STEMdiff™心室心肌细胞分化试剂盒

人PSCs向心室心肌细胞分化的无血清培养基和人PSCs来源的心肌细胞的长期维持

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产品号 #（选择产品）

产品号 #05010_C

人PSCs向心室心肌细胞分化的无血清培养基和人PSCs来源的心肌细胞的长期维持

产品优势

支持整个hscs衍生的心肌细胞工作流程
简单的单层方案在15天内产生心肌细胞
一个无血清试剂盒产生超过5000万个心肌细胞（cTnT+）
在多个hPSC线之间具有最小可变性的稳健性能

产品组分包括

STEMdiff™心肌细胞分化基础培养基，380 mL

STEMdiff™心室心肌细胞分化补充剂A (10X), 10ml

STEMdiff™心室心肌细胞分化补充剂B (10X), 10ml

STEMdiff™心室心肌细胞分化补充剂C (10X), 20ml

STEMdiff™心肌细胞维持基础培养基，490 mL

STEMdiff™心肌细胞维持补充剂（50X）， 10 mL

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概述

STEMdiff™心室心肌细胞分化试剂盒（目录#05010）包括用于将人胚胎干（ES）和诱导多能干细胞（iPS）细胞（人多能干细胞[hPSCs]）分化为心室心肌细胞（心肌肌钙蛋白t阳性[cTnT+]）的培养基，以及用于维持hpsc来源的心肌细胞的培养基。该无血清试剂盒可用于生成源自mTeSR™1（目录#85850），mTeSR™Plus（目录#100-0276），TeSR™-AOF（目录#100-0401）或TeSR™-E8™（目录#05990）中维持的hPSCs的团块培养的心室心肌细胞。这些细胞中超过80%为cTnT+。12孔板的单孔平均可收获1 × 10^6个细胞。

STEMdiff™心肌细胞维持试剂盒（目录#05020）包括维持基础培养基和补充剂；它可以用于长期维持hpsc来源的心肌细胞一个月或更长时间。这些心肌细胞可用于各种下游应用和分析。

注：本产品原名STEMdiff™心肌细胞分化试剂盒；产品本身和制造过程没有改变，但是名称已经更新，以便更准确地反映生成的单元格类型。

Data Figures

Figure 1. Cardiomyocyte Differentiation Protocol

Two days before the differentiation protocol, hPSC colonies are harvested and seeded as single cells at 350,000 cells/well in a 12-well format in TeSR™ medium. After one day (Day -1), the medium is replaced with fresh TeSR™ medium. The following day (Day 0), the TeSR™ medium is replaced with Medium A (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement A) to begin inducing the cells toward a cardiomyocyte fate. On day 2, a full medium change is performed with fresh Medium B (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement B). On days 4 and 6, full medium changes are performed with fresh Medium C (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement C). On day 8, medium is switched to STEMdiff™ Cardiomyocyte Maintenance Medium with full medium changes on days 10, 12 and 14, to promote further differentiation into cardiomyocyte cells. Small beating areas of cardiomyocytes can be seen as early as day 8, progressing to a full lawn of beating cardiomyocytes that can be harvested as early as day 15.

Figure 2. Morphology of hPSC-Derived Cardiomyocytes

Representative images of (A) hES (H9) cells and (B) hiPS (WLS-1C) cells on day 15 of differentiation to cardiomyocytes using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. Differentiated cells exhibit typical cardiomyocyte morphology as an adherent, tightly packed web-like monolayer of beating cells. (C) Representative confocal microscopy image of a single hPSC-derived cardiomyocyte generated with the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit and stained with cTnT (green) and DAPI (blue).

Figure 3. Efficient and Robust Generation of cTnT-Positive Cardiomyocytes

hES and hiPS cells were cultured for 15 days in single wells of 12-well plates using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. At the end of the culture period, cells were harvested and analyzed by flow cytometry for expression of cardiac troponin T (cTnT). (A) Histogram analysis for cardiomyocyte cell marker cTnT for cultures of hES (H9) and hiPS (WLS-1C and STiPS-M001) cells. (Filled = sample; blank = secondary antibody only control) (B,C) Percentages and total numbers of cells expressing cTnT in cultures of hES or hiPS cells are shown. Data shown as mean ± SEM; n=3.

Figure 4. hPSC-Derived Cardiomyocytes Exhibit a Robust and Stable Excitability Profile

Microelectrode array (MEA) voltage recordings of cardiomyocytes (day 27) derived from human pluripotent stem cells generated and maintained with the STEMdiff™ Cardiomyocyte Differentiation and Maintenance Kits. The hPSC-derived cardiomyocytes have a characteristic electrical profile and stable beat rate. A large depolarization spike followed by a smaller repolarization deflection is observed.

Microelectrode array and flow cytometry of human ES and iPS cells maintained in mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds) and differentiated to cardiomyocytes using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit.

Figure 5. Generation of Cardiomyocytes from hPSCs Maintained in mTeSR™ Plus

Human ES (H9) and iPS (WLS-1C) cells were maintained in mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds) and differentiated to cardiomyocytes using the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit. At the end of the differentiation period, cells were harvested and analyzed by microelectrode array (MEA) and flow cytometry. (A) Representative MEA voltage recordings of cardiomyocytes (day 20) demonstrate a characteristic electrical profile and stable beat rate. (B) Percentages of cells expressing cTNT and (C) total number of viable cells harvested are shown. Data are expressed as the mean (± SEM); n=2.

Microscopy images of iPSCdirect cells and differentiated ventral cardiomyocytes, and a video of coordinated contraction or beating behavior of cardiomyocytes in a culture dish

Figure 6. iPSCdirect™ SCTi003-A Human Pluripotent Stem Cells Can Successfully Differentiate into Ventricular Cardiomyocytes

Ventricular cardiomyocytes were generated from iPSCdirect™ SCTi003-A cells using STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (Catalog #05010). (A) 48 hours after thawing and plating in mTeSR™ Plus and CloneR™2, iPSCdirect™ cells reached the desired confluency and are ready for Day 0 of differentiation according to the STEMdiff™ Ventricular Cardiomyocyte Product Information Sheet. (B) By Day 15 of differentiation, monolayer cultures show iPSC-derived ventricular cardiomyocytes that (C) exhibit coordinated beating behavior.

Applications

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Publications (14)

Role of Blood Oxygen Saturation During Post-Natal Human Cardiomyocyte Cell Cycle Activities. L. Ye et al. JACC. Basic to translational science 2020 may

Abstract

Blood oxygen saturation (SaO2) is one of the most important environmental factors in clinical heart protection. This study used human heart samples and human induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) to assess how SaO2 affects human CM cell cycle activities. The results showed that there were significantly more cell cycle markers in the moderate hypoxia group (SaO2: 75{\%} to 85{\%}) than in the other 2 groups (SaO2 {\textless}75{\%} or {\textgreater}85{\%}). In iPSC-CMs 15{\%} and 10{\%} oxygen (O2) treatment increased cell cycle markers, whereas 5{\%} and rapid change of O2 decreased the markers. Moderate hypoxia is beneficial to the cell cycle activities of post-natal human CMs.

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Extracellular Vesicles from Skeletal Muscle Cells Efficiently Promote Myogenesis in Induced Pluripotent Stem Cells. D. Baci et al. Cells 2020 jun

Abstract

The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches."

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Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells. N. C. Leite et al. Cell reports 2020 jul

Abstract

Understanding the root causes of autoimmune diseases is hampered by the inability to access relevant human tissues and identify the time of disease onset. To examine the interaction of immune cells and their cellular targets in type 1 diabetes, we differentiated human induced pluripotent stem cells into pancreatic endocrine cells, including $\beta$ cells. Here, we describe an in vitro platform that models features of human type 1 diabetes using stress-induced patient-derived endocrine cells and autologous immune cells. We demonstrate a cell-type-specific response by autologous immune cells against induced pluripotent stem cell-derived $\beta$ cells, along with a reduced effect on $\alpha$ cells. This approach represents a path to developing disease models that use patient-derived cells to predict the outcome of an autoimmune response.

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Species	Human
Formulation Category	Serum-Free

STEMdiff™心室心肌细胞分化试剂盒

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产品号 #05010_C

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STEMdiff™心室心肌细胞分化试剂盒

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产品号 #05010_C

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