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Figure 1. Generation of Hematopoietic Progenitors Using STEMdiff™ APEL™2 in a Spin EB Protocol

(A) Embryoid bodies can be generated by seeding 8000 hPSCs/well of a 96-well plate or (B) 500 hPSCs per microwell of AggreWell™400 24-well plates using STEMdiff™ APEL™2 Medium supplemented with Rho Kinase Inhibitor. Images shown were taken 24 hours after seeding. For hematopoietic differentiation using STEMdiff™ APEL™2 Medium, EBs in 96-well plates were generated following a modified Zhu and Kaufman protocol (STEMdiff™ APEL™2 supplemented with Human Recombinant SCF, ACF, Human Recombinant VEGF-165, ACF, Human Recombinant BMP-4, Rho Kinase Inhibitor IV, and Human Recombinant bFGF). At Day 6, EBs were dissociated, and cell counts and flow cytometry were performed. Hematopoietic progenitors were generated in one hESC (H9) and two hiPSC (SCTi003-A and WLS- 1C) lines. Cells were gated on singlets and viability. (C) Representative flow plot of WLS-1C. (D) Quantification of CD34+ cells at Day 6 and (E) quantification of yield of viable CD34+ cells generated per 96-well plate. Error bars are shown as mean +/- SEM, n = 3.

Figure 2. EBs Generated with STEMdiff™ APEL™2 Medium Can Differentiate to NK Cells at High Efficiency

Following 6 days of hematopoietic differentiation in STEMdiff™ APEL™2 Medium (Figure 1), 16 EBs were transferred into each well of a gelatin-coated 6-well plate and cultured for 28 days in NK cell differentiation medium described in the protocol. After 28 days, floating cells were harvested, and cell counts and flow cytometry were performed using a panel of NK markers: Anti-Human CD56 (NCAM) Antibody, Clone HCD56 (APC), Anti-Human CD45 Antibody, Clone HI30 (PE), Anti-Human CD16 Antibody, Clone 3G8 (FITC), KIR (clone HP-MA4), NKG2D, NKp44, and NKp46. (A) Cell surface marker expression on pluripotent stem cell (PSC)-derived CD56+ NK Cells. Cells were gated on singlets and viability. NK cells were generated in 2 cell lines: ES cell line (H9) and human iPS cell line (WLS-1C). (B) Quantification of CD45+CD56+ cells at day 28 and (C) quantification of yield of viable CD56+ cells generated per 6-well plate. Error bars are shown as mean +/- SEM, n=3.

Figure 3. EBs Generated with STEMdiff™ APEL™2 Medium Can Differentiation Into Functional NK Cells

Following 6 days of hematopoietic differentiation in STEMdiff™ APEL™2 Medium (Figure 1), EBs were transferred to gelatin-coated plates and cultured for 28 days in NK differentiation media following the . protocol. (A) After 28 days, hPSC-derived CD56+ NK cells were co-cultured with K562 target cells labeled with eBioscience™ Cell Proliferation Dye eFluor™ 670 for 5 hours at effector to target (E/T) ratios of 1:1 or 1:3. Positive controls were freshly isolated peripheral blood (PB) NK cells pre-cultured in ImmunoCult™ NK Cell Base Medium prior to co-culture with K562 target cells. K562 cells were cultured in the absence of NK cells as a negative control. The average percent target killing by hPSC-derived NK cells at a 1:1 E/T ratio ranged between 46% and 62%. For degranulation and IFN-γ production experiments, hPSC-derived CD56+ NK cells were co-cultured with K562 targets for 6 hours at an E/T ratio of 1:3, or left unstimulated in the absence of target cells. Co-cultures were set up in the presence of CD107a antibody, and monensin was added after 1 hour of co-culture. Cultures were stained with GloCell™ Fixable Viability Dye Red 780 and an anti-human CD56 antibody at the end of co-culture. (B) To measure degranulation, surface CD107a was assessed using flow cytometry. IFN-γ production was assessed following fixation and permeabilization of cells and staining with an antibody specific to human IFN-γ (clone 4S.B3). (C) Representative flow plots and (D) quantification show gating on fluorescently labeled CD56+CD107a+ NK cells. Upon stimulation, hPSC-derived CD56+ NK cells are able to degranulate, as shown by surface expression of CD107a (56 - 66% for K562 stimulation) and secrete IFN-γ (18 - 27% for K562 stimulation). Data are shown as mean among 2 - 3 independent experiments.

Figure 4. Generation of hPSC-Derived Hematopoietic Lineages Cultured in STEMdiff™ APEL™2 in 2D

STEMdiff™ APEL™2 Medium can be used to generate multiple hematopoietic lineages from hPSCs in 2D when supplemented with appropriate cytokines. For example, . used STEMdiff™ APEL™2 Medium plus cytokines to generate CD34+/KDR+/CDH5+ hemogenic endothelial cells (not shown) and downstream erythroblasts. (A) By Day 18 of differentiation, red-colored erythroblasts were observed, and (B) by Day 30, flow cytometry showed a high proportion of cells were GlyA+/CD71- , markers of a mature erythrocyte population. . used STEMdiff™ APEL™2 Medium, StemSpan™-ACF Erythroid Expansion Medium, and Iscove's MDM plus cytokines to generate CD34+/CD31+ hemogenic endothelial cells (not shown) and downstream megakaryocyte cells. (C) Flow cytometry at Day 16 of differentiation demonstrated a CD41a+/CD42b+ cell population, markers of megakaryocyte cells. (D) By Day 21 of differentiation, immunostaining revealed expression of megakaryocyte markers and morphology typical of pro-platelets, indicated by the arrow. Adapted from . and ., both available under a . In an alternative protocol, CD34+/CD45+ hematopoietic progenitors were generated by seeding hPSCs onto Matrigel® and culturing for 12 days in STEMdiff™ APEL™2 Medium supplemented with cytokines. On Day 12, hematopoietic progenitors were harvested from the culture supernatant, and cell counts and flow cytometry were performed. (E) Representative image of a hiPSCl line (WLS-1C) at Day 12 and (F) flow cytometry plot for hematopoietic progenitor markers CD34+CD45+. In this protocol, hematopoietic progenitors were generated from 3 cell lines; hESC line (H9) and hiPSC lines (SCTi003-A & WLS-1C). (G) Hematopoietic progenitor marker expression and (H) yield of live cells expressing CD34+CD45+ markers at Day 12 are shown.

Figure 5. Generation of hPSC-Derived Endothelial Cells Using STEMdiff™ APEL™2 Medium

hPSCs were plated at 50,000 cells/cm² and cultured for 6 days in STEMdiff™ APEL™2 plus cytokines (BMP4, CHIR, VEGF) to generate endothelial cells based on the published . 2D protocol. On Day 6, cells were harvested, and cell counts and flow cytometry were performed. Endothelial differentiation was performed in 3 cell lines, one hESC line (H9) and two hiPSC lines (SCTi003-A and WLS-1C ). (A) Representative image and (B) flow cytometry data for endothelial markers CD31+CD144+ of H9 at Day 6. Cells were gated on singlets and viability. (C) Quantification of CD31+CD144+ cells and (D) yield of viable CD31+CD144+ cells per cm².