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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 m.......

Discussion

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.......

Materials

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