Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Summary

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Abstract

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Introduction

A fundamental objective of regenerative medicine is the generation of new, functional cells that can be used to repair or replace damaged, degenerated tissues. Remaking easily available adult cells into new ones, by converting them from one cell type to another, is a particularly appealing approach, especially when the required cell population is not abundant or difficult to access. However, adult cells are remarkably stable. They acquire their differentiated state through a gradual restriction in their options and, once they reach the mature terminal specialization, they stably retain it ¹.

In the last years a number of protocols have been developed, that enable the reprogramming to pluripotency of a somatic cell (iPS) achieved through the forced expression of a set of transcription factors ^2,3. Alternatively, cell conversion can be obtained by direct lineage transdifferentiation, introducing a single ⁴ or a combination of transcription factors ^5-7. This strategy does not involve the transition through a de-differentiated state but requires high expression of the specific transcription factors ⁸.

We have recently developed a conversion protocol based on the brief exposure of adult cells to the demethylating properties of the cytidine analog 5-azacytidine (5-aza-CR), a well-characterized DNA methyltransferase inhibitor. The demethylation step is immediately followed by a specific differentiation protocol ^9-11 that allows to obtain the required terminal phenotype. This method is able to convert mature, differentiated cells into cells of a different lineage and has the substantial advantage to avoid both the use of viral vectors and the transfection of any exogenous transcription factors. The acquisition of a stable pluripotent state, and the related increased susceptibility to cell instability is also avoided.

The detailed protocol that allows the conversion of adult human skin fibroblasts into fully functional insulin-secreting cells is presented here. However, it is worth noting that the technique has been applied to different cell types and has generated positive results, when addressing cells towards various differentiation pathways. Furthermore, epigenetic conversion has been successfully used in the human and porcine species ^9-13 as well as in the dog (manuscript submitted) suggesting a wide efficacy and robustness of the approach.

Protocol

Note: All the procedures described below must be performed under laminar flow hood in sterile conditions. Make sure that all culture procedures are carried out on thermostatically controlled stages and cells are maintained at 37 °C throughout their handling.

1. Skin Fibroblast Isolation

1. Prepare Culture Dish Coating Solution
   1. Dissolve 0.1 g of porcine gelatin in 100 ml of water (final concentration 0.1%). Sterilize solution with autoclave.
   2. Add 1.5 ml of sterile 0.1% porcine gelatin to 35 mm Petri dishes. Wait 2 hr to coat, maintaining them at room temperature.
      Note: Human skin biopsies are collected by excision under local anesthesia from an avascular area of the anterior aspect of the forearm and stored in Dulbecco's Phosphate Buffered Saline (PBS) supplemented with 2% antibiotic antimycotic solution at +4 °C prior to use.
2. Wash biopsies with new PBS supplemented with 2% antibiotic antimycotic solution.
3. Place biopsies in a 100 mm Petri dish and cut into approximately 2 mm³ fragments with sterile scalpels.
4. Remove the coating solution excess immediately prior to plating fragments.
5. Place 5-6 skin fragments into the pre-coated 35 mm Petri dish.
6. Prepare fibroblast culture medium: 77% Dulbecco's Modified Eagle Medium (DMEM) high glucose, 20% Fetal Bovine Serum (FBS), 1% L-Glutamine solution and 2% antibiotic antimycotic solution.
7. Add a droplet of fibroblast medium over each fragment (usually 100 µl per fragment) and culture them at 37 °C in 5% CO₂.
8. After 24 hr, add 500 µl of fibroblast culture medium over the fragments to keep them wet at 37 °C in 5% CO₂.
9. Change the medium with a pipette at least once every 48 hr.
10. After 6 days of incubation, remove tissue fragments carefully and discard them.
    Note: After 6 days, fibroblasts start to grow out of the tissue fragments and begin to form a cell monolayer.
11. Refresh medium, add 2 ml of fibroblast culture medium and continue cell monolayer culture at 37 °C in 5% CO₂ incubator.

2. Fibroblast Culture

1. Culture fibroblasts at 37 °C in 5% CO₂ until 80% confluence
2. For passaging, aspirate fibroblast culture medium from tissue culture dishes. Wash cells three times with 4 ml of PBS supplemented with 1% antibiotic antimycotic solution.
3. Add a thin layer (10% of the culture medium volume) of trypsin-EDTA solution (0.5 g/L porcine trypsin and 0.2 g/L EDTA) and incubate at 37 °C until cell monolayer begins to detach from the bottom of the tissue culture dish and cells dissociate.
4. Dilute cell suspension with 9 parts of fibroblast culture medium to neutralize trypsin action. Centrifugation is not necessary.
5. Plate cells in new culture dishes (without gelatin) and culture at 37 °C in 5% CO₂ incubator. Keep the passage ratio between 1:2 and 1:4, depending on growth rate.
6. When cells reach around 80% confluence, passage them (usually twice per week).

3. Fibroblast Plating for Epigenetic Conversion

1. Add 0.26 ml/cm² of 0.1% porcine gelatin (prepare as described in 1.1) to cell culture dishes. Wait 2 hr to coat.
2. Remove the coating solution excess 10-30 min prior to plating fibroblasts.
3. Remove fibroblast culture medium from culture dishes. Wash cells three times with PBS supplemented with 1% antibiotic antimycotic solution.
4. Add a thin layer (10% of the culture medium volume) of trypsin-EDTA solution (0.5 g/L porcine trypsin and 0.2 g/L EDTA) and incubate at 37 °C until cell monolayer begins to detach from the bottom of the tissue culture dish and cells dissociate.
5. Dilute cell suspension with 9 parts of fibroblast culture medium to neutralize trypsin action.
6. Count cells using a counting chamber under a microscope at room temperature. Calculate the required volume of fibroblast culture medium to resuspend cells, to obtain a cell concentration of 7.8 x 10⁴ fibroblasts/cm². This will depend on the specific type of chamber used.
   Cells/µl = Average number of cell per small grid x 90 (multiplication factor) x dilution
7. Centrifuge cell suspension at 150 g for 5 min at room temperature. Remove supernatant and resuspend cells with the previously calculated volume of fibroblast culture medium.
8. Plate cells on 0.1% gelatin pre-coated dishes and culture them for 24 hr at 37 °C in 5% CO₂ incubator.

4. Increase Cell Plasticity Using the De-methylating Agent 5-aza-CR

1. Day 0
   1. Prepare 5-aza-CR stock solution by dissolving 2.44 mg of 5-aza-CR in 10 ml of DMEM high glucose medium. Sterilize by filtration. Prepare 5-aza-CR stock immediately prior to use.
   2. Dilute 1 µl of 5-aza-CR stock solution in 1 ml of fibroblast culture medium (final concentration 1 µM).
   3. To increase cell plasticity, 24 hr after cell plating (subheading 3.8), remove culture medium from seeded fibroblasts and add 1 µM 5-aza-CR stock solution and culture for 18 hr at 37 °C in 5% CO₂ incubator.
2. Day 1
   1. Prepare fresh Human Pluripotent (HP) medium as described in Table 1.
   2. After incubation with 1 µM 5-aza-CR, remove medium and wash cells three times with PBS to ensure that 5-aza-CR is rinsed away.
   3. Incubate 5-aza-CR treated fibroblasts with HP medium for 3 hr (recovery period) at 37 °C in 5% CO₂.
   4. After the recovery period, remove medium, wash three times with PBS.
   5. To monitor the efficiency of 5-aza-CR treatment, check in this step for the presence of morphological changes (detailed in Results section). Cells lose the typical elongated morphology of fibroblasts and acquire a round or oval shape, becoming smaller in size, with enlarged nuclei.
   6. Proceed with pancreatic differentiation.

5. Pancreatic Differentiation Protocol

1. Days 1-6
   1. Prepare Pancreatic Basal Medium as described in Table 2.
   2. Prepare activin A stock solution: dissolve 5 µg of activin A recombinant human protein in 166.6 µl of sterile water.
   3. Culture 5-aza-CR treated fibroblasts in 0.26 ml/cm² of Pancreatic Basal Medium, supplemented with 1 µl/ml activin A stock solution (see 5.1.2) for 6 days at 37 °C in 5% CO₂ incubator. Change medium daily.
2. Days 7-8
   1. Prepare retinoic acid stock solution by adding 16.6 ml of dimethyl sulfoxide (DMSO) to 50 mg of retinoic acid.
   2. Culture cells in 0.26 ml/cm² of Pancreatic Basal Medium supplemented with 1 µl/ml activin A stock solution (see 5.1.2) and 1 µl/ml retinoic acid stock solution (see 5.2.1) for 2 days at 37 °C in 5% CO₂. Change medium daily.
3. Days 9-36
   1. Culture cells in 0.26 ml/cm² of Pancreatic Basal Medium supplemented with 1% (v/v) Insulin-Transferrin-Selenium (ITS), 2% (v/v) B27 and 0.1% (v/v) Recombinant Human FGF basic (bFGF) stock solution (see Table 1) at 37 °C in 5% CO₂ incubator. Change the medium daily for the first 15 days.
   2. From day 16 onward, refresh medium every other day, under a microscope, since forming aggregate cells may detach from the bottom of the culture dish.

Results

Establishment of primary culture from skin biopsies
Skin biopsies were cut in small fragments and placed in gelatin pre-coated dishes. After 6 days, fibroblasts started to grow out of the tissue fragments and formed a cell monolayer (Figure 1A). Cells showed a typical elongated shape and, as expected, displayed a uniform immune-positivity for the fibroblast specific marker vimentin (Vim, Figure 1B).

Discussion

The present manuscript describes a method that allows the conversion of human skin fibroblasts into insulin-producing cells, through a transient and brief exposure to 5-aza-CR, followed by a tissue specific induction protocol. This approach allows a switch from mesoderm to endoderm related cells, without the forced expression of transcription factors or microRNAs nor the acquisition of a stable pluripotent state, that makes cells more unstable and prone to mistakes ¹⁴.

In the first ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Phosphate Buffered Saline	Sigma	D5652	PBS; for cell wash and solution preparation
Antibiotic Antimycotic Solution	Sigma	A5955	Component of Fibroblast, HP and Pancreatic media
100 mm Petri dish	Sarstedt	83.3902	For Fibroblast isolation
Porcine Gelatin	Sigma	G1890	For dish coating
Water	Sigma	W3500	For solution preparation
35 mm Petri dishes	Sarstedt	83.39	For Fibroblast isolation
DMEM, high glucose, pyruvate	Life Technologies	41966052	For Fibroblast culture medium
Fetal Bovine Serum	Life Technologies	10500064	FBS; Component of Fibroblast and HP media
L-Glutamine solution	Sigma	G7513	Component of Fibroblast, HP and Pancreatic media
Trypsin-EDTA solution	Sigma	T3924	For Fibroblast dissociation
KOVA GLASSTIC SLIDE 10 WITH GRIDS	Hycor Biomedical	87144	Cell counting
5-Azacytidine	Sigma	A2385	5-aza-CR, for increrase cell plasticity in fibroblasts
Ham's F-10 Nutrient Mix	Life Technologies	31550031	For HP medium
DMEM, low glucose, pyruvate	Life Technologies	31885023	For HP medium
KnockOut Serum Replacement	Life Technologies	10828028	Component of HP medium
MEM Non-Essential Amino Acids Solution	Life Technologies	11140035	Component of HP and Pancreatic Basal media
2-Mercaptoethanol	Sigma	M7522	Component of HP and Pancreatic Basal media
Guanosine	Sigma	G6264	Nucleoside mix stock component of HP medium
Adenosine	Sigma	A4036	Nucleoside mix stock component of HP medium
Cytidine	Sigma	C4654	Nucleoside mix stock component of HP medium
Uridine	Sigma	U3003	Nucleoside mix stock component of HP medium
Thymidine	Sigma	T1895	Nucleoside mix stock component of HP medium
Millex-GS 0,22 µm	Millipore	SLGS033SB	For sterilizing of solution
FGF-Basic (AA 1-155) Recombinant Human Protein	Life Technologies	PHG0261	bFGF; Component of HP and Pancreatic Basal medium
Bovine Serum Albumin	Sigma	A3311	BSA; Component of Pancreatic Basal medium
DMEM/F-12	Life Technologies	11320074	For Pancreatic Basal medium
B-27 Supplement Minus Vitamin A	Life Technologies	12587010	Component of Pancreatic medium
N-2 Supplement	Life Technologies	17502048	Component of Pancreatic Basal medium
Activin A Recombinant Human Protein	Life Technologies	PHG9014	For Pancreatic medium
Retinoic Acid	Sigma	R2625	For Pancreatic medium
Dimethyl sulfoxide	Sigma	D2650	DMSO; for Retinoic Acid stock preparation
Insulin-Transferrin-Selenium	Life Technologies	41400045	ITS; for Pancreatic Final medium
Anti-Vimentin antibody	Abcam	ab8069	For immunocytochemical analisys. Working dilution 1:100
4′,6-Diamidino-2-phenylindole dihydrochloride	Sigma	32670	DAPI. For immunocytochemical analisys. Working dilution  1 µg/ml
5-Methylcytidine	Eurogentec	MMS-900P-B	For immunocytochemical analisys. Working dilution 1:500
Anti-C Peptide antibody	Abcam	ab14181	For immunocytochemical analisys. Working dilution 1:100
Anti-PDX1 antibody	Abcam	ab47267	For immunocytochemical analisys. Working dilution 1:500
Mercodia Insulin ELISA	Mercodia	10-1113-10	For insulin release detection