Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Podsumowanie

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Streszczenie

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Wprowadzenie

Cell reprogramming represents a novel technology to transform every somatic cell of the body into a pluripotent stem cell, known as iPSC¹. The possibility of reprogramming an adult somatic cell back to a pluripotent and undifferentiated state has overcome the limits imposed by the poor availability and ethical issues related to the use of pluripotent cells, previously only derivable from human embryos (embryonic stem cells or ESC)^2,3,4. In 2006, Kazutoshi Takahashi and Shinya Yamanaka conducted a pioneering study achieving the first conversion of adult somatic cells from skin into pluripotent cells by artificially adding four specific genes (Oct4, Sox2, Klf4, c-Myc)⁵. A year later, work conducted in Thomson's laboratory led to the successful reprogramming of somatic cells into iPSCs by transduction of a different combination of four genes (Oct4, Sox2, Nanog, Lin28)⁶.

iPSCs offer a number of opportunities to scientists and researchers of different fields, such as regenerative medicine and pharmacology, being an excellent platform to study and treat different diseases along with a genotypic reflection of the characteristics of the patient they are derived from. The use of iPSCs provides several advantages including: the reduced risk for immune response due to a completely autologous origin of cells; the possibility of creating a cell library, an important tool to predict response to new drugs and their side effects, as they are able to continuously self-renew and generate different cell types; and the chance to develop a customized approach for drug administration^7,8,9.

Diverse techniques are known at present, to induce the expression of the reprogramming factors and they are included in two major categories: non-viral and viral vector-based methods^10,11,12,13. Non-viral methods include mRNA transfection, miRNA infection/transfection, PiggyBac, minicircle vectors and episomal plasmids and exosomes^10,11,12,13. Viral-based methods include non-integrating viruses, such as Adenovirus, Sendai virus and proteins, and integrating viruses like Retrovirus and Lentivirus^10,11,12,13.

According to several studies, no significant differences have been noticed among these methods in terms of effectiveness of cell reprogramming, hence, the choice of the suitable method strictly depends on the cell type used and on the subsequent applications of the iPSCs obtained^14,15. All the mentioned methods show disadvantages, for example, the Sendai virus is effective on all cell types, but requires a lot of passages to obtain iPSCs; reprogramming by episomes is excellent for blood cells but needs modification of standard culture conditions for fibroblasts; the PiggyBac method could represent an attractive alternative but studies in human cells are still limited and weak^10,11,12,13. Exosomes are nano-vesicles physiologically secreted into all body fluids by cells. According to recent studies, they are responsible for intercellular communication and can have a role in important biological processes, such as cell proliferation, migration and differentiation. Exosomes can transport and transfer mRNA and miRNA to recipient cells with a completely natural mechanism, as they share the same composition of the cell membrane¹⁶. Therefore, exosomes are a promising new generation technique for reprogramming, but their potential to reprogram somatic cells by their content is still under investigation. Viral vectors-based methods use viruses modified in order to convey reprogramming genes to recipient cells. This technique, despite the high efficiency of reprogramming, is not considered safe, as the integration of the virus within the cell can be responsible for infection, teratomas and genomic instability¹⁷.

The following protocol to generate iPSCs colonies combines the Yamanaka's and Thompson's reprogramming cocktail and is based upon the use of a method requiring NM-RNAs and immune evasion factors with the possibility to perform it in xeno-free conditions. The rationale behind the use of this method is to spread, within the scientific community, a protocol allowing a rapid, simple and highly effective reprogramming of adult human fibroblasts from abdominal skin into iPSCs¹⁸.

The strengths of the proposed method are, in fact, the ease of performance and the short time needed to obtain iPSCs. Furthermore, the method avoids cellular defense mechanisms and the use of viral vectors, responsible for relevant issues.

With respect to the standard protocol, the following modifications were made: (1) Confluent fibroblasts were synchronized at passage 4 by being placing in 0.1% serum for 48 h before the trypsinization; (2) The cellular density for culture and the volume of reagents were adjusted for the utilization on a 24-well multi-well plate instead of a 6-well plate; (3) The reprogramming experiment was performed using a 5% CO₂ incubator instead of an incubator with atmospheric (21% O₂) or hypoxic (5% O₂) conditions.

Protokół

The specimens from human tissue were collected according to the Declaration of Helsinki while observing University Hospital Federico II guidelines. All patients involved in this study provided written consent.

1. Preparation of Supplies and Culture Media

1. Clean and autoclave one large pair of surgical scissors, two sets of fine forceps, two pairs of microdissecting scissors, 1 L sterile bottle, 500 mL sterile bottle, and a 250 mL sterile bottle.
2. Prepare 100 mm plates, 60 mm plates, 35 mm plates, disposable scalpels, 50 mL sterile tubes, 15 mL sterile tubes, and a 100 mm glass plate. Store instruments under sterile conditions until ready for use.
3. Clean and sterilize 22 mm x 22 mm cover glasses before use. For this, place cover glasses in a glass plate, cover them with 70% ethanol and wait for a few seconds before aspirating the ethanol. Let cover glasses dry in an oven at 37 °C and then autoclave them.
4. Prepare 1 L of Hank's balanced salt solution (HBSS) at pH 7.4 by dissolving commercially available powdered salts in double-distilled sterile water and adding 0.35 g of sodium bicarbonate. Sterilize by filtration under a sterile hood and store at +4 °C until use.
5. Prepare 500 mL of sterile 1x phosphate-buffered-saline (PBS) by dissolving 0.1 g of potassium phosphate monobasic, 0.1 g of potassium chloride, 4.0 g of sodium chloride and 0.575 g of sodium phosphate dibasic in sterile double-distilled water. Check the pH value (7.4) and sterilize under a sterile hood by filtration. Store at +4 °C until use.
6. Prepare 50 mL of trypsin stop solution (TSS) by adding 5 mL of 10% fetal bovine serum (FBS) to 45 mL of HBSS under a sterile hood. Store at +4 °C until use.
7. Prepare the Dulbecco's modified Eagle medium for fibroblast isolation (I-DMEM) using 250 mL of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS and 0.5% penicillin and streptomycin (pen/strep) under a sterile hood. Store at +4 °C until use.
8. Prepare the DMEM for fibroblasts synchronization (S-DMEM) using 250 mL of DMEM supplemented with 0.1% FBS and 0.5% pen/strep under a sterile hood. Store at +4 °C until use.

2. Isolation of Human Dermal Fibroblasts

NOTE: Steps 2.1 to 2.3 reported below must be performed under a sterile hood. A cylindrical sample measuring about 0.8 cm in diameter yields 2 x 10⁶ fibroblasts at passage 1.

1. Wash the freshly obtained sample of human skin from abdomen in a 100 mm dish with HBSS solution. Gently shake to remove blood and other biological fluids. Repeat the step three times, changing the HBSS solution and the plate at each wash.
2. Place the sample in a 100 mm plate, remove hair and fat using fine forceps and scissors, and dissect it with a scalpel to obtain 2 mm x 1 mm fragments (for a total of 16 fragments) avoiding scars, dirty (e.g., as resulting from drawings with dermographic pen) or burned areas of the tissue.
3. Place 4 small fragments in each 35 mm dish, cover with a sterile 22 mm x 22 mm cover glass. Adjust by the means of fine forceps. Add 1.5 mL of I-DMEM.
4. Incubate the plates at 37 °C in 5% CO₂ for about 15 days or until cells reach 85% confluence. Change the culture medium every 3 days and check the outgrowth of cells using an inverted phase-contrast microscope daily.

3. Expansion of Human Skin Fibroblasts

NOTE: The steps reported below must be performed under a sterile hood except the steps performed in the incubator.

1. Lift the cover glasses with fine forceps, place them upside-down in one 100 mm dish and wash with 1x sterile PBS.
2. Remove fragments of the samples and discard them, wash plates with 1x sterile PBS.
3. Remove 1x PBS and add 3 mL and 1 mL of trypsin-EDTA (Ethylenediaminetetraacetic acid) to cover glasses placed in the 100 mm dish and 35 mm dishes, respectively. Incubate for 5 min at 37 °C with 5% CO₂.
4. Block the trypsinization by adding 5 mL of TSS to the 100 mm dish and 2 mL of TSS to each 35 mm dish. Collect the suspension into 15 mL sterile tubes, centrifuge at 400 x g at 4 °C for 5 min.
5. Aspirate the supernatant and resuspend the pellet in 12 mL of I-DMEM. Split the cell suspension into 3 mL aliquots for each 60 mm plate.
6. Incubate at 37 °C with 5% CO₂. Change the medium daily. Keep cells in culture until they reach a confluence of 75%.
7. Aspirate the medium from the plates and quickly wash in 1x PBS.
8. Repeat trypsinization (steps 3.3 and 3.4) three times to obtain cells at passage 4. Use 1.5 mL of trypsin-EDTA and 3 mL of TSS in each 60 mm dish. Split the cells 1:3.
9. Synchronize the cells by replacing the I-DMEM with S-DMEM and incubate for 48 h at 37 °C with 5% CO₂.
10. Prepare the following (during the cell synchronization).
    1. Prepare fibroblast expansion medium by adding 5.0 mL of FBS and 0.5 mL of L-glutamine to 44.5 mL of advanced DMEM (A-DMEM), store at +4 °C.
    2. Prepare total NM-RNA-reprogramming cocktail for fibroblast reprogramming by combining the following in each of 9 sterile RNase-free tubes: 32 µL of OSKMNL NM-RNA, 24 µL of EKB NM-RNA, 5.6 µL of NM-microRNAs (61.6 µL final volume for 9 aliquots).
    3. Divide each tube of 61.6 µL of total NM-RNA-reprogramming cocktail for fibroblast reprogramming into four 15.4 µL single-use aliquots in sterile, RNase-free tubes and store at -80 °C (15.4 µL final volume for 36 aliquots).

4. Reprogramming of Dermal Fibroblasts to iPSCs

1. Day 0: Cell seeding
   NOTE: Steps 4.1.1 to 4.1.3 and 4.1.8 to 4.1.9 must be performed under a sterile hood. Basement membrane matrix (BMM) gels rapidly at room temperature. Therefore, it is strongly recommended to thaw BMM overnight on ice in a refrigerator, dilute it with ice-cold serum-free medium and use pre-cooled pipets, tips, and tubes.
   1. Prepare a 24-well plate by coating each well with 100 µL of BMM and incubate for at least 1 h at 37 °C before seeding the cells.
   2. Aspirate the medium from plates with cells in culture and quickly wash in 1x PBS.
   3. Repeat trypsinization (steps 3.3 and 3.4) to obtain cells at passage 5. Aspirate the supernatant and resuspend the pellet in an appropriate volume of fibroblasts expansion medium (see step 3.10).
   4. Prepare and clean the hemocytometer with 70% alcohol.
   5. Prepare a solution by gently mixing 10 µl of trypan blue and 10 µL of cell suspension in 1.5 mL tube, and incubate for 1–2 min at room temperature. Be careful not to incubate longer than 5 min.
   6. Pipette 10 µL of the solution into the hemocytometer place it on the stage of an inverted phase contrast microscope to count. Non-viable cells will be blue, while viable cells will be unstained.
   7. Count the cells in at least 2 squares of the hemocytometer chamber. Apply the following calculation to obtain the total number of cells:
      total number of cells = (total number of counted cells/square number) x 2 x 10 x total volume of cell suspension
   8. Perform a dilution to obtain a density of 2.5 x 10⁴ cell per 500 µL of fibroblasts expansion medium, based on the total number of counted cells. Resuspend the pellet in an appropriate volume of fibroblasts expansion medium.
   9. Pipette 500 µL of the cell suspension into each well of the 24-well plate. Incubate cells overnight at 37 °C and 5% CO₂.
2. Day 1: Transfection
   NOTE: All working surfaces and supplies (e.g., gloves, bottles, sterile hood surfaces, pipettors) MUST be cleaned with cleaning reagent for removing RNase before starting the procedure. Perform steps 4.2.2, 4.2.4–4.2.8 under a sterile hood.
   1. Warm up xeno-free, serum-free,low growth factor human ESC/iPSC culture medium (XF/FF culture medium) in a 37 °C water bath.
   2. Remove the old medium from each well and replace it with 500 µL of pre-warmed XF/FF culture medium.
   3. Incubate at 37 °C under 5% CO₂ for at least 6 h.
   4. Thaw five 15.4 µL aliquots of total NM-RNA reprogramming cocktail at room temperature then place it on ice.
   5. Add 234.6 µL of reduced-serum medium to each aliquot, gently pipette 3–5 times and label each one as TUBE A (RNA + reduced-serum medium).
   6. Label 5 sterile, RNase-free 0.5 mL tubes as TUBE B and mix 6 µL of synthetic siRNA transfection reagent with 244 µL of reduced-serum medium (RNA-transfection reagent + reduced-serum medium).
   7. Add the contents of each TUBE B to a TUBE A dropwise (to obtain 500 µL final volume of NM-RNA transfection complex solution). Mix by tapping the bottom of the tube. Incubate at room temperature for 15 min.
   8. Add 125 µL of NM-RNA transfection complex solution from each of the five aliquots of 500 µL to 4 wells (to reach a total of 20 wells), tilting the plate and pipetting dropwise into the medium. Mix by rocking gently. Use the remaining 4 wells as a reference.
   9. Incubate for 15 h at 37 °C, 5% CO₂.
3. Day 2–4: Complete the transfection
   1. Aspirate the medium. Repeat the transfection procedure as on day 1 under the sterile hood.
4. Day 5–10: Media changes
   1. Aspirate the medium and exchange with fresh, pre-warmed XF/FF culture medium under a sterile hood.
   2. Incubate overnight at 37 °C, 5% CO₂.
   3. Keep in culture until observing the formation of iPSC colonies monitoring daily by a phase-contrast microscope.

Wyniki

The aim of the protocol was to reprogram dermal fibroblasts isolated from abdominal skin using non-integrating reprogramming method based on NM-RNAs to induce the expression of specific factors. To achieve this goal, human dermal fibroblasts were isolated from skin specimens of patients undergoing tummy tuck surgery and iPSCs were generated introducing Oct4, Sox2, Klf4, cMyc, Nanog, Lin28 reprogramming factors and E3, K3, B18 immune evasion factors by a commercial ready-to-use reprogramming kit that combines NM-RNA and m...

Dyskusje

iPSCs are rapidly emerging as the most promising cell candidate for regenerative medicine applications and as a tremendously useful tool for disease modeling and drug testing^3,8. The protocol presented here describes the generation of human iPSCs from a sample having the size of a skin punch biopsy with a simple and efficient procedure that does not require any specific equipment or previous experience with reprogramming technology.

It...

Materiały

Name	Company	Catalog Number	Comments
10 mL serological pipet	Falcon	357551	Sterile, polystyrene
100 mm plates	Falcon	351029	Treated, sterile cell culture dish
15 mL sterile tubes	Falcon	352097	Centrifuge sterile tubes, polypropylene
24-well plates	Falcon	353935	Clear, flat bottom, treated multiwell cell culture plate, with lid, sterile
25 mL serological pipet	Falcon	357525	Sterile, polystyrene
35 mm plates	Falcon	353001	Treated, sterile cell culture dish
5 mL serological pipet	Falcon	357543	Sterile, polystyrene
50 mL sterile tubes	Falcon	352098	Centrifuge sterile tubes, polypropylene
Advanced DMEM (Dulbecco's Modified Eagle Medium)	Gibco	12491-015	Store at 2-8 °C; avoid exposure to light
DMEM (Dulbecco's Modified Eagle Medium)	Sigma- Aldrich	D6429-500ml	Store at 2-8 °C; avoid exposure to light
Fetal Bovine Serum	Sigma- Aldrich	F9665-500ml	Store at -20 °C. The serum should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles
Hank's Balanced Salt Solution	Sigma- Aldrich	H1387-1L	Powder
L-glutamine	Lonza	BE17-605E	Store at -20 °C. It should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles
Lipofectamine RNAiMAX Transfection Reagent	INVITROGEN	13778-030	Synthetic siRNA Transfection Reagent; store at 2-8 °C
Matrigel	CORNING	354234	Basement Membrane Matrix, store at -20 °C. Avoid multiple freeze-thaws.
Neubauer Chamber	VWR	631-1116	Hemocytometer
NutriStem XF Culture Medium	Biological Industries	05-100-1A-500ml	Xeno-free, serum-free, low growth factor human ESC/iPSC culture medium. Store at -20 °C. Upon thawing, the medium may be stored at 2-8 °C for 14 days. Media should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles.
Opti-MEM	Gibco	31985-062-100ml	Reduced-Serum Medium; store at 2-8 °C; avoid exposure to light
Penicillin and Streptomycin	Sigma- Aldrich	P4333-100ml	Store at -20 °C. The solution should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles
Potassium Chloride	Sigma- Aldrich	P9333	Powder
Potassium Phosphate Monobasic	Sigma- Aldrich	P5665	Powder
RNase-free 0.5 mL tubes	Eppendorf	H0030124537	RNase-free sterile, microfuge tubes, polypropylene
RNase-free 1.5 mL tubes	Eppendorf	H0030120086	RNase-free sterile, microfuge tubes, polypropylene
RNaseZAP	INVITROGEN	AM9780	Cleaning agent for removing RNase
Sodium Bicarbonate	Sigma- Aldrich	S5761	Powder
Sodium Chloride	Sigma- Aldrich	S7653	Powder
Sodium Phosphate Dibasic	Sigma- Aldrich	94046	Powder
StemRNA 3rd Gen Reprogramming Kit	Reprocell	00-0076	Third Generation NM-RNAs-based Reprogramming Kit for Cellular Reprogramming of Fibroblasts, Blood, and Urine. Store at or below -70 °C.
Trypsin-EDTA	Sigma- Aldrich	T4049-100ml	Store at -20 °C. It should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles