Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Summary

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Abstract

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3β inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Introduction

Endothelial cells (ECs) are a heterogeneous population of cells that perform multiple functions throughout the human body and in engineered tissues. In addition to supporting and regulating other cell types (i.e., cardiomyocytes¹, osteoblastic cells²), these functions include forming a selective barrier between blood and tissues and assisting in tissue formation³. Differentiation of mature ECs during normal development requires diverse signaling pathways. Primordial ECs are derived from mesoderm progenitors, and are then specified toward mature arterial, venous, capillary and lymphatic phenotypes<....

Protocol

1. Reagents and reagent preparation

NOTE: A list of reagents is provided in Table of Materials.

1. Obtain the human pluripotent stem cell lines: H1-hESC, H9-Fucci-hESC.
   NOTE: The generation of hemogenic ECs may be more efficient in the H1 cell line.
2. Prepare matrix protein stocks: Aliquot the matrix protein into pre-chilled 1.5 mL tubes (on ice) so that each tube contains 1 mg of matrix protein. 1 mg of matrix protein is enough to coat all wells of two 6-well plates (12 wells total). Store the aliquots at -20 °C until use.
   NOTE: Perform all the steps involving matrix protein on ice or at....

Results

A schematic outlining the specification of primordial ECs and hemogenic ECs from hESCs, and a representative image of cells 24 h after plating are shown in Figure 1. Following specification, primordial ECs and hemogenic ECs are FACS purified on days 5 and 8, respectively. Primordial ECs are defined as CD31⁺ CD45^- and hemogenic ECs are defined as CD31⁺ KDR⁺ c-Kit⁺ CD34⁺ VE-Cadherin^- CD45^-. A representative.......

Discussion

Herein, the steps for producing hemogenic endothelial cells from human embryonic stem cells in approximately 1 week using a murine feeder- and serum-free 2D culture system (Figure 1) are outlined. This protocol expands on a method described by Sriram et al. (2015) to obtain primordial ECs³⁸. The primordial nature and specification potential of the CD31⁺ CD45^- ECs is demonstrated by culturing these cells on DLL4-coated plates and observing gene ex.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 cm dishes	Corning	430599	tissue culture treated
35 mm dishes	Corning	430165	tissue culture treated
6-well plates	Corning	3516	tissue culture treated
Antimicrobial reagent Brand Name: Normocin	Invitrogen	ant-nr-1
bFGF	R&D systems	233-FB-025	use at 50 ng/mL
BMP4	BioLegend	595202	use at 25 ng/mL
Bovine Serum Albumin (BSA)	Fisher Scientific	BP1600-1
Cell Detatchment Solution Brand Name: Accutase	Stemcell Technologies	7920
Dimethyl Sulfoxide (DMSO)	Sigma Aldrich	D2650-100mL
Dispase	Stemcell Technologies	7913
DLL4	R&D systems	1506-D4/CF	recombinant human; use at 10 μg/mL
DMEM:F12	Gibco	11320-033
Dulbecco's Phosphate Buffered Saline (PBS)	Gibco	14190144
Endothelial cell growth medium Brand Name: EGM-2 Endothelial Cell Growth Medium-2 BulletKit (EGM-2)	Lonza	CC-3162
FACS tubes	Corning	352235	polystyrene round bottom with filter cap
Fetal Bovine Serum (FBS)	Gemini Bio	100-106
Fibronectin	ThermoFisher Scientific	33016015	use at 4 μg/cm2
GSK3i/CHIR99021	Stemgent	04-0004-02	10 mM stock; use at 5 μM
Hanks Balanced Salt Solution (HBSS)	Gibco	14175-095
Hydrochloric Acid (HCl)	Fisher Scientific	A144S-500
Matrix protein  Brand Name: Matrigel	Corning	356230	Growth factor reduced. Refer to the Certificate of Analysis for the lot to determine the protein (Matrigel) concentration. This concentration is required to calculate the volume of Matrigel that contains 1 mg of protein.
Methylcellulose-based medium Brand Name: MethoCult H4435 Enriched	Stemcell Technologies	4435
Pluripotent stem cell differentiation medium Brand Name: STEMdiff APEL 2	Stemcell Technologies	5270
Pluripotent stem cells: H1, H9, H9-FUCCI	WiCell	WA09 (H9), WA01 (H1)	human; H9-FUCCI were obtained from Dr. Ludovic Vallier's lab at Cambridge Stem Cell Institute
Protein-Free Hybridoma Medium (PFMH)	Gibco	12040077
Retinoic Acid	Sigma Aldrich	R2625-50mg	use at 0.5 μM
Reverse transcription master mix Brand Name: iScript Reverse Transcription Supermix	BioRad	1708840
RNA extraction kit Brand Name: RNeasy Mini Kit	Qiagen	74104
Sodium Hydroxide (NaOH)	Fisher Scientific	SS255-1
Stem cell growth medium Brand Name: mTeSR1	Stemcell Technologies	85850
SYBR Green master mix Brand Name: iTaq Universal SYBR Green Master Mix	BioRad	1725121
Trypsin-EDTA	Gibco	25299956	0.25%
VEGF165 (VEGF-A)	PeproTech	100-20	use at 50 ng/mL
α-CD31-FITC	BioLegend	303104	2 μg/mL*
α-CD34-Pacific Blue	BioLegend	343512	2 μg/mL*
α-CD45-APC/Cy7	BioLegend	304014	2 μg/mL*
α-c-Kit-APC	BioLegend	313206	2 μg/mL*
α-Flk-1-PE/Cy7	BioLegend	359911	2 μg/mL*
α-VE-Cadherin-PE	BioLegend	348506	2 μg/mL*
* Antibody fluorescent conjugates should be optimized based on the cell sorter used. Presented here are the final concentrations utilized in this study.