Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Summary

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Abstract

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

Introduction

The discovery of human induced pluripotent stem cells (hiPSCs) has revolutionized the field of regenerative medicine, providing a feasible method for generation of patient-specific stem cells ^1-3. hiPSCs have been successfully generated from various somatic cell types, including fibroblasts ^4,5, hematopoietic cells ^6,7, renal epithelial cells from urine ⁸ and keratinocytes ^9,10. To date, skin fibroblasts and hematopoietic cells represent the most commonly used cell sources for generating patient-specific iPSCs. Arguably, this is due to the fact that skin biopsies and blood collection are routine medical procedures....

Protocol

The collection of human hair sample from individuals requires ethical approval by the human research ethics committee in the host institutions and should be done in compliance with the institutional guidelines.

1. Isolation of Keratinocytes from Plucked Hair

1. Thaw Extracellular Matrix (ECM) solution (i.e., Matrigel) on ice O/N.
2. Using pre-chilled pipette tips, add 200 µl ECM solution to 12 ml of chilled DMEM/F12 medium. Coat a 12-well plate with 1 ml of diluted ECM solution into each well. Incubate the coated plate O/N at 37 °C. Wash the coated plate with pre-warmed DMEM/F12 medium befor....

Results

The hair goes through 3 different phases of growth cycle: anagen (the growth phase), catagen (the regression phase) and telogen (the rest phase) ^20,21. The anagen hair follicle contains multiple layers of epithelium; these layers include the ORS, IRS and the hair shaft (Figure 1). Anagen hair eventually undergoes transition to the catagen phase, which is marked by apoptosis of the ORS and termination of hair shaft differentiation. Finally, catagen hair transition to the telogen phase, where ap.......

Discussion

Generation of patient-specific hiPSCs offers a unique approach for studying pathogenesis in the diseased cell types in vitro, and also provides a platform for drug screening to identify novel molecules that can rescue the disease phenotypes. This disease modeling approach using hiPSCs has yielded promising results for a variety of diseases, including Long QT syndrome, Huntington’s disease, Parkinson’s disease and amyotrophic lateral sclerosis ²². Several initiatives are already underway to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibiotic Mix:
250 ng/ml Antimycotic amphotericin B	Sigma	A2942-20ml	Antibiotic mix is made up in PBS.
1X Penicillin/Streptomycin	Invitrogen	15140-122
PBS (-)	Invitrogen	14190-144
Knockout Serum Replacement (KSR) medium:			KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use.
20% knockout serum replacement (KSR)	Invitrogen	10828-028
DMEM/F12 with glutamax	Invitrogen	10565-042
1× MEM non-essential amino acid	Invitrogen	11140-050
0.5× Penicillin/Streptomycin	Invitrogen	15140-122
0.1 mM β-mercaptoethanol	Invitrogen	21985
bFGF (10 ng/ml, added fresh)	Millipore	GF003
Keratinocyte medium:
EpiLife with 60 µM Calcium	Invitrogen	M-EPI-500-CA
1× Human keratinocyte growth supplement (HKGS)	Invitrogen	S-001-5
Fetal Bovine Serum (FBS) medium:			FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use.
10% fetal bovine serum (FBS)	Invitrogen	26140079
DMEM	Invitrogen	11995-073
0.5x Penicillin/Streptomycin	Invitrogen	15140-122
2 mM L-glutamine	Invitrogen	25030
0.25% trypsin-EDTA	Invitrogen	25200-056
Extracellular Matrix (ECM):
Matrigel	Corning	354234	Aliquot Matrigel stock and store in -80°C following manufacturer’s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200µl matrigel for coating a 12-well plate (~150µg/well).
Coating Matrix Kit	Invitrogen	R-011-K
Plasmids:			Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.
-          pCXLE-eGFP	Addgene	27082
-          pCXLE-hOct3/4-shP53F	Addgene	27077
-          pCXLE-hSK	Addgene	27078
-          pCXLE-hUL	Addgene	27080
Transfection reagent Fugene HD	Promega	E231B
Gelatin (from porcine skin)	Sigma	G1890	Make up 0.1% gelatin in distilled water. Autoclave before use.
Reduced Serum medium: OPTI-MEM	Invitrogen	31985062
Accutase	Sigma	A6964-100ml
Mouse embryonic fibroblast (MEF) feeder			MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16.
26G needle	Terumo	NN2613R
6-well plate (tissue culture treated)	BD Biosciences	353046
12-well plate (tissue culture treated)	BD Biosciences	353043
10 cm dish (tissue culture treated)	BD Biosciences	353003
Dispase	Invitrogen	17105-041	Use at 10 mg/ml
Collagenase IV	Invitrogen	17104-019	Use at 1 mg/ml
TRA-160 antibody	Millipore	MAB4360	Use at 5 µg/ml
OCT4 antibody	Santa Cruz	SC-5279	Use at 5 µg/ml
NANOG antibody	R&D Systems	AF1997	Use at 10 µg/ml
MycoAlert Detection kit	Lonza	LT07-418