Figure 1. Hematopoietic Differentiation Protocol

For 2D culture, start with harvesting and seeding human hESC/hiPSC colonies as small aggregates on Matrigel®-coated plates in mTeSR™1, mTeSR™ Plus, TeSR™-E8™ on Day -1, or in eTeSR™ on Day -2. For 3D culture, seed the aggregates in matrix-free suspension culture in TeSR™-AOF 3D on Day -1 using 6-well plates/Nalgene™ bottles on orbital shakers, or in PBS-MINI MagDrive Bioreactors. For both protocols, replace the TeSR™ medium with Medium A (STEMdiff™ Hematopoietic Basal Medium containing Supplement A) on Day 0 to begin inducing the cells toward a mesoderm-like state. On Day 2, a half-medium change is performed with fresh Medium A. On Day 3, the medium is changed to Medium B (STEMdiff™ Hematopoietic Basal Medium containing Supplement B) with half-medium changes on Days 5, 7 and 10, to promote further differentiation into hematopoietic cells. Typically, by Day 12, large numbers of HPCs can be harvested from the culture supernatant. For the hematopoietic differentiation protocol in 3D suspension culture, refer to this technical bulletin for step-by-step instructions.

Figure 2. Morphology of hPSC-Derived HPCs

Representative images of (A) hES (H1) cells and (B) hiPS (WLS-1C) cells on Day 12 of differentiation to HPCs using the STEMdiff™ Hematopoietic Kit. Differentiated cells exhibit typical HPC morphology as round cells that float freely in suspension.

Figure 3. Efficient and Robust Generation of CD34+CD45+/CD43+ HPCs

hES and hiPS cells were cultured for 12 days in single wells of 12-well plates using the STEMdiff™ Hematopoietic Kit. At the end of the culture period, cells in suspension were harvested and analyzed by flow cytometry for expression of hematopoietic cell surface markers: CD34, CD45 and CD43. (A,B) Example flow cytometry plots for hematopoietic cell surface-marker analysis of cultures of hES (H1 and H9) and hiPS (STiPS-M001) cells. (C,D) Percentages and total numbers of CD34+CD45+ cells in cultures of hES (H1 and H9) or hiPS (WLS-1C, STiPS-F016, STiPS-M001 and STiPS-B004) cells are shown. Data shown as mean ± SEM; n ≥ 3.

Figure 4. hPSC-Derived HPCs Produce Colonies of Multiple Lineages

Cells in suspension were harvested from the cultures on Day 12 of the hematopoietic differentiation protocol and assessed in colony-forming unit (CFU) assays using MethoCult™ H4435 Enriched (Catalog #04435) methylcellulose-based medium. (A) CFU frequencies in cultures of 6 different hPSC lines. (Data shown as mean ± SEM; n ≥ 3.) The CFU frequencies were variable between the different cell lines, with on average approximately 120 CFU per 10,000 hPSC-derived HPCs plated. (B) The progenitor cell types observed included granulocyte/macrophage (CFU-M, CFU-G and CFU-GM), erythroid (BFU-E and CFU-E) and occasional mixed (CFU-GEMM) colonies. Representative colony images are shown at 40X magnification.

Figure 5. Generation of Hematopoietic Progenitor Cells from hPSCs Maintained in mTeSR™ Plus

Human ES (H1, H9) and iPS (STiPS-M001, WLS-1C) cell lines maintained in mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds) were differentiated to hematopoietic progenitor cells using the STEMdiff™ Hematopoietic Kit. At the end of the differentiation period, cells were harvested from the supernatant and analyzed by flow cytometry for co-expression of CD34+ and CD45+ . (A) Representative density plots showing CD34+ and CD45+ expression, (B) percentage of cells co-expressing CD34+ and CD45+ , and (C) total number of viable cells harvested are shown. Data are expressed as the mean (± SEM); n=4.