Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Summary

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Abstract

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Introduction

Since the first derivation of hiPS cell lines by Takahashi et al.^1,2, hiPS cells have provided a useful tool for disease modeling, drug discovery and as source material for generating cell therapies in regenerative medicine³. hiPS cell culture has long been dependent on co-culture with fibroblast feeder cells^4,5 or on Matrigel⁶ and with media formulations containing fetal bovine serum (FBS). Batch-to-batch variances are a common consequence of the undefined nature of these culture conditions, res....

Protocol

The collection of patient material and derivation of hiPS cells is approved by the Ethics Review Board, Stockholm, March 28, 2012, Registration number: 2012/208-31/3. Cell culture steps should be performed in biosafety cabinets unless otherwise mentioned. Always practice sterile handling techniques when working with cells. Allow media, plates and reagents to reach room temperature before starting. Incubate cells at 37 ᵒC, 5% CO₂ in high humidity.

1. Isolation of Human Fibroblasts from Dermal Biopsy

1. Preparations of culture medium, digestive enzymes, coating of dishes and dissecting tools.
   ....

Results

From biopsy to hiPS cells

The entire process from biopsy to established hiPS cells, clear of reprogramming vector and ready for characterization, takes approximately 16 weeks (Figure 1). A more detailed timeline is specified in Figure 2A. Approximately 4 weeks is needed to establish and expand fibroblast cultures. The first hiPS cell colonies started to emerge about three-.......

Discussion

The expected result of this protocol is the successful generation of several clonally derived hiPS cell lines. Importantly, the method for the maintenance of and expansion of established hiPS cells here described is reliable and can be performed with little prior experience of stem cell culture. Enzymatic single cell passaging with ROCKi together with the LN-521 matrix is known to maintain cells as karyotypically normal, pluripotent and readily able to differentiate while avoiding induced heterogeneity that colony based .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dispase	Life Technologies	171105-041	Biopsy digestion
Collagenase	Sigma	C0130	Biopsy digestion
Gelatin	Sigma	G1393	Fibroblast matrix
IMDM	Life Technologies	21980002-032	Fibroblast medium
FBS	Invitrogen	10270106	Fibroblast medium
Penicillin/Streptomycin	Life Technologies	15070-063	Antibiotic
Essential 8 media	ThermFisher Scientific	A1517001	iPS cell culture media
LN-521	Biolamina	LN521-03	iPS cell culture matrix
TrypLE select 1X	ThermoFisher Scientific	12563011	Dissociation reagent
Rho-kinase inhibitor Y27632	Millipore	SCM075	Rho-kinase inhibitor (ROCKi)
CytoTune – iPS 2.0 reprogramming kit	Life Technologies	A1377801	Sendai virus reprogramming vector
35 mm tissue culture dish	Sarstedt	83.3900.300	Cell culture
60 mm tissue culture dishes	Sarstedt	83.3901.300	Cell culture
24 well tissue culture plates	Sarstedt	83.3922.300	Cell culture
T25 tissue culture flasks	VWR	734-2311	Cell culture
15 mL tubes	Corning	430791	Centrifuge tubes
1.5 mL tube	Eppendorf	0030123.301	1.5 mL tube
CoolCell cell LX	Biocision	BCS-405	Freezing container
DMSO	Sigma	D2650-100ml	Fibroblast freezing
Cryovials 1.8 mL	VWR	479-6847	Cryovials
PSC Cryopreservation Kit	Gibco	A2644601	PSC CryomediumRevitaCell supplement
Trypsin-EDTA (0.05%)	ThermoFisherScientific	25300054	Fibroblast dissociation enzyme
DMEM/F12	Life Technologies	31331-028	Digestive enzymes dilutant
DPBS	Life Technologies	14190-094	PBS
HBSS	ThermoFIsher Scientific	14025-050	Biopsy preparation
Haemocytometer	Sigma	BRAND, 718920	Cell counting
Parafilm	VWR	291-1213	Sealing plates for storage