Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

Summary

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Abstract

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

Introduction

Stem cells have been one of the most attractive materials in clinical therapy for the last several decades¹. The attractive properties of stem cells are pluripotency and the ability to self-renew. In 1981, the first embryonic stem cells (ESCs) were isolated from the mouse embryo². However, when the technique was applied to human embryos, it faced several ethical issues.

In 2006, when Dr. Yamanaka and his team reprogrammed the first pluripotent cell from mouse somatic cells, the stem cell field regained its possibility and interest was rekindled³. By delivering several defined factors, pluripotent stem cells ....

Protocol

Ethics Statement: This study protocol was approved by the institutional review board of The Catholic University of Korea (KC12TISI0861).

1. Isolation of Monocytic Cells from Blood

1. Isolation of monocytic cells (Day -5)
   1. Obtain at least 10 ml of fresh blood from a blood draw in a cell preparation tube (CPT).
   2. Transfer the blood to a new 50-ml conical tube and dilute it with sterile phosphate-buffered saline (PBS) at a 1:4 ratio.
      ​NOTE: A higher ratio of dilution can be used for higher purity.
   3. Add 10 ml of density gradient media to a new 50-ml conical tube and carefully layer the diluted blood on top of the....

Results

This protocol presents a simple method to reprogram PBMCs isolated from blood. Using the combination of serial plating and centrifugation, iPSCs were successfully generated. With this method, iPSCs could be generated with a small amount of whole blood cells without isolating or expanding a specific cell type. We successfully generated iPSCs from only 1x10⁴ cells in a small cell culture plate.

Before reprogramming, blo.......

Discussion

Since embryonic stem cells (ESCs) showed several shortcomings, the need of an alternative tool was required. Therefore, the development of induced pluripotent stem cells (iPSCs) by Yamanaka came under the international spotlight. It has been almost a decade since Yamanaka discovered that pluripotency can be induced by adding only four genes into adult somatic cells. Since iPSCs are "induced" from mature somatic cells, they can evade ethical issues that had once been the concern relating to ESCs. Unlike ESCs, iPSC.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plasticware
100 mm Dish	TPP	93100
6-well Plate	TPP	92006
50 ml Cornical Tube	SPL	50050
15 ml Cornical Tube	SPL	50015
10 ml Disposable Pipette	Falcon	7551
5 ml Disposable Pipette	Falcon	7543
12-well Plate	TPP	92012
24-well Plate	TPP	92024
PBMC Isolation Materials
DPBS	Life Technologies	14190-144
Ficoll	GE Healthcare	17-1440-03
StemSpan	STEMCELL Technologies	9805	Blood cell media
CC110	STEMCELL Technologies	8697	Blood cell media supplement (100x)
iPSC Generation and Culture Materials
CytoTune-iPSC Sendai Reprogramming Kit	Life Technologies	A16518
TeSR-E8 Media	STEMCELL Technologies	5940	iPSC media
Vitronectin	Life Technologies	A14700
ROCK Inhibitor	Sigma Aldrich	Y0503
TrypLE express (TrypLE)	Life Technologies	12604-039
ReleSR	STEMCELL Technologies	12604-039	Colony detaching solution
Quality Control Materials
18 mm Cover Glass	Superior	HSU-0111580
4% Paraformaldyhyde	Tech & Innovation	BPP-9004
Triton X-100	BIOSESANG	9002-93-1
Bovine Serum Albumin	Vector Lab	SP-5050
Anti-SSEA4 Antibody	Millipore	MAB4304
Anti-Oct4 Antibody	Santa Cruz	SC9081
Anti-TRA-1-60 Antibody	Millipore	MAB4360
Anti-Sox2 Antibody	Biolegend	630801
Anti-TRA-1-81 Antibody	Millipore	MAB4381
Anti-Klf4 Antibody	Abcam	ab151733
Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody	Molecular Probe	A11029
Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody	Molecular Probe	A11037
DAPI	Molecular Probe	D1306
Prolong gold antifade reagent	Invitrogen	P36934
Slide Glass, Coated	Hyun Il Lab-Mate	HMA-S9914
Trizol	Invitrogen	15596-018
Chloroform	Sigma Aldrich	366919
Isoprypylalcohol	Millipore	109634
Ethanol	Duksan	64-17-5
RevertAid First Strand cDNA Synthesis kit	Thermo Scientfic	K1622
i-Taq DNA Polymerase	iNtRON BIOTECH	25021
UltraPure 10X TBE Buffer	Life Technologies	15581-044
loading star	Dyne Bio	A750
Agarose	Sigma-Aldrich	9012-36-6
1kb (+) DNA ladder marker	Enzynomics	DM003
Alkaline Phosphatase	Millipore	SCR004
Tris base	Fisher Scientific	BP152-1	Rinse Buffer
Sodium Chloride	Duchefa Biochemie	S0520.1000	Rinse Buffer
Tween-20	BIOSESANG	T1027	Rinse Buffer
Hydrochloric Acid	Duksan	1129	Rinse Buffer