Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

Summary

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Abstract

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

Introduction

The unique ability of human pluripotent stem cells (hPSCs) to self-renew and to differentiate into cells of the three germ layers, including blood, make them a valuable tool for the mechanistic studies of hematopoietic development, modeling of blood diseases, drug screening, toxicity studies, and the development of cellular therapies. Because blood formation in the embryo proceeds from hemogenic endothelium (HE) through an endothelial hematopoietic transition^1,2, the generation of HE in cultures would be essential to study the molecular mechanisms regulating the endothelial to hematopoietic transition and hematopoietic specification. Current methods for stu....

Protocol

1. Virus Preparations and Transcription Factor Combinations

1. Prepare solutions with pSIN-EF1α lentiviral expression plasmid containing protein-coding DNA for ETV2, GATA1, GATA2, TAL1 and LMO2 (Table 1).
2. Measure the concentration and purity of plasmid preparations used for lentiviral production by recording UV absorption with a spectrophotometer at 230 nm, 260 nm, and 280 nm. Note: DNA preparations demonstrating A260/280 and A260/230 values greater than 1.8 are generally considered good quality. Lower A260/280 values may indicate protein contamination, while lower A260/230 values indicate impurities with salts or ....

Results

The schematic diagram of HE and blood induction from hPSCs by overexpression of transcription factors is shown in Figure 1. ETV2 with GATA1 or GATA2 combination induces pan-myeloid hematopoiesis, while a GATA2, TAL1 +/- LMO2 combination induces predominantly erythro-megakaryocytic hematopoiesis. Both TF combinations directly induced HE cells that subsequently transformed into blood progenitors with a distinct spectrum of hematopoietic differentiation. Differentiation of hPSCs from the pluripotent state t.......

Discussion

The above-described method for hematopoietic differentiation of hPSCs by overexpression of TFs, represents a rapid and efficient approach for the generation of HE and myeloid and erytho-magakaryocytic progenitors from hESCs and iPSCs, thereby allowing the production of up to 30 millions blood cells from one million pluripotent stem cells¹⁰. This method exhibited consistent differentiation in multiple hESC and iPSCs lines¹⁰. During differentiation by ETV2 and GATA2, GATA1 factors as well as GATA2 and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Table 1. Induction of hPSCs differentiation with trancription factors and analysis of hemogenic endothelium and blood cells.
pSIN4-EF1a-ETV2-IRES-Puro	Addgene	Plasmid #61061	Lentiviral Vector
pSIN4-EF1a-GATA2-IRES-Puro	Addgene	Plasmid #61063	Lentiviral Vector
pSIN4-EF1a-GATA1-IRES-Puro	Addgene	Plasmid #61062	Lentiviral Vector
pSIN4-EF1a-TAL1-IRES-Puro	Addgene	Plasmid #61062	Lentiviral Vector
pSIN4-EF1a-LMO2-IRES-Puro	Addgene	Plasmid #61064	Lentiviral Vector
Hexadimethrine bromide (Polybrene)	Sigma-Aldrich	107689-10G	Cationic polymer used to increase the efficiency of infection
Y-27632 (Dihydrochloride) ROCK inhibitor	STEMCELL Technologies	72302	RHO/ROCK pathway inhibitor Inhibits ROCK
StemPro Accutase Cell Dissociation Reagent	Life Technologies	A11105-01	Cell Dissociation Reagent
Incomplete (growth factor- free) culture medium                                              mTeSR1 Custom formulation	WiCell Research Institute (Madison, WI)	MCF	Serum-free mTeSR1 medium without bFGF and TGFb
human SCF	Peprotech	300-07	Premium grade
human TPO	Peprotech	300-18	Research grade
human FGF-basic	Peprotech	100-18B	Premium grade
CD144 (VE-cad) FITC	BD Biosciences	560411	Endothelial marker (FACS)
CD226 PE	BD Biosciences	338305	Hematopoietic (FACS)
CD43 PE	BD Biosciences	560199	Hematopoietic (FACS)
CD73 APC	R&D Systems	FAB5795A	Endothelial marker (FACS)
CD45 APC	BD Biosciences	555485	Hematopoietic (FACS)
7AAD	Life Technologies	A1310	Live/Dead assay (FACS)
Paraformaldehyde	Sigma-Aldrich	P6148-500G	Cell fixation
Triton X-100	Sigma-Aldrich	T9284-500ML	Permeabilization
FBS	Fisher Scientific	SH3007003	Fetal bovine serum
Mouse anti-human CD43	BD Biosciences	551457	Pure, primary antibody for Immunofluorescence (IF) staining
Rabbit anti-human VE-cadherin	BenderMedSystem	BMS158	Primary (IF)
Anti-rabbit Alexa Fluor 488-conjugated	JacksonResearch	715-486-152	Secondary (IF)
Anti-mouse Alexa Fluor 594-conjugated	JacksonResearch	715-516-150	Secondary (IF)
DAPI nucleic acid stain	Life Technologies	D1306	Live/Dead assay (IF)
Clonogenic medium MethoCult H4435 Enriched	STEMCELL Technologies	4435	CFC-assay
Wright Stain solution	Sigma -Aldrich	32857	Staining cytospins
Table 2. hPSCs culture.
Human Pluripotent Stem Cells (hPSCs)	WiCell Research Institute (Madison, WI)	hESCs (WA01, WA09) human Embryonic Stem cells; iPSCs (DF-19-9-7T, DF-4-3-7T) transgene-free induced Pluripotent Stem Cells	hPSCs are able to self-renew and to differentiate into cells of three germ layers
Complete serum-free medium for culture of hPSCs                                                    mTeSR1 media	WiCell Research Institute (Madison, WI)	M500	Serum-free  medium with growth factors for feeder free culture of ESC/iPSCs
Matrigel	BD Biosciences/ Corning	356234	Matrix for maintenance of human ESC/iPSCs
DMEM/F-12, powder	Life Technologies	12500-062	Basal Medium
HyClone Dulbecco's PBS powder	Fisher Scientific	dSH30013.04	PBS
Dispase II, powder	Life Technologies	17105-041	Neutral protease, Cell dissociation
Table 3. Primers for detection of virus genomic integration.
Primer's Name	Forward (Fwd)  5’ -->3’	Reverse (Rev) 5’-->3’	Discription
pSIN EF1a Fwd	TTC CAT TTC AGG TGT CGT GA	--	EF1a promoter sequence
GATA1 Rev	--	TCC CTG TAG TAG GCC AGT GC	Coding Region
GATA2 Rev	--	GGT TGG CAT AGT AGG GGT TG	Coding Region
TAL1 Rev	--	AGG CGG AGG ATC TCA TTC TT	Coding Region
LMO2 Rev	--	GGC CCA GTT TGT AGT AGA GGC	Coding Region
ETV2 Rev	--	GAA CTT CTG GGT GCA GTA AC	Coding Region