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 were successfully "induced" from adult somatic cells, and were thus named "induced pluripotent stem cells (iPSCs)." In 2007, this technique was applied to human cells⁴, yielding cells with the exact characteristics of ESCs but none of the ethical debate. Theoretically, iPSCs can be generated from any cell type obtained from any individual or patient. Patient-specific iPSCs are rising as a potential tool that can simulate the disease phenotypes and epigenetic conditions of each individual patient. Using gene editing or other methods that can reverse the pathogenic condition, patient-specific iPSCs can also be used in personalized medicine⁵. Moreover, iPSCs are less associated with immune rejection because they have the same immune identity as the donor, making auto-transplantation more feasible⁶. Therefore, iPSCs have become the most promising platform in disease modeling, drug screening, and regenerative therapies. Given these benefits, improved protocols that can give purer and higher yields in the least amount of time from the smallest cell source are constantly under development. One major consideration of finding the most efficient protocol for future application is the primary cell type. Most of the early iPSC generation protocols are optimized for adherent cells since the original iPSC lines were induced from skin fibroblasts⁴. However, the isolation and preparation of these cells are labor intensive. Also, the isolation of skin fibroblasts includes invasive surgical procedures that can become a major shortcoming for broader application.

Therefore, for the further use of iPSCs, a cell source with convenient acquisition is required. Blood is regarded as an ideal cell source since it is obtained through a rather minimally invasive procedure^7-9. In this study, we developed a simple modification to the protocol generating hiPSCs from peripheral blood mononuclear cells (PBMCs). Without the difficult expansion process of a specific cell type, such as CD34+ cells, whole blood cells or PBMCs were serially plated onto matrix-coated plates by centrifugation after transduction with Sendai virus containing Yamanaka factors. This method reduced the time required for the attachment of transduced floating cells and decreased the loss of reprogrammed cells that were not able to attach on their own.

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 density gradient media. Centrifuge at 750 x g for 30 min at room temperature (RT) without a centrifugation brake.
   4. Carefully transfer the buffy layer to a new 50-ml conical tube, add 30 ml of PBS to the tube, and wash the cells.
   5. Centrifuge the cells at 515 x g for 5 min at RT.
   6. Discard the PBS and resuspend the cells in 0.5 ml of blood cell media.
   7. Count the cells and plate 1 x 10⁶ cells per well of a 24-well plate. Add PBS to the surrounding wells to prevent evaporation.
   8. Stabilize the cells for 5 days at 37 °C in 5% CO₂ before transduction. Add an additional 0.5 ml of fresh blood cell media on days 3-4 without disturbing the cells.

2. Transduction by Sendai Virus

1. Transduction (Day 0)
   1. Collect and transfer the blood cells to a 15-ml conical tube and count them using a hemocytometer.
   2. Prepare 3 x 10⁵ cells per transduction and centrifuge the cells at 515 x g for 5 min at RT.
   3. Discard the supernatant by suction and resuspend the cells in 0.5 ml of blood cell media.
   4. Transfer the cells to a well of a non-coated 24-well plate.
   5. Thaw the Sendai virus mixture in ice and add it to the suspended cells. Add Sendai virus to the cells based on the manufacturer's recommendations.
   6. Seal the plate with a sealing film and centrifuge it at 1,150 x g for 30 min at 30 °C.
   7. After centrifugation, incubate the cells at 37 °C in 5% CO₂ overnight (O/N).
2. Cell transfer to feeder matrix (Day 1)
   1. The next day, coat a 24-well plate with vitronectin. Dilute the vitronectin solution in PBS for a final 5 µg/ml concentration. Add 1 ml of vitronectin to a well of a 24-well plate and incubate it at RT for at least 1 hr. Remove the coating solution before use. Coated plates can be stored in RT for 3 days.
   2. Transfer all the media containing the cells and the virus to the coated well.
   3. Collect the remaining cells with an additional 0.5 ml of fresh blood cell media and add it to the cell-containing well.
   4. Centrifuge the plate at 1,150 x g for 10 min at 35 °C.
   5. After centrifugation, remove the supernatant, add 1 ml of iPSC media, and maintain the cells at 37 °C in 5% CO₂ O/N.
3. Second cell transfer (Day 2)
   1. Coat the wells of a new 24-well plate with 5 µg/ml vitronectin, as described in step 2.2.1. Use one well of the plate for each transduction.
   2. Transfer the cell suspension from the first plate to the newly coated vitronectin plate.
      ​NOTE: If not needed, suspension cells can be discarded. The procedure mentioned in step 2.3 can be repeated 2-3 times with the suspension cells. If the steps will be repeated, harvest the cells.
   3. Meanwhile, add 1 ml of iPSC media to the well of the first plate, for maintenance, and incubate it at 37 °C in 5% CO₂ O/N.
   4. Maintain the attached cells at 37 °C and 5% CO₂ and perform a daily media change with fresh iPSC media. Colonies will appear on days 14-21 after transduction.
   5. Centrifuge the newly coated plate containing the suspension cells at 1,150 x g and 35 °C for 10 min.
   6. After centrifugation, incubate the cells at 37 °C in 5% CO₂ O/N.
   7. The next day, remove the supernatant and replace it with fresh iPSC media.
      ​NOTE: The procedure mentioned in step 2.3 can be repeated 2-3 times with the suspension cells. If the steps will be repeated, harvest the cells in the supernatant and repeat step 2.3.
   8. Maintain the attached cells with daily media changes until 80% confluency is reached.

3. Reprogrammed Cell Maintenance

1. Early maintenance after the 24-well plate culture
   1. 7-10 days after transduction, once the cells are confluent, prepare a vitronectin-coated, 60-mm dish. Dilute vitronectin solution in PBS for a final 5 µg/ml concentration. Add 1 ml of vitronectin to the dish and incubate it at RT for at least 1 hr.
   2. Wash the cells with PBS and add 1 ml of PBS/1 mM EDTA to detach the cells.
   3. Incubate them at 37 °C and 5% CO₂ for 2 min.
   4. Harvest the cells and centrifuge them at 250 x g and RT for 2 min. Remove the supernatant and resuspend the cells in 3 ml of fresh iPSC media.
   5. Plate all resuspended cells onto the newly coated 60-mm dish.
   6. Add 10 mM RHO kinase inhibitor to the cells and maintain them at 37 °C in 5% CO₂ until the cells are 80% confluent.
2. Split for colony appearance (Subcloning Preparation)
   1. Prepare a vitronectin-coated, 100-mm dish, as described in step 3.1.2.
   2. Wash the cells with PBS and add 1 ml of PBS/1 mM EDTA to detach the cells.
   3. Incubate them at 37 °C and 5% CO₂ for 2 min.
   4. Harvest the cells and centrifuge them at 250 x g and RT for 2 min.
   5. Count the cells using a hemocytometer and prepare 1 x 10⁴ cells per dish.
   6. Centrifuge the cells at 250 x g and RT for 2 min.
   7. Resuspend 1 x 10⁴ cells in 6 ml of iPSC media, plate them onto the coated 100-mm dish, and add 10 mM RHO kinase inhibitor to the media.
   8. Incubate the cells at 37 °C in 5% CO₂ for a week until large colonies appear.
      ​NOTE: Maintain and expand the colonies for subcloning. Subcloning is usually done in under 5 passages.
3. Colony picking using iPSC colony detaching solution
   1. A week before colony picking, seed 1 x 10⁴ cells in a vitronectin-coated, 100-mm dish, as mentioned in step 3.2.7.
   2. Prepare a vitronectin-coated, 60-mm dish by adding 2 ml of vitronectin solution and incubating at RT for at least 1 hr.
   3. By observing through a microscope (40X or 100X magnification), mark colonies with clear boundaries using a marker pen. Remove the vitronectin solution from the new plate made in step 3.3.2 and add 6 ml of iPSC media supplemented with 10 mM RHO kinase.
   4. Remove the culture medium from the cells and wash them with 3 ml of PBS.
   5. Add 1 ml of iPSC colony detaching solution and incubate them for 30 s at RT.
   6. Remove the solution from the plate and incubate it at RT for additional 30 sec.
   7. Using a P200 pipette, draw 200 µl of media from the plate and detach the targeted colonies by pipetting. Transfer the scattered colonies to the new 100-mm dish.
   8. Incubate and maintain the cells at 37 °C in 5% CO_2.
      NOTE: After obtaining a pure iPSC colony, cells were maintained until they reached passage 10. Characterization was done after at least 10 passages. Antibody dilutions and primer information are shown in Tables 1 and 2.

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

Name	Company	Catalog Number	Comments
Plasticware
100mm 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