Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Summary

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Abstract

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.

Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Introduction

Human embryonic stem cells (hESCs) have a capacity to self-renew in vitro and have pluripotency, which could be potentially useful for disease modeling, for drug screening, and to develop cell-based therapies to treat disease and tissue injuries. However, hESCs have a limitation for cell replacement therapy because of immunological, oncological and ethical barriers, and to study disease related genes, disease-specific hESCs could be isolated through pre-implantation genetic diagnosis (PGD) approaches, but it is still technically challenging and the embryo donations are pretty rare. These issues are related to the progress in stem cell biology, which has ....

Protocol

1. Preparation of Cell and Media (Day 1)

1. Culture and expand human fibroblast with DMEM media containing 10% FBS.
2. Plate human fibroblasts (Figure 1) onto a 24-well plate at the appropriate density per well on the day before transduction.
   NOTE: The following serial dilutions are recommended (200K, 100K, 50K, 25K, 12.5K and 6.25K) because different cell types have different attachment ability.
3. Incubate the cells for one more day in a 37 °C, 5% CO₂ incubator, ensuring the cells have fully adhered and extended.

2. Perform Transduction (Day 2)

....

Results

Usually infected fibroblasts do not show any morphological changes in several days after Sendai virus transduction, but five days later, they start to have different shapes (Figure 1). As described in a right panel of Figure 1, cells do not have typical fibroblast morphology any more. They have a round shape and bigger nucleus than cytoplasm. Even when transduction is performed in 80% cellular confluency, it looks like they are less confluent in the well after they start to reprogram. If.......

Discussion

Reprogramming human somatic cells to hiPSCs holds unprecedented promises in basic biology, personal medicine, and transplantation¹². Previously, human iPSC generations required DNA virus that has integration risk into the host genome, which can create undesirable genetic mutations that limit further clinical applications such as drug development and transplantation therapies¹³. With this reason, many studies have been reported to generate vector- and transgene-free system human iPSCs by several alte.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CytoTune-iPS Reprogramming Kit	Invitrogen	A1378002
CF-6,MEFs, neomycin-resistant, mitomycin C treated	Global stem	GSC-6105M	5 x 105/6 cm or 12.5 x 105/24-well plate
Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X	Invitrogen	25200114
DMEM/F-12 medium	Invitrogen	11330-032
24-well Cell Culture Plate, flat-bottom with lid	BD	353935
Y-27632	TOCRIS	1254	10 μM (Stock: 10 mM)
basic fibroblast growth factor	LIFE TECHNOLOGIES	PHG0263	10 ng (Stock : 100 ug)
Knock-out serum replacement	Gibco	10828028
Dulbecco's Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)	Invitrogen	11965118
Fetal bovine serum	Thermo Scientific Fermentas	SH30071.03
L-Glutamine-200 mM (100X), liquid	GIBCO	25030-081	1/100
MEM Non-Essential Amino Acids Solution, 100X	LIFE TECHNOLOGIES	11140050	1/100
2-Mercaptoethanol (1,000X), liquid	GIBCO	21985023	1/1,000
Hausser Phase Contrast Hemacytometers	Hausser Scientific	02-671-54
EmbryoMax 0.1% Gelatin Solution	Millipore	ES-006-B
SSEA-4	DSHB	MC-813-70	1/200
anti-Tra-1-81	Cell Signaling	4745S	1/200
mouse monoclonal Oct4 antibody	Santa Cruz	SC-5279	1/1,000
Nanog	R&D	AF1997	1/1,000
Alexa Flouor 488 goat anti-mouse	Invitrogen	948492	1/2,000
DPBS (Dulbecco's Phosphate-Buffered Saline), 1X without calcium & magnesium	Cellgro	21-031-CV
QuantiTect Reverse Transcription Kit	QIAGEN	205313
PCR Master Mix [2X]	Thermo Scientific Fermentas	K0171
Trizol	Invitrogen	15596018
picking hood	NuAire	NU-301
dissecting scope	Nikon	SMZ745