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Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.
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.
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<....
1. Reagents and reagent preparation
NOTE: A list of reagents is provided in Table of Materials.
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.......
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.......
This work was partially supported by NIH grants HL128064 and U2EB017103. Further support was provided by CT Innovations 15-RMB-YALE-04 grant.
....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: vAccutase | 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 mg/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. |
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