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, avoiding the need of any transgenic and/or viral vector^{1,2,3,4,5,6,7,8,9,10}. Numerous studies have recently shown that it is possible to switch cells from one phenotype to another by providing specific biochemical and biological stimuli that induce the reactivation of hypermethylated genes^{11,12,13,14,15}. These demethylating events allow for the conversion of terminally differentiated cells into a primitive progenitor, a multipotent or a high plasticity/pluripotent cell^{1,2,3,4,5,6,7,8,9,10}.

In parallel, many studies have been recently focussing on the understanding of mechanosensing-related cues and, more specifically, on the possibility to use mechanical forces to directly influence cell plasticity and/or differentiation^16,17,18,19. Indeed, it has been clearly demonstrated that the extracellular matrix (ECM) plays a key role in the control of cell fate. In particular, the biomechanical and biophysical signals produced by ECM directly regulate molecular mechanisms and signaling pathways, influencing cell behavior and functions^20,21. These recent data have paved the way to the development of novel 3D culture systems that more closely mimic the in vivo cell microenvironment, replicating mechanical and physical stimuli driving cell behaviour.

We here describe a two-step protocol that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. In the first step, cells are incubated with the demethylating molecule 5-aza-cytidine (5-aza-CR). This agent is able to induce a significant global DNA demethylation through a combined effect of the direct ten-eleven translocation 2 (TET2)-mediated action^8,10 and the indirect inhibition of the DNA methyltransferases (DNMT)^22,23. This step induces the removal of the epigenetic blocks with a subsequent re-activation of pluripotency-related gene expression and, therefore, the generation of high plasticity cells^1,2,3,8,10, hereinafter referred as “epigenetically erased cells”. In the second step, cells are encapsulated in a 3D culture system. To this end, the non-reactive hydrophobic synthetic compound polytetrafluoroethylene (PTFE; with particle size of 1 μm) is used as micro-bioreactor, that permits the creation of a cellular microenvironment unachievable through the use of traditional 2D culture systems¹⁰. The PTFE powder particles adhere to the surface of the liquid drop in which cells are re-suspended and isolate the liquid core from the supporting surface, while allowing gas exchange between the interior liquid and the surrounding environment²⁴. The “PTFE micro-bioreactor” thus obtained, also known as “Liquid Marble”, encourages cells to freely interact with each other, promoting 3D cell rearrangement^25,26,27, and extends and stably maintains the acquired high plasticity state though bio-mechanosensing-related cues¹⁰.

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 fibroblast isolation

NOTE: All the procedures described below can be applied to fibroblasts isolated from different mammalian species, including mouse, porcine, and human. Murine cells were isolated from 7-week-old male mice and porcine skin tissue were collected at local slaughterhouse. Human cells were isolated from adult patients, after written informed consent.

1. Prepare 0.1% porcine gelatin solution.
   1. Weigh 0.1 g of porcine gelatin and dissolve it in 100 mL of water. Sterilize gelatin solution by autoclaving before use.
   2. Coat 35 mm Petri dish with 0.1% porcine gelatin by adding 1.5 mL of the prepared solution. Incubate for 2 h at room temperature.
2. Cut mammalian (mouse, porcine, and human) skin biopsies of approximately 2-5 cm in length and place them in Dulbecco's Phosphate Buffered Saline (PBS) containing 2% antibiotic antimycotic solution. Store at + 4 °C until use.
   NOTE: Biopsy collections must be carried out in agreement and after the Ethical Committee’s approval, in accordance with the established guidelines.
3. Extensively wash the collected biopsies three times in fresh sterile PBS containing 2% antibiotic antimycotic solution.
4. Collect biopsies from the last wash and place them into a sterile 100 mm Petri dish. Use a sterile scalpel to cut them into pieces of approximately 2 mm³ size.
5. At the end of 2 h incubation, remove the excess of gelatin solution from the 35 mm Petri dish (described in step 1.1.2) and, using a sterile surgical tweezer, immediately place 5-6 skin fragments into each pre-coated culture dish.
6. Wet the fragments by adding 100 µL droplets of fibroblast isolation medium (Table 1) over each of them. Culture at 37 °C in 5% CO₂ incubator.
   NOTE: To prevent the medium from evaporation, place the 35 mm Petri dish within a 100 mm or bigger Petri dish containing sterile water. Ensure to cap both Petri dishes.
7. After 24 h of culture, check the quantity of the medium in the 35 mm culture Petri dish. If needed, add 500 µL of fibroblast isolation medium to keep wet the fragments.
8. Carefully remove the medium and refresh it at least every 2 days of culture using a pipette.
9. When fibroblasts start to grow out of the skin fragments placed in the 35 mm Petri dish (described in step 1.5.) and begin to form a cell monolayer (usually 6 days). Remove skin pieces using a sterile surgical tweezer and culture in 2 mL of fibroblast isolation medium.
10. Continue to culture the cell monolayer at 37 °C in 5% CO₂ incubator until 80% confluence and refresh medium every other day.

2. Fibroblast primary cell line culture

1. When fibroblasts reach 80% confluence, carefully remove fibroblast isolation medium and wash cells three times with 3 mL of PBS containing 1% antibiotic antimycotic solution.
2. For cell detaching, add 600 µL of 0.25% trypsin-EDTA solution in the culture dish and incubate at 37 °C for 3-5 min.
3. Add 5.4 mL of fibroblast culture medium to neutralize trypsin when cells start to detach from the culture dish (Table 1).
4. Dislodge cells by repeated and gentle pipetting. Plate cells in new culture dishes (without gelatin), keeping the passage ratio between 1:2 and 1:4 (depending on growth rate).
   NOTE: Centrifugation is not necessary.
5. Maintain cells in culture and change medium every 2 days, until they have reached 80% confluency and passage them.
   NOTE: Propagate fibroblasts twice a week to maintain vigorous growth.

3. Fibroblast exposure to 5-aza-CR

1. Prepare fresh 1 mM 5-aza-CR stock solution.
   1. Weigh 2.44 mg of 5-aza-CR and dissolve it in 10 mL of DMEM high glucose. Resuspend the powder by vortexing. Sterilize the solution with 0.22 µm filter.
      NOTE: 5-aza-CR stock solution must be prepared immediately before use.
   2. Prepare 5-aza-CR working solution by diluting 1 µL of 5-aza-CR stock solution (3.1.1.) in 1 mL of fibroblast culture medium.
      NOTE: The concentration of 5-aza-CR working solution is 1 μM^1,2,3,8,9.
2. Trypsinize cells as previously described (2.1.-2.3.) and dislodge cells by repeatedly and gently pipetting.
3. Collect the cell suspension and transfer it into a conical tube.
4. Count cells using a counting chamber under an optical microscope at room temperature. Calculate the volume of medium needed to re-suspend cells to obtain 4 x 10⁴ cells in 30 μL of fibroblast culture medium supplemented with 1 μM 5-aza-CR (see step 3.1.2.).
   NOTE: The formula to be used depends on the specific type of chamber.
   
5. Centrifuge the cell suspension at 150 x g for 5 min at room temperature. Remove the supernatant and resuspend pellet with the fibroblast culture medium supplemented with 1 μM 5-aza-CR (see step 3.1.2.). For the volume of the fibroblast culture medium to be used see step 3.4.
   NOTE: As a negative control, resuspend cells ad the same concentration in fibroblast culture medium without 5-aza-CR and proceed with cell encapsulation in PTFE powder (step 4.1.-4.13.).

4. Fibroblast encapsulation in PTFE micro-bioreactors

1. Fill a 35 mm Petri dish with polytetrafluoroethylene (PTFE) powder to produce a bed (Figure 1A).
   NOTE: Use 35 mm bacteriology Petri dishes to avoid liquid marble adhesion. In order to obtain a thin hydrophobic and porous shell, use a PTFE powder with an average particle size of 1 μm and produced with a maximum grind of 2.0 NPIRI. This allows for the creation of gas-permeable liquid marbles. Furthermore, the translucent coating facilitates the observation of cell aggregation processes in real-time Larger particle size leads to high polydispersity that can cause elevated evaporation, deformity and loss of the spherical shape, and the premature dissolution of the micro-bioreactors.
2. Dispense 30 μL single droplet containing 4 x 10⁴ cells (see steps 3.4.- 3.5.) onto the powder bed (Figure 1B).
3. Gently rotate the 35 mm Petri dish in a circular motion to ensure that PFTE powder entirely cover the surface of the liquid drop to form a liquid marble micro-bioreactor (Figure 1C).
4. Pick up the liquid marble micro-bioreactor using a 1,000 μL pipette tip, cut at the edge, to accommodate the diameter of the marble (Figure 1D,E). Plate the liquid marble micro-bioreactor onto a clean bacteriology Petri dish to stabilize it (Figure 1F).
   NOTE: To create a friction to grip the marble inside the tip, cut the pipette tips with a diameter approximately slightly less than the liquid marble diameter.
5. Transfer the liquid marble micro-bioreactor from the Petri dish into a 96 well plate (one marble/well) (Figure 1G).
6. Slowly add 100 μL of media from the margin of the well. The micro-bioreactor starts to float on top of the media (Figure 1H).
   NOTE: The micro-bioreactor breaks in direct liquid contact, due to the disruption of PTFE hydrophobicity. As an alternative approach, the liquid marble micro-bioreactors can be individually placed in a 35 mm bacteriology culture dish. In this case, in order to prevent liquid marble evaporation, the 35 mm Petri dish containing the micro-bioreactor must be inserted in a 100 mm Petri dish, previously aliquoted with sterile water
7. Incubate liquid marble micro-bioreactor for 18 h at 37 °C in 5% CO₂ incubator^1,2,3,8,9.
   NOTE: The PTFE particle size of 1 μm can ensure an optimal gas exchange between the interior liquid and the surrounding environment.
8. After 5-aza-CR incubation for 18 h, collect the liquid marble micro-bioreactor using a 1,000 μL pipette tip cut at the edge (see step 4.5).
9. Place the micro-bioreactor in a new 35 mm bacteriology Petri dish (Figure 1D-F).
10. Use a needle to puncture the liquid marble and break it.
11. Recover formed spheroids with a 200 μL pipette tip, cut at the edge, under a stereomicroscope (Figure 1I,J).
    NOTE: Epigenetically erased cells encapsulated in PTFE form a 3D spherical structure (one aggregate in each liquid marble).
12. To assess the acquisition of pluripotent state in response to 5-aza-CR, check the onset of the pluripotency- related gene expression, OCT4, NANOG, REX1, and SOX2, by qualitative PCR (Table 2).
13. Proceed with the second step of the protocol as described below.

5. Culture in PTFE micro-bioreactors of epigenetically erased cells

1. Prepare fresh ESC culture medium (Table 1).
2. Transfer organoids in a Petri dish containing ESC medium for washing 5-aza-CR residuals (see steps 5.1.-5.2.).
3. Prepare a new 35 mm bacteriology Petri dish containing PTFE powder bed (see also step 4.1.).
4. Dispense a single organoid in a droplet of 30 μL ESC culture medium onto the powder bed using a 200 μL pipette tip, cut at the edge (see steps 4.9.; 5.3.).
5. Gently rotate the 35 mm Petri dish in a circular motion to form a new liquid marble micro-bioreactor, pick up it using a 1,000 μL pipette tip, cut at the edge, and place the newly formed micro-bioreactor into a well of 96-well plate (one marble/well) (see steps 4.3.-4.6.).
6. To float the micro-bioreactors, add 100 μL of media from the margin of the well to slowly bathe the marble (see note 4.7.).
7. Culture liquid marble micro-bioreactors at 37 °C in 5% CO₂ incubator for as long as required. Change medium every other day, following the procedure described in 5.3.-5.7.
   NOTE: In the present manuscript, results obtained with organoids culture for 28 days are provided. However, if needed longer culture period can be performed.

Results

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...

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

List of materials used in this article

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