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STEMdiff™ 单核细胞试剂盒

用于将人多能干细胞（hPSCs）分化为单核细胞
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产品号 #05320_C

用于将人多能干细胞（hPSCs）分化为单核细胞

产品优势

  • 无血清、无饲养层配方
  • 单的单层培养方案，在悬浮液中生成单核细胞，便于收获
  • 14 - 23 天内生成 CD14⁺ 单核细胞
  • 在多个 hPSC 细胞系中稳定生成单核细胞

产品组分包括

  • STEMdiff™ 造血基础培养基，120 mL
  • STEMdiff™ 造血补充剂 A (200X)，225 μL
  • STEMdiff™ 造血补充剂 B (200X)，225 μL
  • STEMdiff™ 单核细胞分化添加剂 (100X)，3 x 1 mL（产品号 #05324）
  • StemSpan™ SFEM II，3 x 100 mL
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总览

使用无饲养层、无血清的 STEMdiff™ 单核细胞试剂盒，可将人多能干细胞（hPSCs）分化为表达 CD14 的单核细胞。

该简便的实验方案在二维贴壁培养中进行。在前 3 天，培养基 A 诱导细胞向中胚层分化。在接下来的 4 天，使用培养基 B 进一步诱导中胚层细胞向造血谱系分化。在第 7 天，将培养基更换为单核细胞分化培养基，以促进其向单核细胞分化。CD14 +单核细胞最早可从第 14 天开始从培养上清液中直接收集，并可在后续培养过程中多次收集。CD14 +细胞的峰值频率通常在 60% - 80%之间。

hPSC 衍生的单核细胞可分别使用ImmunoCult™ 树突状细胞培养试剂盒或ImmunoCult™-SF 巨噬细胞培养基进一步分化为树突状细胞或巨噬细胞。

为了方便起见，用于配制单核细胞分化培养基所需的成分StemSpan™ SFEM II和STEMdiff™ 单核细胞分化添加剂 (100X)也可单独购买。

亚型
专用培养基
 
细胞类型
树突状细胞（DCs），巨噬细胞，单核细胞，髓系细胞，多能干细胞
 
种属
人
 
应用
细胞培养，分化，扩增
 
品牌
STEMdiff
 
研究领域
疾病建模，免疫，干细胞生物学
 
制剂类别
无血清
 

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Data Figures

Differentiation of hPSC-Derived CD34+ Cells into CD14+ Monocytes

Figure 1. Monocyte Differentiation Protocol

One day prior to differentiation, human pluripotent stem cell (hPSC) colonies are harvested and seeded as small aggregates (100 - 200 μm in diameter) at 10 - 20 aggregates/cm2 in mTeSR™1, TeSR™-E8™, or mTeSR™ Plus media. After one day, the medium is replaced with Medium A (STEMdiff™ Hematopoietic Basal Medium + Supplement A) to induce mesodermal specification (stage 1). On day 3, the medium is changed to Medium B (STEMdiff™ Hematopoietic Basal Medium + Supplement B) to promote hematopoietic specification (stage 2). On day 7, the medium is replaced with Monocyte Differentiation Medium (StemSpan™ SFEM II + STEMdiff™ Monocyte Differentiation Supplement) to promote the production of CD14+ monocytes (stage 3). Monocyte Differentiation Medium is used for all medium changes for the remaining culture period. CD14+ cells can be detected in suspension starting after day 14, and their frequency gradually increases until day 17 - 23. CD14+ cells can be harvested directly from the culture supernatant during medium changes.

hPSC-Derived CD14+ Monocyte Characterization, Frequency and Yield

Figure 2. Robust and Efficient Generation of CD14⁺ Monocytes Using STEMdiff™ Monocyte Kit

hPSCs were differentiated to monocytes using the 2D culture system described in Figure 1. Between days 17 and 23, cells were harvested every 2 - 3 days and analyzed by flow cytometry for CD14 expression. Representative flow cytometry plots are shown for (A, B) iPS (WLS-1C)-derived cells and (C, D) ES (H9)-derived cells. (E) The average frequency of viable CD14+ monocytes at the peak harvest was 61 - 78%. The average yield of CD14+ monocytes produced per 6-well plate at the peak harvest was between 1.6 x 10^6 and 7.1 x 10^6 cells. Data are shown as mean ± SEM (n = 3 - 14).

Characterization and Phagocytosis Analysis of Macrophages

Figure 3. STEMdiff™ Monocyte Kit Generates Monocytes That Are Capable of Differentiation to Macrophages

hPSC-derived monocytes were harvested after 21 days of culture. These were then differentiated to macrophages using ImmunoCult™-SF Macrophage Medium (Catalog #10961) with 100 ng/mL M-CSF for 4 days. Macrophages were then incubated for an additional 2 days with either 10 ng/mL of LPS and 50 ng/mL of IFN-γ, or 10 ng/mL IL-4, to become polarized to M1 or M2a macrophages, respectively. Representative flow cytometry plots of (A) M1 and (B) M2a macrophages produced from the WLS-1C iPS cell line are shown. (C) To measure phagocytosis, PSC-derived M2a macrophages and peripheral blood (PB) monocyte-derived M2a macrophages (primary M2a macrophages), were incubated with pHrodo™ Red Zymosan A BioParticles® Conjugate and incubated at 37°C for 8 hours. Images were acquired using the IncuCyte® ZOOM every 30 minutes and analyzed for internalization of pHrodo™ Red Zymosan A BioParticles® (measured as red object/mm2). hPSC-derived and primary M2a macrophages show similar phagocytic activity.

Characterization of Dendritic Cells

Figure 4. STEMdiff™ Monocyte Kit Generates Monocytes That Can Be Differentiated to Dendritic Cells

hPSCs were differentiated into monocytes, harvested after 21 days, and differentiated to dendritic cells using ImmunoCult™ Dendritic Cell Culture Kit (Catalog #10985). Half of the dendritic cells were harvested on day 7 and examined for CD14 and CD83 expression to identify CD14⁻CD83⁻/lo immature dendritic cells. The remaining dendritic cells were activated for 2 days and assessed for the presence of CD14⁻CD83⁺ mature dendritic cells at day 7. Representative cultures initiated with ES (H9) cells are shown for production of (A) immature dendritic cells and (B) mature dendritic cells.

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™ Monocyte Kit
Catalog #
05320
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
STEMdiff™ Monocyte Kit
Catalog #
05320
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
STEMdiff™ Monocyte Kit
Catalog #
05320
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
STEMdiff™ Monocyte Kit
Catalog #
05320
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
STEMdiff™ Monocyte Kit
Catalog #
05320
Lot #
All
Language
English
Document Type
Safety Data Sheet 5
Product Name
STEMdiff™ Monocyte Kit
Catalog #
05320
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

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Species Human
Formulation Category Serum-Free
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