StemSpan™ Leukemic Cell Culture Kit

总览

StemSpan™白血病细胞培养试剂盒适用于恶性细胞的体外培养和扩增，并已在慢性髓性白血病（CML）和急性髓性白血病（AML）样本中进行了测试。该优化方案允许您扩增、培养恶性细胞并将其用于药物筛选。StemSpan™白血病细胞培养试剂盒包括无血清培养基SFEM II（产品号 #09605），StemSpan™CD34+扩增添加物（10X；产品号 #02691）和小分子UM729（产品号 #72332）。

分类
基础培养基，添加剂

细胞类型
癌细胞及细胞系，造血干/祖细胞，白血病/淋巴瘤细胞

种属
人

应用
细胞培养，扩增，细胞毒性检测

品牌
StemSpan

研究领域
癌症，药物发现和毒性检测

制剂类别
无血清

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实验数据

The experiments in this Technical Bulletin were performed using cryopreserved cells, however similar results are expected when using fresh samples. Additionally in place of UM729, the small molecule UM171 was used to generate data in Figures 4 - 7. UM171 is no longer licensed for sale by STEMCELL Technologies, however similar results are expected when using UM729 prepared to a final concentration of 1 μM (data not shown). Further titration may be necessary to optimize cell fold expansion in specific conditions.

For more information including data comparing UM171 and UM729, see Fares et al. 2014.

Figure 1. Isolation of Leukemic CD34+ Cells

Cryopreserved CML or AML PBMCs and BMMCs were thawed and prepared for isolation. CD34+ cells were isolated using EasySep™ Human Cord Blood CD34 Positive Selection Kit II. The percentage of CD34+ cells before (A, C) and after (B, D) CD34+ cell isolation was measured by flow cytometry. Dead cells were excluded by light scatter profile and viability staining. In this example the purity of CD34+ cells increased from 3% to 82% (CML) and from 16% to 93% (AML).

Figure 2. Expansion of CD34+ CML Cells

CD34+ CML cells were cultured in StemSpan™ SFEM II containing CD34+ Expansion Supplement (Exp) without or with UM171. After 7 and 14 days, the cultured cells were stained with fluorescently labeled antibodies against CD45, CD34, CD90, CD45RA, and with ALDEFLUOR™ (Catalog #01700) to measure ALDH activity, and analyzed by flow cytometry. Sequential gates were used to determine the percentages of viable CD45+, CD45+CD34+ and CD45+CD34+CD90+CD45RA- cells(based on “Fluorescence Minus One” (FMO) controls), and ALDHbr cells (based on DEAB control). (A) Representative flow cytometry profiles at day 7 are shown. The (B,D) frequency and (C,E) cell numbers of these subsets per initial CD34+ cell on (B,C) day 7 and (D,E) day 14 are shown. StemSpan™ SFEM II supplemented with CD34+ Expansion Supplement supports the expansion of CML cells in culture. The addition of UM171 enhances expansion of all subsets shown (~10-fold expansion of CD34+ progenitor cells at day 7 and ~20-fold at day 14 compared to cultures without UM171). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.0001). All six CML samples tested were able to expand in culture.

Figure 3. Expansion of CD34+ AML Cells

CD34+ AML cells were cultured in StemSpan™ SFEM II containing CD34+ Expansion Supplement (Exp) alone, or with UM171. After 7 and 14 days, the cultured cells were stained with fluorescently labeled antibodies and with ALDEFLUOR™ Reagent as described in Figure 2. (A) Representative flow cytometry profiles at day 7 are shown. The (B, D) frequency and (C, E) cell numbers of these subsets per initial CD34+ cell on (B, C) day 7 and (D, E) day 14 are shown. SFEM II supplemented with CD34+ Expansion Supplement supports the expansion of AML cells in culture. The addition of UM171 further enhances expansion of all subsets shown (~3-fold expansion of all subsets at day 7 and ~7-fold at day 14 compared to cultures without UM171). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05; **P < 0.01). Six out of ten AML samples tested were able to expand in culture.

Figure 4. Colony-Forming Potential of CD34+ CML Cells is Maintained During Culture

CML cells were assayed in colony assays using MethoCult™ H4435 Enriched medium directly after CD34+ cell isolation (day 0) or after 7 or 14 days of expansion without or with UM171 (as described in Figure 2). After 14 days of culture in StemSpan™ SFEM II with CD34+ expansion supplement (Exp) with or without UM171, colonies were (A) imaged with STEMvision™ and counted manually from digital images. (B) CFU output expressed as the total number of colonies per original input CD34+ cell. Numbers above each of the individual bars indicate the proportion of BCR-ABL positive colonies, measured by qRT-PCR on individual plucked colonies across 6 different samples (8-12 colonies were plucked for each sample per condition). SFEM II supplemented with CD34+ Expansion Supplement (Exp) supports the expansion of colony-forming progenitor cells in culture. UM171 further promotes colony forming progenitor cell output (~3.5-fold expansion at day 7 and ~8-fold at day 14). Single-colony qRT-PCR analysis revealed that colonies generated from samples on day 0, and colonies generated from cells expanded for 7 and 14 days, were predominantly BCR-ABL+ but also that normal BCR-ABL- progenitor cells were present at low frequencies. Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05).

Figure 5. Colony-Forming Potential of CD34+ AML Cells is Maintained During Culture

AML cells, after CD34+ cell isolation (day 0) or after 7 or 14 days of expansion without or with UM171 (as described in Figure 3), were plated in colony assays with MethoCult™ H4435 Enriched medium. After 14 days of incubation, colonies were (A) imaged with STEMvision™ and counted manually from digital images. (B) CFU output expressed as the total number of colonies per original input CD34+ cell. SFEM II supplemented with CD34+ Expansion Supplement (Exp) supports the expansion of colony-forming progenitor cells in culture. Addition of UM171 further promotes colony-forming progenitor cell output (~3-fold expansion at day 7 and ~4-fold at day 14). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05).

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产品说明书及文档

请在《产品说明书》中查找相关支持信息和使用说明，或浏览下方更多实验方案。

Document Type

Product Name

Catalog #

Lot #

Language

Document Type

Product Information Sheet

Product Name

StemSpan™ Leukemic Cell Culture Kit

Catalog #

09720

Lot #

All

Language

English

Document Type

Safety Data Sheet 1

Product Name

StemSpan™ Leukemic Cell Culture Kit

Catalog #

09720

Lot #

All

Language

English

Document Type

Safety Data Sheet 2

Product Name

StemSpan™ Leukemic Cell Culture Kit

Catalog #

09720

Lot #

All

Language

English

应用领域

本产品专为以下研究领域设计，适用于工作流程中的高亮阶段。探索这些工作流程，了解更多我们为各研究领域提供的其他配套产品。

Research Area

Workflow Stages

Workflow Stages for Hematopoietic Stem and Progenitor Cell Research

Cell Sourcing and Isolation

Expansion and Differentiation

Analysis

Workflow Stages for Myeloid Cell Research

Sample Preparation

Cell Sourcing

Cell Isolation

Cell Differentiation & Maturation

Cell Characterization

相关材料与文献

Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization S. Kaito et al. Nature Communications 2024 Aug

Abstract

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies,although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein ( TOPORS ),which encodes a ubiquitin/SUMO E3 ligase,augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis,suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks,which undergo SUMOylation,followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain,which mediates ubiquitination,is responsible for HMA resistance. In TOPORS knockout cells,DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination,resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1,thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently,the ubiquitination inhibitor,TAK-243,and the SUMOylation inhibitor,TAK-981,show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks. Subject terms: Myelodysplastic syndrome,Myelodysplastic syndrome

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PLK1 Inhibition Induces Synthetic Lethality in Fanconi Anemia Pathway–Deficient Acute Myeloid Leukemia A. S. Sheth et al. Cancer Research Communications 2025 Apr

Abstract

Overall survival of acute myeloid leukemia (AML) remains limited. Inhibitors of the master mitotic kinase PLK1 have emerged as promising therapeutics,demonstrating efficacy in an undefined subset of patients with AML. However,the clinical success of PLK1 inhibitors remains hindered by a lack of predictive biomarkers. The Fanconi anemia (FA) pathway,a tumor-suppressive network comprised of at least 22 genes,is frequently mutated in sporadic AML. In this study,we demonstrate that FA pathway disruption sensitizes AML cells to PLK1 inhibition. Mechanistically,we identify novel interactions between PLK1 and both FANCA and FANCD2 at mitotic centromeres. We demonstrate that PLK1 inhibition impairs recruitment of FANCD2 to mitotic centromeres,induces damage to mitotic chromosomes,and triggers mitotic collapse in FANCA-deficient cells. Our findings indicate that PLK1 inhibition targets mitotic vulnerabilities specific to FA pathway–deficient cells and implicate FA pathway mutations as potential biomarkers for the identification of patients likely to benefit from PLK1 inhibitors. This work demonstrates that FA pathway mutations,which are frequently observed in sporadic AML,induce hypersensitivity to PLK1 inhibition,providing rationale for a novel synthetic lethal therapeutic strategy for this patient population.

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Heme oxygenase 1 confers gilteritinib resistance in FLT3-ITD acute myeloid leukemia in a STAT6-dependent manner T. Zhang et al. Cancer Cell International 2025 Apr

Abstract

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. We previously discovered that heme oxygenase 1 (HO1) is crucial for chemoresistance in AML,but the detailed molecular mechanism of that remains unclear. RNA sequencing was conducted to assess transcriptomic changes in three pairs of AML cells after regulating the expression of HO1. The molecular mechanism by which HO1 induces gilteritinib resistance in FLT3-ITD (FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD)) AML was evaluated by quantitative real-time PCR (qRT-PCR),CCK-8,flow cytometry,and western blotting. FLT3-ITD AML mouse models were established to investigate the effects of HO1 expression on gilteritinib resistance in vivo. In these three pairs of AML cells,we discovered that HO1-mediated drug resistance is connected to the interleukin-4-mediated signaling pathway (specifically STAT6) only in MV4-11 cells with the FLT3-ITD mutation. Further findings revealed that HO1 overexpression confers gilteritinib resistance in FLT3-ITD AML cell lines and primary individual specimens. While suppression of HO1 sensitized FLT3-ITD AML cell lines and primary individual specimens to gilteritinib. Mechanistically,western blotting and flow cytometry confirmed that HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines and primary individual specimens. Moreover,tumor-bearing mice were employed to determine that HO1 overexpression conferred gilteritinib resistance in vivo. Collectively,these studies illustrate that HO1 may act as a successful treatment target for gilteritinib-resistant FLT3-ITD AML patients. The online version contains supplementary material available at 10.1186/s12935-025-03757-3.

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StemSpan™白血病细胞培养试剂盒

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物种	人
配方	无血清

StemSpan™白血病细胞培养试剂盒

产品号 #09720

产品号 #（选择产品）

产品号 #09720_C

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实验数据

产品说明书及文档

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