STEMdiff™ 中胚层诱导培养基

用于早期中胚层分化的成分明确、无异种成分的诱导培养基

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产品号 #05220_C

用于早期中胚层分化的成分明确、无异种成分的诱导培养基

产品优势

成分明确且无异种成分
可在 2–4 天内快速诱导中胚层
高效且可重复地分化多种人ES和iPS细胞系
生成的早期中胚层细胞可进一步分化为多种下游细胞类型

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Need a high-quality cell source? Use the hiPSC SCTi003-A (female) or SCTi004-A (male) control lines, manufactured with mTeSR™ Plus.

要查看实验方案所需的所有配套产品，请参阅《实验方案与技术文档》。

TeSR™-E8™
Feeder-free, animal component-free culture medium for maintenance of human ES and iPS cells
Gentle Cell Dissociation Reagent
cGMP, enzyme-free cell dissociation reagent
Y-27632 (Dihydrochloride)
RHO/ROCK pathway inhibitor; Inhibits ROCK1 and ROCK2
mTeSR™1
cGMP, feeder-free maintenance medium for human ES and iPS cells
Labeling Antibodies
Compatible antibodies for purity assessment of isolated cells

总览

实验数据

产品说明书及文档

应用领域

总览

STEMdiff™ 中胚层诱导培养基（MIM）是一种成分明确、无异种成分的培养基，适用于从人胚胎干细胞（ES）和诱导多能干细胞（iPS）中生成早期中胚层细胞。由于中胚层分化的常规方法常常操作复杂且结果不一致，因此推荐使用简短且操作简便的 STEMdiff™ MIM 单层培养方案来诱导人多能干细胞（hPSC）分化。STEMdiff™ MIM 是一种完全培养基，可产生富含早期中胚层的细胞群，其特征为 Brachyury (T) 和 NCAM 标记物的阳性表达。作为人多能干细胞培养流程的一部分，STEMdiff™ MIM 可高效诱导在TeSR™培养基中培养的hPSC进行分化。通过定向诱导，使用 STEMdiff™ MIM 所获得的早期中胚层细胞可进一步分化为多种特化细胞类型，如成骨细胞、软骨细胞、脂肪细胞或内皮细胞。

亚型
专用培养基

细胞类型
中胚层，PSC衍生，多能干细胞

种属
人

应用
细胞培养，分化

品牌
STEMdiff

研究领域
干细胞生物学

制剂类别
无血清，Xeno-Free

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实验数据

Figure 1. Schematic of Mesoderm Induction Medium Differentiation Timeline

On day 0, hPSC colonies are harvested and seeded as single cells at 5 x 10 4 /cm 2 in mTeSR™1 or TeSR™-E8™ and supplemented with 10 µM Y-27632. TeSR™ medium is replaced on day 1 with STEMdiff™ Mesoderm Induction Medium when cells are at approximately 20 - 50% confluency. Cells are then fed daily and cultured in STEMdiff™ MIM (days 2-4). Cells can be transferred to downstream differentiation conditions between days 3 - 5 or collected on day 5 for analysis.

Figure 2. STEMdiff™ MIM Generates a Homogenous Population of T + OCT4 - Early Mesoderm

(A) Data showing marker expression characteristic of the early mesoderm (positive Brachyury (T) expression and negative OCT4 and SOX 17 expression) on day 5 of the protocol. Data is expressed as a mean percentage of cells expressing each marker ± SD, n = 33 (T, OCT4), n = 5 (SOX17). (B) Expression of undifferentiated cell markers (OCT4, SOX2, NANOG) and early mesoderm markers (T, MIXL1, NCAM), measured by quantitative PCR (qPCR) and normalized to levels in undifferentiated cells, n = 2.

Figure 3. Mesoderm Differentiation and Cell Expansion are Efficient and Comparable Across Multiple hPSC Cell Lines

Graphs show mesoderm formation in multiple human ES (H1 and H9) and iPS (WLS-4D1, WLS-1C, STiPS-M001 and STiPS-F016) cell lines as measured by expression of Brachyury (T) and absence of OCT4. Cells maintained in (A) mTeSR™1 or (B) TeSR™-E8™ medium were differentiated using STEMdiff™MIM. (A, n = 2 - 10 per cell line, B, n = 3, data are expressed as a mean percentage ± SD) (C) Mesoderm differentiation on Corning® Matrigel® or Vitronectin XF™ is comparable. (n = 5, data are the mean percentage ± SD) (D) Average fold expansion of cells cultured in STEMdiff™MIM, as determined by cell yield / cells seeded. (n = 3 - 13. Error bars indicate SEM)

Figure 4. Phenotype of Cells Treated with STEMdiff™ MIM is Consistent with Early Mesoderm

Representative flow cytometry plots showing the switch from (A) EpCAM + NCAM -/low in hPSCs cultured in mTeSR™1 to (B) EpCAM -/low NCAM + expression in STEMdiff™ MIM-treated cells (day 5). EpCAM -/low NCAM + expression is characteristic of the early mesoderm. Expression of PDGFRα and KDR are low in both (C) hPSCs cultured in mTeSR™1 and (D) early mesoderm cells derived with STEMdiff™ MIM.

Figure 5. Mesenchymal Stem Cells Derived from Early Mesoderm Cells Can Be Further Differentiated in In Vitro Assays

(A) Early mesoderm cells generated with the 5-day STEMdiff™ MIM protocol and subsequently cultured with MesenCult™-ACF develop mesenchymal stem cell (MSC)-like morphology, 40X magnifi cation. MSC-like cells can subsequently differentiate into (B) adipocytes (Oil Red O staining), 200x magnification, (C) chondrocytes (Alcian Blue staining), 100X magnification, and (D) osteogenic cells (Fast Red and Silver Nitrate staining), 40X magnification.

Figure 6. Robust Endothelial Differentiation of STEMdiff™ MIM-Generated Early Mesoderm Cells

On day 3 of the STEMdiff™ MIM protocol, early mesoderm cells were switched to a downstream endothelial differentiation protocol based on Tan et al. (A) Differentiated cells display characteristic endothelial cell morphology and (B) are able to uptake Dil-Ac-LDL (red). Representative flow cytometry plots showing (C) 85.5% CD144 + CD31 + and (D) 87.6% CD105 + KDR + expression in differentiated endothelial cells.