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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.
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.
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....
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.......
The present protocol describes all the steps to be performed to generate and stably maintain mammalian pluripotent cells from adult somatic cells. This method has been successful with fibroblasts isolated from different mammalian species, namely mouse, porcine and human. The representative results here reported are obtained from all cell lines, irrespectively of the species of origin.
Morphological analyses show that, after 18 h incubation with the demethylating agent 5-aza-CR, fibroblasts enc.......
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.......
This work was funded by Carraresi Foundation and MiND FoodS Hub ID: 1176436. All the authors are members of the COST Action CA16119 In vitro 3-D total cell guidance and fitness (CellFit).
....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 |
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