STEMdiff™ APEL™2 Medium for PSC Differentiation

概述

STEMdiff™APEL™2 Medium是一种完全定义的、无血清和无动物来源的培养基，用于人类胚胎干细胞（ES）和诱导多能干细胞（iPS）的分化。它是基于Andrew Elefanty博士发表的APEL配方，缺乏未定义的成分，如无蛋白杂交瘤培养基。

STEMdiff™APEL™2可用于贴壁或基于胚状体（EB）的方案，如AggreWell™。它可以与多种不同的诱导因子或细胞因子一起使用，以支持沿外胚层，中胚层和内胚层谱系的分化。

Subtype
Specialized Media

Cell Type
Pluripotent Stem Cells

Species
Human

Application
Cell Culture, Differentiation

Brand
STEMdiff

Area of Interest
Drug Discovery and Toxicity Testing, Stem Cell Biology

Formulation Category
Animal Origin-Free

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Data Figures

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 3 cell lines; ES cell line (H9) and human iPS cell lines (STCi-003-A and WLS-1C). 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 Zhu and Kaufman 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 Zhu and Kaufman 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, Jeon et al. 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. Krisch et al. 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 Jeon et al. and Krisch et al., both available under a Creative Commons 4.0 License. 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 hiPS cell 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; ES cell line (H9) and human iPS cell 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 Tan et al. 2D protocol. On Day 6, cells were harvested, and cell counts and flow cytometry were performed. Endothelial differentiation was performed in 3 cell lines: ES cell line (H9) and human iPS cell lines (STCi-003-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.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type

Product Name

Catalog #

Lot #

Language

Document Type

Product Information Sheet

Product Name

STEMdiff™ APEL™2 Medium

Catalog #

05275, 05270

Lot #

All

Language

English

Document Type

Safety Data Sheet

Product Name

STEMdiff™ APEL™2 Medium

Catalog #

05275, 05270

Lot #

All

Language

English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area

Workflow Stages

Workflow Stages for Human Pluripotent Stem Cell Research

Reprogramming

Maintenance

Differentiation

Workflow Stages for hPSC-Derived Endoderm Cell Research

Endoderm Induction

Characterization and Isolation

Downstream Differentiation

Workflow Stages for hPSC-Derived Mesoderm Cell Research

Mesoderm Induction

Downstream Differentiation

Resources and Publications

Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity. H. Zhu et al. Cell stem cell 2020 jun

Abstract

Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here, we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently, CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration, and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together, these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity.

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Generation of human vascularized brain organoids M. T. Pham et al. NeuroReport 2018

Abstract

The aim of this study was to vascularize brain organoids with a patient's own endothelial cells (ECs). Induced pluripotent stem cells (iPSCs) of one UC Davis patient were grown into whole-brain organoids. Simultaneously, iPSCs from the same patient were differentiated into ECs. On day 34, the organoid was re-embedded in Matrigel with 250 000 ECs. Vascularized organoids were grown in vitro for 3-5 weeks or transplanted into immunodeficient mice on day 54, and animals were perfused on day 68. Coating of brain organoids on day 34 with ECs led to robust vascularization of the organoid after 3-5 weeks in vitro and 2 weeks in vivo. Human CD31-positive blood vessels were found inside and in-between rosettes within the center of the organoid after transplantation. Vascularization of brain organoids with a patient's own iPSC-derived ECs is technically feasible.

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Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience. P. A. De Sousa et al. Stem cell research 2017 APR

Abstract

A fast track Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement

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