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.
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.
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 administration7,8,9.
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 methods10,11,12,13. Non-viral methods include mRNA transfection, miRNA infection/transfection, PiggyBac, minicircle vectors and episomal plasmids and exosomes10,11,12,13. Viral-based methods include non-integrating viruses, such as Adenovirus, Sendai virus and proteins, and integrating viruses like Retrovirus and Lentivirus10,11,12,13.
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 obtained14,15. 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 weak10,11,12,13. 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.
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
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.
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.
4. Reprogramming of Dermal Fibroblasts to iPSCs
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 microRNA technology. The timeline of the protocol is summarized in Figure 1.
Human fibroblasts outgrew from samples of abdominal skin within one week of culture (Figure 2A) and reached 85% confluence within two weeks (Figure 2B). Cells were characterized by adhesive growth on plastic culture dishes and their morphology varied from elongated and spindle-shaped (Figure 2C) to flattened and star-shaped (Figure 2D). Fibroblast morphology and arrangement in culture dramatically changed after seeding on BMM, when they acquired an elongated morphology and arranged to form thin branched structures (Figure 3A). Small colonies were already visible in culture at day 1 from first transfection (Figure 3B) and their size grew progressively over time, while their number increased till day 7 and then remained stable between day 7 and day 14 (Figure 3C,D), probably as a result of smaller colonies merging to form larger colonies.
Since the reprogramming procedure is performed using antibiotic-free media, microbial contamination is a major issue and it may occur (Figure 4) if sterile conditions are not guaranteed Adopt standard procedures to prevent contamination (e.g., wearing gloves, remove dust from all surfaces, clean all surfaces and equipment with 70% ethanol, avoid talking during steps with uncovered cell culture plates).
Figure 1: Protocol timeline. Timeline of isolation and reprogramming of human fibroblasts from abdominal skin. Please click here to view a larger version of this figure.
Figure 2: Isolation and culture of dermal fibroblasts. Representative images of fibroblasts outgrowth from the skin fragments (A). Fibroblasts reached confluence in about 14 days (B) and showed spindle-shaped (C) and star shaped (D) phenotype. Skin fragments are indicated by a white star. Scale bar = 200 μm. Please click here to view a larger version of this figure.
Figure 3: Skin fibroblast reprogramming progression. Cells seeded on BMM showed a marked change in arrangement and morphology (A). Small colonies of iPSCs formed as early as 24 h after the first transfection (B) and their size increased progressively at day 7 (C), and 14 (D), while their number increased between day 1 and day 7 but remained stable between day 7 and 14 from first transfection. Scale bar = 500 μm. Please click here to view a larger version of this figure.
Figure 4: Microbial contamination during reprogramming procedure. Representative image using phase-contrast microscopy showing a microbial contamination of fibroblasts culture during the reprogramming procedure. Scale bar = 500 μm. Please click here to view a larger version of this figure.
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 testing3,8. 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 is of primary importance to optimize fibroblast isolation and culture to increase chance of success, as plating density and proliferation rate impact reprogramming efficiency. Further, it has been recently reported that dermal fibroblasts respond differently to reprogramming technology and, specifically, fibroblasts isolated from the skin of the abdominal region are more easily and readily reprogrammed than dermal fibroblasts isolated from other body regions18. Therefore, it is important to accurately select the somatic cells to reprogram and define the plating density on the basis of cell proliferation rate. To this aim, the present protocol also instructs on how to isolate dermal fibroblasts from abdominal skin and how to propagate them in vitro to ease and accelerate the procedure.
Prior to reprogramming, fibroblasts were propagated to reach passage 5 to erase cell memory18 and comply with reported evidence that passaging could accelerate the induction of pluripotent state19. Moreover, according to our experience, fibroblasts in culture exhibit variable proliferative properties, thus we modified the protocol by introducing an additional step to synchronize fibroblasts and reduce variability in the cell population.
Using a commercial NM-RNAs-based kit that introduces a combination of reprogramming factors resulting from Yamanaka's and Thomson's approach5,6, along with miRNA that have been proven to improve mRNA-based reprogramming20, human dermal fibroblasts can be successfully reprogrammed to iPSC and small colonies become apparent as early as 24 h after the first transfection. The main advantages of mRNA-based reprogramming are, indeed, the early emergence of colonies even when a low number of cells is reprogrammed20 along with a very low aneuploidy rate and the complete absence of integration that make iPSCs generated with this method safe for a use in regenerative medicine. Moreover, the commercial kit used for this protocol co-delivers immunomodulating factors that are known to improve the efficiency of reprogramming by preventing the cell death caused by cytotoxic and immunogenic NM-RNAs21,22.
Although the kit used for reprogramming was designed for 6-well multi-well plate, we optimized cellular density and adjusted the volume of reagents for the utilization on a 24-well multi-well plate to make the protocol effective for the generation of human iPSCs from a skin punch biopsy. Moreover, even though if the need for a tissue culture incubator with O2 control is reported by several authors20,23 and recommended by the kit manufacturer, following the protocol described here we reprogrammed human dermal fibroblasts from abdominal skin in a standard 5% CO2 incubator. Therefore, the procedure can be performed in any cell culture laboratory without the need for atmospheric (21% O2) or hypoxic (5% O2) culture conditions, although these might further improve reprogramming efficiency24,25.
Nonetheless, we used non-human animal-derived reagents to derive from small skin specimens iPSCs that can be used for research purposes. Although the most attractive application is for the regeneration of tissues and organs, iPSCs have been used for modeling different diseases and then both investigate on underlying molecular mechanisms and develop specific drugs and therapies3,26.
Remarkably, substituting animal-derived reagents for appropriate xeno-free reagents, the same protocol allows the reprogramming of adult human fibroblasts into iPSCs in a complete xeno-free culture environment that warrants their clinical use.
However, the heavy workload needs to be taken into consideration. In our opinion, it is critical to plan the experiment carefully well in advance, including steps that need to be performed during weekends, and to prepare all transfection reagents during the synchronization step. Indeed, transfection is to be performed every day for four days and, afterwards, cells need to be monitored every day and medium is to be replaced on a daily basis.
Furthermore, the accurate identification of iPSC colonies cannot be based solely upon morphological criteria. Hence, newly derived iPSCs need to be characterized searching for the expression of multiple pluripotent markers through cellular and molecular analyses. Widely accepted markers include NANOG, OCT4, SOX2, TRA-1-60, TRA-1-81 and SSEA4, which may be identified by immunocytochemical analysis and by gene expression analysis using semi-quantitative or quantitative RT-PCR. Since alkaline phosphatase activity has been shown to be upregulated in pluripotent stem cells, the detection of such enzymatic activity can be easily carried out to identify iPSCs and verify the occurrence of reprogramming27,28.
The authors have nothing to disclose.
The authors have no acknowledgments.
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 |