STEMdiff™ Endothelial Differentiation Kit

Figure 1. Schematic Workflow of Endothelial Induction Using the STEMdiff™ Endothelial Kit

In Phase 1, human embryonic stem (ES) or induced pluripotent stem (iPS) cells are cultured in a TeSR™ maintenance medium (mTeSR™ Plus, mTeSR™1, or TeSR™-E8™). On Day 1 (Phase 2) of the protocol, cells are ready for induction into early mesoderm progenitor cells by replacing TeSR™ medium with STEMdiff™ Mesodermal Induction Medium (MIM). By Day 3 (Phase 3), STEMdiff™ Mesoderm Induction Medium is replaced with STEMdiff™ Endothelial Induction Medium to derive endothelial cells. On Day 7, cells are passaged 5 - 6 times onto cultureware pre-coated with Animal Component-Free Cell Attachment Substrate in STEMdiff™ Endothelial Expansion Medium (Phase 4).

Figure 2. A Representative Flow Cytometric Analysis of Endothelial Marker Expression in hPSC-Derived Endothelial Cells

Human pluripotent stem cell (hPSC; H9 cell line)-derived endothelial cells were obtained at Day 7 using STEMdiff™ Endothelial Induction Medium. Greater than 85% of the cells were CD34+ and had high levels of CD31 and CD144 expression. With subsequent passages, the proportion of cells expressing endothelial markers (CD34+, CD31, and CD144) increased up to passage 5.

Figure 3. STEMdiff™ Endothelial Differentiation Kit Generates Functional hPSC-Derived Endothelial Cells

Endothelial cells generated from hPSCs (F016 cell line) using the STEMdiff™ Endothelial Differentiation Kit take up acetylated LDL when plated at 10,000 cells/cm2. Cells are able to form tubular networks in vitro in a tube formation assay when plated at 20,000 cells/well in a 96 well-plate for 24 hrs.

Figure 4. Endothelial Cells Expand Faster in STEMdiff™ Endothelial Expansion Medium Compared to Serum-Containing Medium

STEMdiff™ Endothelial Expansion Medium (A) sustains expansion rate in later passages and leads to (B) superior expansion of hPSC (C1 cell line)-derived endothelial cells when compared to serum-containing medium.

Figure 5. hPSC-Derived Endothelial Cells Generated Using the STEMdiff™ Endothelial Differentiation Kit Express High Levels of ACE2

(A) hPSC (C1 cell line)-derived endothelial cells were generated using the STEMdiff™ Endothelial Differentiation Kit and expanded in STEMdiff™ Endothelial Expansion Medium for 6 passages at 10,000 cells/cm2. (B) The cells were then analyzed for expression of angiotensin-converting enzyme 2 (ACE2). 85% of cells expressed high levels of ACE2.