serum Replacement	Thermo Fisher Scientific	10828028	Component of ESC medium
KOVA glasstic slide 10 with grids	Hycor Biomedical	87144	For cell counting
Leica MZ APO Stereo Microscope	Leica	NA	For organoid observation
L-Glutamine solution	Sigma-Aldrich	G7513	Component of fibroblast and ESC media
MEM Non-Essential Amino Acids Solution (100X)	Thermo Fisher Scientific	11140035	Component of ESC medium
Millex-GS 0.22 µm pore filters	Millipore	SLGS033SB	For solution sterilization
M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant	Promega	M3681	mRNA reverse transcription
Multiskan FC Microplate Photometer	Thermo Fisher Scientific	51119000	For ELISA plate reading
Nikon Eclipse TE300 Inverted Phase Contrast Microscope	Nikon	NA	For cell observation
Perkin Elmer Thermal Cycler 480	Perkin Elmer	NA	Thermal cycler for reverse transcription
Poly(tetrafluoroethylene) 1 μm particle size	Sigma-Aldrich	430935	For generating micro-bioreactor
PureLink Genomic DNA Mini Kit	Thermo Fisher Scientific	K182001	Genomic DNA estraction
TaqMan Gene Expression Cells-to-CT Kit	Thermo Fisher Scientific	AM1728	Quantitative PCR
Thymidine	Sigma-Aldrich	T1895	Component of nucleoside mix for ESC medium
Tissue Culture Dish 100X20 mm, Standard	Sarstedt	833902	For fibroblast isolation
Tissue Culture Dish 35X10 mm, Standard	Sarstedt	833900	For Fibroblast isolation
Tissue Culture Dish 35X10 mm, Suspension	Sarstedt	833900500	Bacteriology petri dish for liquid marble culture
Tissue Culture Plate 96 Well,Standard,F	Sarstedt	833924005	For liquid marble culture
Trypsin-EDTA solution	Sigma-Aldrich	T3924	For fibroblast dissociation
Tube 15ml, 120x17mm, PS	Sarstedt	62553041	For cell suspension centrifugation
Uridine	Sigma-Aldrich	U3003	Component of nucleoside mix for ESC medium