Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Summary

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Abstract

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Introduction

iPSCs are derived from somatic tissues via ectopic expression of a minimal set of pluripotency genes. This technique was initially demonstrated by retroviral transduction of human fibroblasts with OCT4, SOX2, KLF4, and cMYC, which are highly expressed in the pluripotent state¹. These transiently expressed “reprogramming factors” alter the target cell’s epigenetic landscape and gene expression profile analogous to human embryonic stem cells². Once created, iPSCs can potentially be differentiated into any tissue type for further investigation. Thus, they hold promise for use in regenerative medicine, dis....

Protocol

Written informed consent, as approved by the Institutional Review Boards of the Fred Hutchinson Cancer Research Center and the Children"s Hospital of Philadelphia, was obtained from patients before collecting peripheral blood samples. All institutional guidelines were observed. All animal experiments including MEF generation and teratoma formation were approved by the Institutional Animal Care and Use Committee.

1. Ficoll Separation of PBMCS and Expansion of Erythroblasts – Day 0

1. Dilute peripheral blood 1:1 with Dulbecco’s Phosphate Buffered Saline (DPBS). In a 15 ml round-bottom polystyrene tube, carefully layer....

Results

Three days after nucleofection and before plating the nucleofected cells onto iMEFs, the efficiency of successful nucleofection should be estimated by fluorescence microscopy for eGFP. Figure 1 shows a typical nucleofection experiment with approximately 5-10% of the total cell population expressing eGFP.

Reprogrammed iPSC colonies will begin to appear approximately two weeks after nucleofection. The colonies are generally circular with well-defined borders and may be identifie.......

Discussion

For successful iPSC generation when using this protocol, there are several important caveats that should be considered. During the erythroblast expansion stage, the media change schedule should be strictly followed, as deviations may lead to inefficient stimulation of the target progenitor cell population and a lower efficiency of iPSC generation. It is important to make new expansion medium with fresh dexamethasone with each media change; and the QBSF-60 base medium and dexamethasone should be protected from light durin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ficoll-Paque PLUS	Fisher Scientific	45-001-750	Store at 4 °C. Warm to room temperature before use.
DPBS	Life Technologies	14190-250	Store at 4 °C.
RPMI 1640	Life Technologies	11875-093	Store at 4 °C.
fetal bovine serum (FBS)	Life Technologies	10437028	Store at -20 °C until needed. Thaw, aliquot, store at 4 °C.
dimethyl sulfoxide (DMSO)	Sigma-Aldrich	154938	Store at room temperature.
QBSF-60 serum-free medium	Fisher Scientific	50-983-234	Store at 4 °C.
penicillin/streptomycin	Life Technologies	15140122	Store at 4 °C.
Cell Line Nucleofector Kit V	Lonza	VCA-1003	Store at 4 °C.
2% gelatin solution	Sigma-Aldrich	G1393	Store at 4 °C, liquify in 37 °C water bath before use.
Hank's Balanced Saline Solution (HBSS)	Life Technologies	14175-103	Store at 4 °C.
Dulbecco's Modified Eagle Medium (DMEM)	Life Technologies	11965-092	Store at 4 °C.
Iscove's Modified Dulbecco's Medium (IMDM)	Life Technologies	12440-061	Store at 4 °C.
L-glutamine, 200 mM	Life Technologies	25030-081	Aliquot, freeze at -20 °C.
non-essential amino acids (NEAA), 100X	Life Technologies	11140-050	Store at 4 °C, away from light.
DMEM/Ham's F12, 1:1	Fisher Scientific	SH30023.02	Store at 4 °C.
KnockOut Serum Replacement	Life Technologies	10828-028	Aliquot, freeze at -20 °C.
sodium pyruvate, 100 mM	Life Technologies	11360-070	Store at 4 °C.
sodium bicarbonate, 7.5%	Life Technologies	25080094	Store at 4 °C.
basic fibroblast growth factor (bFGF)	Life Technologies	PHG0263	Make 10 mg/ml stock in DPBS, aliquot, freeze at -20 °C. Once thawed, store at 4 °C.
sodium butyrate	Sigma-Aldrich	B5887-1G	Make 2000x stock (400 mM) in DPBS, aliquot, freeze at -20 °C. Once thawed, store at 4 °C.
hES Cell Cloning & Recovery Supplement	Stemgent	01-0014-500	Store at -20 °C until needed. Once thawed, store at 4 °C.
ESC-qualified BD matrigel	BD Biosciences	35-4277	Thaw overnight on ice at 4 °C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20 °C until needed. Thaw at 4 °C, use immediately.
StemSpan SFEM	STEMCELL Technologies	9650	Aliquot, freeze at -20 °C.
ascorbic acid, powdered	Sigma-Aldrich	A4403-100MG	Make 5 mg/ml stock in DPBS, sterile filter, store at 4 °C.
recombinant human stem cell factor (SCF)	R & D Systems	255-SC-010	Make 100 μg/m stock in SFEM, aliquot, freeze at -20 °C. Once thawed, store at 4 °C.
recombinant human interleukin 3 (IL-3)	R & D Systems	203-IL-010	Make 100 μg/ml stock in SFEM, aliquot, freeze at -20 °C. Once thawed, store at 4 °C.
erythropoietin (EPO)	R & D Systems	287-TC-500	Make 1000 U/ml stock in SFEM, aliquot, freeze at -20 °C. Once thawed, store at 4 °C.
recombinant human insulin-like growth factor 1 (IGF-1)	R & D Systems	291-G1-200	Make 100 μg/ml stock in SFEM, aliquot, freeze at -20 °C. Once thawed, store at 4 °C.
β-mercaptoethanol	Sigma-Aldrich	M7522	Make 1000x stock (100 mM) in DPBS.
dexamethasone	Sigma-Aldrich	D4902-25MG	Make 50x stock (50 μM) in DPBS, sterile filter, store at 4 °C.
SAHA (vorinostat)	Cayman Chemical	149647-78-9	Make 2,000x stock (400 mM) in DPBS, aliquot, freeze at -20 °C. Once thawed, store at 4 °C.
12 well tissue culture plate	Fisher Scientific	08-772-29
15 ml conical tube	Sarstedt	62553002
1.5 ml Eppendorf tube	Fisher Scientific	05-408-129
6 well tissue culture plate	Fisher Scientific	08-772-1B
35 mm tisue culture plates	BD Biosciences	353001
10 ml disposable serological pipettes	Fisher Scientific	13-675-20
5 ml disposable serological pipettes	Fisher Scientific	13-675-22
2 ml disposable serological pipettes	Fisher Scientific	13-675-17
20 μl pipette tips, barrier tips	Genessee	24-404
glass Pasteur pipettes	Fisher Scientific	13-678-20D
pipette aid	Fisher Scientific	13-681-15
pCXLE-hOCT3/4-shp53-F	Addgene	27077
pCXLE-hSK	Addgene	27078
pCXLE-hUL	Addgene	27080
pCXLE-EGFP	Addgene	27082
pCXWB-EBNA1	Addgene	37624