Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells
March 31st, 2021
Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.
This protocol details a method to generate a well-characterized population of human hemogenic endothelial cells. These cells can be utilized to study the molecular regulation and endothelial-to-hematopoietic transition. Begin by culturing human embryonic stem cells for four days to differentiate them into primordial endothelial cells.
On day five, aspirate the medium above the cells. Then gently wash the cells with one milliliter of DMEM/F-12 per well. Add one milliliter of freshly prepared hemogenic endothelial cell differentiation medium per well and incubate the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide.
On day six and day seven, replace the medium above the cells with one milliliter of freshly prepared homogenic endothelial cell differentiation medium per well, and incubate the cells for another 24 hours. On day eight. after detaching and washing the cells, divide them evenly into microcentrifuge tubes on ice, each containing a minimum of 600 microliters of cells at a density of 1 times 10 to the 5th cells per milliliter.
Add antibodies to the tubes containing cells as appropriate and incubate on ice protected from light for 30 minutes. Pellet the cells by centrifugation at 1000 times G and 4 degrees Celsius for five minutes. Remove the supernatant and resuspend the pellets in 600 microliters of ice-cold sorting buffer.
Strain the samples through the mesh filter cap of five milliliter FACS tubes and store the cells on ice protected from light for immediate cell sorting. To isolate hemogenic endothelial cells by FACS, first gate the CD45-negative cell population. Then within the CD45-negative population, gate the CD31-positive cells.
Next within the CD31-positive population, gate for the VE-cadherin-negative cell population. And then within the VE-cadherin-negative population, gate for the c-Kit-positive cells. From the c-Kit-positive population, gate the CD34-positive cell population.
And finally from the CD34-positive cell population, identify and collect the KDR-positive cells. After seeding hemogenic endothelial cells in a Methylcellulose-based medium formulated for growth of Hematopoietic Progenitor Cells, Colony Forming Unit-Erythroid colonies, and Blast Forming Unit-Erythroid colonies are counted on day eight. Colony-Forming Unit-Granulocyte-Macrophage and Colony-Forming Unit-Granulocyte, Erythroid, Macrophage, and Megacaryocyte.
Multipotent hematopoietic progenitor colonies are counted on day 14. Per 1000 hemogenic endothelial cells plated, approximately 20 colony forming units are generated. Cells with endothelial cell morphology are also visible in the cultures.
These are the hemogenic endothelial cells that give rise to multilineage hematopoietic progenitors on a single-cell level. This protocol is a 2D serum and murine feeder-free culture system that can generate human hemogenic endothelial cells in roughly one week.
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Overview
This article presents a detailed protocol for deriving hemogenic endothelial cells from human embryonic stem cells in approximately one week. The method enables researchers to generate a well-characterized population of these cells, which are crucial for studying the endothelial-to-hematopoietic transition and have potential applications in regenerative medicine and blood product generation.
Key Study Components
Area of Science
- Stem cell biology
- Vascular biology
- Hematopoiesis
- Tissue engineering
Background
- Blood vessels are essential for tissue function and are lined by vascular endothelial cells.
- Hemogenic endothelial cells are a specialized subtype that give rise to hematopoietic stem and progenitor cells during development.
- Generating these cells in vitro is valuable for studying blood cell development and for potential therapeutic applications.
- Existing protocols for endothelial cell derivation do not yield well-characterized hemogenic endothelial cells from human stem cells.
Purpose of Study
- To establish a reproducible protocol for generating hemogenic endothelial cells from human embryonic stem cells.
- To enable the study of molecular regulation and the endothelial-to-hematopoietic transition in vitro.
- To provide a feeder-free, serum-free system suitable for downstream applications.
Methods Used
- Culture of human embryonic stem cells and stepwise differentiation over eight days.
- Induction of mesoderm and endothelial lineages using specific growth factors and small molecules (e.g., GSK3β inhibitor, bFGF, BMP4, VEGF-A, retinoic acid).
- Flow cytometry (FACS) for sequential gating and isolation of hemogenic endothelial cells based on surface markers (CD45-, CD31+, VE-cadherin-, c-Kit+, CD34+, KDR+).
- Functional assessment via colony-forming assays in methylcellulose-based medium to evaluate hematopoietic potential.
Main Results
- The protocol yields a defined population of hemogenic endothelial cells within one week.
- Approximately 20 colony-forming units per 1000 hemogenic endothelial cells are generated, indicating robust hematopoietic potential.
- Both erythroid and multipotent hematopoietic progenitor colonies are observed.
- Endothelial cell morphology is maintained in culture, confirming cell identity.
Conclusions
- This feeder-free, serum-free protocol efficiently generates human hemogenic endothelial cells from embryonic stem cells.
- The method facilitates in vitro studies of endothelial-to-hematopoietic transition and molecular regulation.
- It provides a platform for potential ex vivo blood product generation and reduces reliance on human donors.
