Macs Stem Cell Research
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Macs Stem Cell Research Document Transcript

  • 1. MACS® Stem Cell ResearchDiscover. Advance. Translate. Embryonic and induced pluripotent stem cells Hematopoietic stem cells Mesenchymal stem cells Cancer stem cells Authorized distributorExcite and Inspire. of Stemgent products
  • 2. Contents Pioneering research needs pioneering technology ell separation technology linical development 6 Embryonic stem (ES) and induced pluripotent stem (iPS) cells 10 Hematopoietic stem cells (HSCs) 12 Mesenchymal stem cells (MSCs) 14 Cancer stem cells (CSCs) 16 Molecular applications For ordering information and the latest news on our products: www.macs-stemcells.com info@macs-stemcells.com References: The proof is in the publications! Visit www.macs-stemcells.com/references for the extensive list of literature on successful research conducted with our stem cell products. Stemgent products are not distributed by Miltenyi Biotec in Israel, and not all Stemgent products are distributed in the US. Please visit www.macs-stemcells.com for product-specific availability in your country. 2
  • 3.  Pioneering research needs pioneering technology Complete workflow solutions Miltenyi Biotec offers a complete portfolio of stem cell products that operates as a comprehensive system for seamless workflow—from sample preparation to downstream applications. Move from stage to stage in your experiment with confidence in a technology that provides for you each step of the way. MACS® Products cover the following fields: Sample preparation •  onvenient, standardized, and time-saving dissociation of embryoid bodies, C tumors, and other tissues with the gentleMACS™ Dissociator • High yields, high cell viability, high reproducibility •  omplete dissociation into single-cell suspensions to support optimal results C during cell separation or flow cytometry Cell separation •  ACS Technology: The gold standard in cell separation—proven in over 14,500 M publications •  ure populations of different adult and pluripotent stem cells in under an hour! P • Enrichment / depletion of stem cell progeny for differentiation experiments •  asy removal of feeder cells from ES and iPS cell cultures E •  anual and automated cell sorting; scalable to serve your needs M • Applicable from basic research to clinical applications Cell analysis •  asily verify cell separation results with MACS Control Cocktails E •  mall footprint with big features: The compact MACSQuant® Analyzer fits on S your benchtop while featuring 9-parameter detection, absolute cell counting, and automated compensation •  range of fluorochrome-conjugates for multi-parameter phenotyping A •  ntibodies and kits for the analysis of various stem and progenitor cells A Cell culture •  xpansion media for ES and iPS cells and MSCs E •  remium- and GMP-grade cytokines to support your stem cell research P •  upplements for the preparation of viable single-cell suspensions from human S ES and iPS cells •  pecifically designed differentiation media for CFU assays S •  mall molecules for easy manipulation of stem cell cultures S Molecular applications •  ensitive one-step mRNA isolation and cDNA synthesis for more reliable S quantitative PCR analysis •  igh-quality microarray services for gene and microRNA expression profiling H •  uperAmp™ Technology allows gene expression profiling even down to a few cells S •  traight-forward isolation of intact mitochondria S3
  • 4. Cell separation technology Advance your stem cell research with MACS® Technology MACS® Technology, the recognized standard in cell MACS Technology separation, has supported research for over 20 years. Magnetic labeling Benefit from MACS Technology: Cells of interest are labeled with MACS MicroBeads in a short •  asily isolate rare cell populations E incubation step. •  ptimal recovery and excellent purity O •  ast, convenient, and reliable, with no complicated flow F sorting needed Magnetic separation •  entle to cells, with flexibility to conduct separation in G Labeled and unlabeled cells media, and less stressful compared to flow sorting are separated on a MACS Column placed in the magnetic •  utomated cell separation with the autoMACS® Pro A field of a MACS Separator. The Separator flow-through can be collected as the non-magnetic, unlabeled •  ompatible with flow cytometry C cell fraction. •  ell separations can easily be scaled up C •  bility to perform your experiments in a sterile setting A •  ridges basic research and clinical applications B •  ave control over your experiments with a technology H that presents you with ease of use. Test the technology for yourself and experience how easily it advances your research! Elution of the labeled cell fraction About MACS MicroBeads: The separation column is •  uperparamagnetic particles S removed from the magnetic field and the retained cells •  olloidal, for easy handling and short incubation times C are flushed out. •  mall (50 nm), non-toxic, and biodegradable: no need to S Both the labeled and unlabeled remove them from cells after the separation process fractions can be recovered and used for downstream app- •  onjugated to highly specific monoclonal antibodies to C lications. ensure optimal specificity To learn more about MACS Technology, including separation strategies and the full range of MicroBeads, Columns, and Separators, visit www.macs-stemcells.com/technology. A B C A B N S Figure 1: MicroBead features. Scanning electron microscopy of a cell isolated with MACS MicroBeads (A) 50 nm MicroBeads are so small they can only be seen on a Transmission Electron Microscope (B). A cross Figure 2: MACS Cell Separation. Cell separation can be performed section of a MACS Column showing the steel ball matrix (gray) with the manually with a MACS Separator (A) or automated with the autoMACS magnetic field in which labeled cells (purple) are retained (C). Pro Separator (B).4
  • 5. Clinical development Bench to bedside Design freeze Basic Pre-clinical Clinical trials Clinic research research Highly regulated Research products Clinical products Figure 3: The highly regulated phases of research to pre–clinical research, clinical trials and towards applications in the clinic. Design freezes are implemented at the pre-clinical stage implying a substantial impact financially and time-wise if changes are made subsequently. From bench to bedside. The progress of research from basic Our experience in bringing research into the clinic began when science through pre-clinical research to clinical research has the CliniMACS® CD34 Separation Kit was CE-marked several crucial phases (fig. 3). in 1997; more than 10,000 patients have been treated in the last five years with cellular products produced using our CliniMACS Miltenyi Biotec works with you as a partner to see you through Technology. the pivotal processes of evolution from pre-clinical research into Phases I, II, and III prior to attaining approval for clinical use. Besides the potential to propel research from basic science to These phases are highly regulated and entail a design freeze of the clinic, Miltenyi Biotec’s products also guarantee the the entire study at the pre-clinical research stage. possibility for scalability in your research, an important factor for consideration. With our products, you can be sure that the By using a portfolio that makes transitions seamless, you can protocols and methodology used in your research can be avoid costly and time-consuming set backs in your work. smoothly transitioned to a clinical setting. With our proven track record of over 14,500 publications, and specifically over 3,500 in the clinical arena, we give you the When deciding which platform to conduct your confidence to move your research forward with products that experiments on, it is crucial to start and advance your are designed to comply with each phase of development. research with a technology you can rely on. Take your research from bench to bedside with no obstacles, using Mindful of this, we have developed an integrated product Miltenyi Biotec’s complete product portfolio. portfolio that provides a smooth transition through each stage of clinical development. For example, our MACS® Cytokine For more information, please visit products are available in research, premium and GMP grades, www.macs-stemcells.com/clinical. ensuring that each step of the process couldn’t be more convenient.5
  • 6. ES and iPS cells Sample preparation and cell separation Dissociation of embryoid bodies Achieve greater purity, higher yields, and save time compared to traditional cell culture selection methods: Complete dissociation of embryoid bodies (EBs) in ES and iPS • MACS Technology cuts days and weeks to cell differentiation protocols is a prerequisite to allow successful under an hour cell separation and analysis. Benefit from Miltenyi Biotec’s • Sterile separation under your own hood. innovative gentleMACS™ Dissociator, which helps streamline and standardize this process for reliable, reproducible results. •  ave time: effortless, efficient, and reliable automated tissue S Mouse/human ESCs or dissociation at the push of a button iPSC on feeder cells •  lexible: prepare viable single-cell suspensions or perform F cell homogenization from EBs Cell harvest, Single-cell suspension •  educe contamination: closed system enables sterile sample R handling •  ersatile: additional programs and protocols for different V A B C cellular and molecular applications Depletion by Sedimentation/ Weaning off by For more details see www.macs-stemcells.com/gentlemacs. MACS Technology selective adhesion serial passaging using Feeder on gelatin Removal MicroBeads B A C Magnetic labeling 15 min 45 min 3 days Figure 4: Complete dissociation of EBs with the gentleMACS Disso- ciator. Differentiation of ES and iPS cells frequently involves embryoid MACS LS bodies (A). Dissociation of these structures with traditional methods is Column often incomplete and can yield high numbers of dead cells (B). gentleMACS 3 min Technology can generate viable single-cell suspensions in a standardized, semi-automated process and thereby ensure reproducible results (C). Visit www.macs-stemcells.com/info to download application note 1 and 45 min 3 days see the protocol used at the NIH. Feeder- Cell separation depleted ESCs/ iPSCs Pure and homogeneous cell populations are essential for investigating ES and iPS cell biology. With MACS MicroBeads and Kits, you can isolate specific cell populations with ease: Figure 5: Efficient depletion of feeder cells. Feeder Removal MicroBeads permit an efficient depletion of feeder cells in less than 30 minutes (A). Puri- •  emove feeder cells such as mouse embryonic fibroblasts for R ties of more than 99% can be achieved. In contrast, traditional methods for better results in downstream applications (fig. 5) the removal of feeder cells include sedimentation and plastic adherence (B) or weaning off over several passages (C). Both are time-consuming, •  eplete unwanted differentiated cells for untouched D incomplete, and can result in a significant loss of pluripotent stem cells. isolation of your target cell population (fig. 6) For detailed information please download poster 2 from •  onduct positive selection of human or mouse pluripotent C www.macs-stemcells.com/info. stem cells using a wide variety of markers, including For our full range of ES and iPS cell separation products, visit TRA-1-60, CD326 (EpCAM), or SSEA-1 (fig. 7) www.macs-stemcells.com/es-ipsc-separate.6
  • 7. ES and iPS cells Improved culture and differentiation Magnetic separation in ES and iPS cell differentia-A B C tion protocols: specifically enrich your target cells 10³ 10³ 10³ •  btain well-defined, homogeneous cell populations O Anti-SSEA-1-APC Anti-SSEA-1-APC 4% 35% 2% Anti-SSEA-1-APC •  arkers available for many differentiation pathways M Anti-SSEA-1-APC Anti-SSEA-1-APC Anti-SSEA-1-APC 10² 10² 10² 10¹ 10¹ 10¹ •  ork with a more standardized procedure compared to W 1 1 1 conventional selection in culture 0 0 0 -1 -1 0 1 10¹ 10² 10³ -1 -1 0 1 10¹ 10² 10³ -1 -1 0 1 10¹ 10² 10³ •  o need for transgenic selection markers; thus compatible N Anti-Diff-PE Anti-Diff-PE Anti-Diff-PE Anti-Diff-PE Anti-Diff-PE Anti-Diff-PE with clinical research requirements •  eparation is further enhanced by pre-dissociation of S Figure 6: Ability to remove early differentiated cells from mouse ES embryoid bodies with gentleMACS Technology cell cultures. Pluripotent mouse ES cells show a broad range of SSEA-1 expression. In contrast, only 4% of these cells show expression of the Visit www.macs-stemcells.com/reference which shows novel marker Anti-Diff (A). A culture which was deprived of LIF for 2 days published examples of ES or iPS cell differentiation that use was used to model spontaneous differentiation. In such a culture the magnetic separation. expression profile of SSEA-1 remains unchanged, while differentiated cells can be detected easily with our new Anti-Diff marker (B). The novel Pluripotent Stem Cell Isolation Kit, mouse, is based on Anti-Diff and allows you to remove early differentiated cells (C). Download poster 3 for more A B C information from www.macs-stemcells.com/info. <0.2% <0.1% 5.2% 5.2% 95% 95% SSC SSC SSC A B C SSC-H SSC SSC CD326-APC CD326-APC CD34-APC CD34-APC CD34 CD326-APC CD326-APC CD34-APC CD34-APC CD34-APC Figure 8: Enrichment of target cells in ES or iPS cell differentiation protocols. Human ES cells were cultured in the absence of growth factors TRA-1-60-PE Anti-Ha-1-60-PE TRA-1-60-PE Anti-Ha-1-60-PE to induce differentiation (A). After 10 days, 5–10% of the culture consisted of CD34-positive progenitors (B). MACS Technology and CD34 MicroBeads allowed rapid isolation of this cell population with high purity (C). Figure 7: Enrichment of pluripotent cells. ES or iPS cell cultures can Up to 108 cells were used per separation. For detailed information visit contain differentiated cells that have down-regulated pluripotency mark- www.macs-stemcells.com/info and download customer report 4 by Wang et al. ers such as CD326 (EpCAM) and TRA-1-60 (A). Cells isolated by magnetic separation using the Pluripotent Stem Cell MicroBeads or the Anti-TRA-1-60 MicroBead Kit do not show unwanted differentiation (B) and maintain Mouse Feeder Enrichment Depletion of Depletion normal karyotypes for more than 6 months (C). removal of ES and iPS pluripotent of early cells cells during differen- differentia-­­ tiated tion cells Cell differentiation Feeder Removal + Human ES and iPS cells can differentiate into virtually any cell MicroBeads type. MACS Technology allows the separation of the cells at Anti-SSEA-1 different time points during differentiation. Target cells can be (CD15) +1 + + isolated by positive selection if the cells express a specific MicroBeads surface marker that distinguishes them from other cells. Human Pluripotent CD34, for instance, was successfully used to isolate hematopoi- Stem Cell + Isolation Kit etic and endothelial progenitors from differentiated human ES cell cultures (fig. 8). If there is no suitable marker for a particular Table 1: Examples of magnetic separation in mouse ES and iPS cell research. cell type, depletion of unwanted cells is a useful alternative for the isolation of the desired cells. 1 Positive selection of ES and iPS cells. 7
  • 8. ES and iPS cells Optimal cell analysis Human Feeder Enrichment Enrichment Enrich- •  se our unique Anti-Feeder antibodies to exclude feeder U removal of iPS cells of ment of cells from your flow cytometric analysis after pluripotent cardiovas- repro- cells during cular pro- •  est-in-class: the MACSQuant Analyzer is a benchtop flow B gramming culture genitors cytometer with absolute cell counting capability Feeder For our full range of ES and iPS cell analysis products, visit Removal +2 MicroBeads, www.macs-stemcells.com/es-ipsc-analysis. mouse • Pre-defined sets of established pluripotency markers Anti- Fibroblast +3 • Live cell staining with Stemgent’s StainAlive MicroBeads, antibodies human • Analysis of surface markers and transcription factors Anti- TRA-1-60 +1 + + MicroBead Kit Marker Mouse Human Pluripotent ES and iPS cells ES and iPS cells Stem Cell +1 + + Alkaline phosphatase + + MicroBeads, human CD324 – + Anti-SSEA-1 CD326 – + (CD15) +4 MicroBeads Nanog + + 1 Positive selection of ES and iPS cells Oct4 + + 2 If using mouse feeder cells Sox2 + + 3 If using human feeder cells 4  uman pluripotent cells are CD15-negative and thereby removed H SSEA-1 + + in this step. General protocols for complete depletion of pluripotent SSEA-3 – + cells are under development. SSEA-4 – + Table 2: Examples of magnetic separation in human ES and iPS research. TRA-1-60 – + TRA-1-81 – + Cell analysis Rex1 + + c-Myc – + MACS Antibodies in combination with the MACSQuant Analyzer Klf4 + + offer you great solutions for cell analysis. We can provide you with even more solutions for stem cell analysis, including Table 3: Markers for mouse and human ES and iPS cell research. Stemgent’s products. The combined portfolio brings you a broad range of high-quality antibodies, ensuring all your needs A B C D are conveniently met. Relative cell number •  ase of use: Evaluate the differentiation status of human E Relative cell number and mouse ES and iPS cells with ease using the detection kit for alkaline phosphatase activity, a phenotypic marker of pluripotent stem cells •  onvenient: Live cell staining antibodies enable real-time C CD324 (E-Cadherin)-APC CD324 (E-Cadherin)-APC immunocytochemistry of viable cells—no fixation required; continued culture possible Figure 9: Easy histochemical staining. Alkaline phosphatase (AP) is ex- •  omprehensive range of fluorochrome conjugates available C pressed at elevated levels in undifferentiated pluripotent cells (A). Stemgent’s (VioBlue®, PerCP, PE, APC, FITC, DyLight™, StainAlive®) to AP Staining Kit II provides an easy immunohistochemical staining protocol meet even multiparameter imaging requirements to assess expression of this phenotypic marker. Detection of additional stem cell surface markers such as CD324 (B), TRA-1-60 (C) and TRA-1-81 complements the phenotypic analysis. This can be done by immunochem- istry or even directly in culture with Stemgent’s StainAlive antibodies (D).8
  • 9. ES and iPS cellsGuaranteed cell cultureCell culture Stemgent, mouse primary iPS and human fibroblast cell lines enhance your reprogramming and differentiationThe right medium is vital for ensuring ES and iPS cell culture. studies in basic and translational research. Stemgent cellOur comprehensive selection in combination with Stemgent’s lines closely mimic the in vivo state and generate moreportfolio provides you with: physiologically relevant data.•  eady-to-use media and cell culture supplements that R For our full range of ES and iPS cell culture products mentioned, guarantee you optimal and standardized culture conditions visit www.macs-stemcells.com/es-ipsc-culture. during expansion and maintenance of pluripotent ES and iPS cell lines•  broad range of recombinant cytokines and growth factors A (e.g. FGF-2, EGF, SHH, LIF, Wnt-3a, Wnt-5a, Activin A) to supplement your media according to your needs and individual differentiation protocols•  way to take your research from bench to bedside with our A cytokines available from research to GMP grades•  unique portfolio of Stemgent small molecules, potent A tools that allow you to manipulate cell reprogramming, self-renewal, and differentiation NutriStem™ XF/FF Culture Medium • Xeno- and feeder-free culture (fig. 10) Figure 10: NutriStem XF/FF Culture Medium: improved human ES and • Low amounts of FGF-2 and TGF-β closer to iPS cell culture. Colonies of the human ES cell line H1 cultured in physiological levels NutriStem XF/FF Culture Medium show typical ES cell morphology and maintain pluripotency and normal karyotypes over long-term culture. • Ideal for human iPS cell generation hES Cell Cloning & Recovery Supplement (Thiazovivin) 2,500 cells 10,000 cells 40,000 cells •  Improves survival of single human ES cells by more than 30-fold after thawing, dissociation, and replating (fig. 11) A • Crucial for preparing viable single-cell suspensions for flow cytometry and cell separation Small molecules with results you want: • Easy cell penetration: simply add them to your culture media B • Low antigenicity • Tested for cytotoxicity on stem cells • Download poster 5 at www.macs-stemcells.com/info to learn how small molecules act Figure 11: hES Cell Cloning and Recovery Supplement—clearly advan- tageous. Human H1 ES cells were trypsinized and single-cell suspensions Save time and effort with Stemgent’s ready-to-use were reseeded at different cell numbers as indicated in NutriStem XF/FF retroviruses for cell reprogramming. Culture Medium (A). Addition of the hES Cell Cloning and Recovery Supplement improves survival of solitary ES and iPS cells over 30-fold (B) and is also advantageous for all applications involving single-cell suspensions, such as separations by MACS Technology and cell analysis. 9
  • 10. HSCs Cell separation—trusted in research and the clinic for more than 15 years Cell separation A B HSC research has contributed a great deal to understanding hematopoiesis for use within clinical settings. Conduct your experiments in a standardized, seamless process with products CD45-APC CD45-APC CD45-APC CD45-APC optimized for direct use with each other: •  irectly isolate HSCs or hematopoietic progenitor cells D (HPCs) with a range of different markers (table 4); fast and easy, from any source CD34-FITC CD34-FITC •  nrich untouched HSCs and HPCs using our Lineage Cell E CD34-FITC CD34-FITC Depletion Kits, mouse or human C D •  erform sequential sorting to define subpopulations based P on several markers (fig. 13) CD34-FITC CD34-FITC •  asily obtain committed cells for comparison using related E CD34-FITC CD34-FITC separation products from our portfolio of mature lineage markers For our full range of HSC separation products, visit www.macs-stemcells.com/hsc-separate. CD33/2-PE CD133/2-PE-A CD33/2-PE CD133/2-PE Marker/ Mouse Human MicroBead Kit Figure 12: Save time on HSC isolation. HSCs represent a minor fraction of human peripheral blood mononuclear cells (PBMCs), identified here as CD341 + CD34+CD45lo (A) or CD34+CD133+ cells (C). Whether in the research or clinical CD1051 + setting, MACS Technology provides a highly efficient way of isolating pure populations of this rare fraction using either CD34 MicroBeads (B) or the CD117 + + CD133 MicroBead Kit (D). CD133 + Sca-1 + A B Linneg + + Ter-119)-Biotin/Anti-Biotin-APC Ter-119)-Biotin/Anti-Biotin-APC Lineage markers (CD5, CD11b, Lineage markers (CD5, CD11b, Linneg/CD34+ + CD45R, anti-Ly-6g, 7-4, CD45R, anti-Ly-6g, 7-4, Linneg-APC Linneg-APC Lin /CD133 neg + + 1 also available with MultiSort option Table 4: Markers for the isolation of HSCs and HPCs. • Decrease processing time and stress to cells with CD117-PE CD117-PE magnetic separation prior to flow sorting CD117-PE CD117-PE • Take advantage of CD133, a unique marker for Figure 13: Efficiently enrich mouse progenitor cells. Mouse HSCs and primitive HSCs early HPCs are contained in the Linneg CD117hi compartment (A). Using the • Available for use in clinical settings: CD34 and CD133 Lineage Cell Depletion Kit, mouse, followed by positive selection with MicroBeads as CE-marked reagents, manufactured CD117 MicroBeads, HSCs and HPCs can be efficiently enriched (B). under GMP conditions * • Proven technology: CD34 MicroBeads have been cited more than 2,000 times since their launch in 1993 *For availability in your country, please contact your local sales representative.10
  • 11. HSCsComprehensive cell culture and analysisCell analysis Cell cultureIn HSC research, characterizing the surface phenotype of Stimulate and culture your HSCs with the MACS Cell Cultureyour target cells is critical to ascertain the cellular subset you portfolio:are working with. Utilize our wide range of products for flow •  ACS HSC-CFU Media, evaluate the number of HSCs in your Mcytometry or microscopic investigations to make the starting material and assess their differentiation potential asphenotyping process easier: colony-forming units. See poster 7 at•  rofit from our collection of fluorochrome-conjugated P www.macs-stemcells.com/info for more information on the hematopoietic markers to analyze stem cell as well as HSC-CFU media lineage markers for your cells by flow cytometry •  ACS Cytokines are reliable supplements for routine culture M•  ACS Antibodies are perfectly suited to our separation M as well as delicate differentiation experiments. Benefit from reagents. Get reproducible staining results and avoid low endotoxin levels, high purity, and tested biological inaccessible epitopes with our recommended staining activity, and supplement your basic media according to your protocols needs•  ur MACS Control Cocktails, available for CD34 and CD133, O For our full range of HSC cell culture products, visit allow easy and convenient analysis of your separation www.macs-stemcells.com/hsc-culture. results (fig. 14) Take your research from bench to bedside with MACSThe entire range of HSC cell analysis products is available at Cytokines, available in research, premium, and GMPwww.macs-stemcells.com/hsc-analysis. grades. Commonly used cytokines in HSC research Endothelial progenitor cells (EPCs) are part of the CD34+ include Stem Cell Factor (SCF), FLT3-Ligand, and population. The EPC Enrichment and Enumeration Kit Thrombopoietin (TPO). utilizes EPC markers for efficient magnetic pre- enrichment by MACS Technology and subsequent sensitive cell enumeration by flow cytometry. Visit www.macs-stemcells.com/info for poster 6 with further information. CFU-GEMM A B P1/P2/P3/P4 P1/P2/P3/P4 CD34-APC CD34-APC CD34-APC CD34-APC CFU-GM BFU-E CD133/2-PE CD133/2-PE CD133/2-PE CD133/2-PEFigure 14: Convenient evaluation of separation results. The MC CD34/CD133 Stem Cell Cocktail was used to evaluate separation results obtainedwith CD133 MicroBeads. Gating on viable CD45+CD34+ cells, primitive CD133+HSCs can be readily enumerated in pre (A)- and post (B)-separation samples.Using the ‘Easymode’ function for automated gating on the MACSQuant CFU-M CFU-G CFU-EAnalyzer makes the protocol even more convenient. Figure 15: Reliable evaluation of human HSC differentiation potential. Schematic diagram showing colony-forming units (CFUs) formed by multipotent and lineage-committed hematopoietic progenitors. 11
  • 12. MSCs Standardize your cell expansion and differentiation potential Cell separation A B Work with purified cell populations to fully explore the potential of MSCs, which hold great promise for research and Relative cell number Relative cell number therapeutic applications. Forward Scatter Forward Scatter •  asily isolate MSCs from human bone marrow, lipoaspirate, E or other tissues using a wide range of markers (table 5) •  btain MSCs directly from tissue for downstream O experiments CD271-PE CD271-PE CD271-PE CD271-PE •  eplete contaminating cells from your human or mouse D MSC cultures C D • Enrich mouse bone marrow MSCs after expansion in culture Cumulative population Number of CFU-F •  se the ready-to-go research toolboxes to isolate and U doubling expand your human MSCs Marker Source tissue CD271 Bone marrow, lipoaspirate PA neg. pos. PA positive MSCA-1 Bone marrow fraction fraction fraction Anti-fibroblast antigen Bone marrow Figure 16: Clinical-scale isolation of CD271+ MSCs. In human bone CD105 Bone marrow marrow, MSCs are contained in the CD271+ compartment (A), which can be CD146 Umbilical cord blood, lipoaspirate, efficiently enriched using the CD271 MicroBead Kit (B). Further characteriza- dental pulp, endometrial tissue tion reveals that MSCs with CFU-F differentiation potential are exclusively present in the CD271+ fraction (C) and have a higher clonogenic potential Table 5: MACS Technology isolates defined human MSC than cells isolated by conventional plastic adherence (PA) (D). To learn more populations with high-purity. Specific markers for specific source tissues. about clinical-scale isolation of CD271+ MSCs download poster 8 at www.macs-stemcells.com/info. “… Our results indicate that CD271 antigen provides a versatile marker for prospective isolation and expansion of multi-potent mesenchymal stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. …” Selim Kuçi et al. CD45-FITC CD45-FITC CD45-FITC CD45-FITC Visit www.macs-stemcells.com/references for the full reference. Anti-MSCA-1 (W8B2)-APC Anti-MSCA-1 (W8B2)-APC Anti-MSCA-1 (W8B2)-APC Anti-MSCA-1 (W8B2)-APC Figure 17: Isolate highly purified MSCs in under an hour. Human bone marrow MSCs also express the novel surface marker MSCA-1. MSCA-1+ cells are shown to be CD45 – and CD271+. Using MSCA-1 MicroBeads, highly purified MSCs can be isolated from bone marrow in under an hour. Understanding mouse MSCs is an ongoing effort. We offer a wide range of MicroBeads and fluorochromes for you to choose from for your mouse MSC research needs. Find out more at www.macs-stemcells.com/msc-separate.12
  • 13. MSCsImprove cell analysis and cultureCell analysis CytoMix-MSC, human is a composition of cytokines for the most efficient and reproducible expansion ofIdentify and enumerate MSCs from bone marrow, lipoaspirate human MSCs, resulting in 50% more cumulativeand other tissues by flow cytometry or fluorescence microscopy population doublings. Full results are in poster 9 atwith our range of antibodies. www.macs-stemcells.com/info.Ensure accurate and reliable phenotyping of MSCs with theMSC Phenotyping Kit, a ready-to-use cocktail containing all sixmarkers for MSC characterization, recommended by the ISCTor with our antibodies for individual markers.For our full range of MSC cell analysis products, visitwww.macs-stemcells.com/msc-analysis. NH stem cell source, MSC enumeration Relative cell number e.g., bone marrow, NH CFU-F Medium lipoaspirate CD73-APC CD105-PE CD105-PE CD90-FITCFigure 18: ISCT guidelines–compliant MSC analysis. MSCs isolated withthe CD271 MicroBead Kit were expanded in culture and analyzed for theexpression of surface markers. The MSC Phenotyping Kit in combination with MSC expansionthe MACSQuant Analyzer guarantees robust staining for ISCT-standardized NH Expansion Mediummarkers and straightforward analysis.Cell cultureExpansion of isolated MSCs and evaluation of their differentiationpotential are critical steps in basic MSC research and a pre-requisite for clinical applications.•  ACS NH Expansion Media are optimized to provide the M most convenient expansion of MSCs from various sources. Combination with our separation products for MSC isolation Adipocytes Chondrocytes Osteoblasts guarantees high expansion rates NH AdipoDiff NH ChondroDiff NH OsteoDiff•  ACS NH CFU-F Medium supports you in enumerating the M Medium Medium Medium number of MSCs in your sample•  ACS NH Differentiation Media allow you to explore the full M Figure 19: Reliable expansion and differentiation of MSCs. In MSC research, expansion and differentiation of MSCs are routinely performed. differentiation capacity of your cells, be it for adipocytic, Expand MSCs in less time with the NH Expansion Media and CytoMix-MSC, chondrocytic, or osteoblastic lineages human cytokine cocktail. MSCs can be differentiated into adipocytes, chondrocytes, or osteoblasts using our special differentiation media.•  ACS Cytokines are available in research, premium and GMP M grades to take you from bench to bedside. Commonly used cytokines in MSC research include FGF-1, FGF-2, EGF, TGF-β, and BMP-2For our full range of MSC cell culture products, visitwww.macs-stemcells.com/msc-culture. 13
  • 14. CSCs Whenever you need it—from tumor tissue to purified CSCs Sample preparation Cell separation Breaking up solid tumor tissue into viable single-cell Understanding the role of different tumor cell types is the key suspensions is a challenge. Consistent, reliable dissociation is to discovering future therapies. Tumors are a heterogeneous even more imperative when patient material is limited and mix of multiple cell types. Isolate CSCs with MACS Technology precious. The gentleMACS Dissociator streamlines and to obtain high purities and yields of your target cells. standardizes sample preparation using a combined mechanical •  ide range of reagents for many markers commonly W and enzymatic process for reliable and reproducible results. expressed on tumor samples (table 6) •  pecialized programs, protocols, and kits that are optimized S •  se indirect magnetic labeling for customized cell U for tumor dissociation separations: any cell, any species, any primary antibody •  fficient generation of single-cell suspensions of viable cells E • solate cells on your own schedule whenever your sample I •  ser-independent mechanical processing ensures high U is ready (fig. 21) reproducibility For our full range of CSC separation products, visit •  fficient, automated tissue dissociation at the push E www.macs-stemcells.com/csc-separate. of a button • Closed system that guarantees sterile sample handling Tumor type Cell surface marker •  ptimal preparation of tissue for subsequent flow O Acute myeloid leukemia (AML) CD34+/CD38 – cytometric analysis or magnetic separation Breast cancer EpCAM (ESA)+/CD44+/CD24 –/ Lineage – For more details see www.macs-stemcells.com/gentlemacs. Ovarian cancer CD133+ CD44+/CD117+ CD24+ Glioblastoma CD133+ CD15+ Medulloblastoma CD133+ CD15+ Small cell and non-small CD133+ cell lung cancer Hepatocellular carcinoma CD45–/CD90+ Prostate cancer CD44+/α₂ β₁hi/CD133+ Colon cancer CD133+ CD44+ CD26+ ERY Melanoma CD20+ ABCB5+ CD271+ TIL Pancreas adenocarcinoma CD44+/CD24+/EpCAM (ESA)+ Renal carcinoma CD133 enhances vascularization Head and neck squamous cell CD44+ TC carcinoma (HNSCC) Table 6: Cell surface markers of CSCs in different types of tumors. . Miltenyi Biotec offers a broad portfolio of antibodies for the analysis of Figure 20: Efficient tumor dissociation. Human melanoma metastases tumor samples and the isolation of CSCs. For the complete portfolio, please were dissociated using the gentleMACS Dissociator tumor dissociation visit www.macs-stemcells.com/csc. program. After dissociation, the single-cell suspensions are composed mainly of tumor cells (TC), tumor-infiltrating lymphocytes (TIL), and erythrocytes (ERY). Download poster 10 for more information at www.macs-stemcells.com/info.14
  • 15. CSCsExtensive markers for cell analysis Cell analysis A C Detect and analyze CSCs and other tumor cells with our antibodies: Forward scatter scatter Forwardscatter Forward scatter •  arge selection of antibodies for the phenotypic L characterization of various cell types Forward •  ariety of dye conjugates for effective multiparameter V flow cytometry or sophisticated multi spectral imaging CD44-PE CD44-PE experiments CD44-PE CD44-PE B D CSCs are characterized by different markers, depending on the tissue source (table 6). Regardless of the type of tissue you work with, we provide antibodies for your analysis. Forward scatter CD44-APC CD44-APC CD44-APC For our full range of CSC analysis products, visit www.macs-stemcells.com/csc-analysis. CD44-PE CD24-FITC CD24-FITC CD24-FITC A BFigure 21: Convenient isolation of CSCs. CSCs can express different mark-ers depending on the tissue source or cell line, e.g. CD44+ (A) or CD24 –/CD44+ Side scatter(B). Isolated CD44+ or CD24 –/CD44+ cells were obtained using either CD44 Side scatterMicroBeads alone (C) or in combination with the CD24 MicroBead Kit (D),respectively. MicroBeads can be used to isolate CSCs with high purity fromcancer cell lines or primary tissue. CD44-PE CD44-PE Figure 22: Phenotyping CSCs. CD44+ CSCs are easily analyzed by flow cytometry (A) or immunofluorescence (B). 15
  • 16. Molecular applications Kits to advance your stem cell research Take your stem cell research and analysis to the subcellular and molecular level. Cells or tissue are lysed using Lysis/Binding Buffer. One-step mRNA isolation and in-column cDNA synthesis Premium mRNA is isolated within 15 minutes directly from cells or tissues. The μMACS™ One-step cDNA Kit combines efficient magnetic isolation of mRNA with revolutionary in-column 5 min cDNA synthesis. Purified cDNA can be generated from just a few to as many as 107 cells (fig. 23). Clearing of lysate using LysateClear Column. ChIP-in-a-column with MACS Technology The μMACS Protein A/G MicroBeads improve standard immunoprecipitation and significantly accelerate the search for 3 min interacting proteins. Chromatin immunoprecipitation (ChIP) protocols also benefit from the higher specificity and lower Addition of µMACS™ Oligo(dT) background binding of μMACS Protein A/G MicroBeads. MicroBeads to the lysate. 0 min Isolation of human mitochondria Lysate is applied to a Achieve higher yields of functional mitochondria as compared µ Column in thermoMACS™ to differential– and ultracentrifugation protocols. The kit’s Separator. Wash of mRNA. procedure relies on the renowned MACS Technology. After cell lysis, an outer mitochondrial membrane molecule is used to magnetically isolate functional organelles. Enhanced transfection of stem cells cDNA synthesis mix is applied Stemfect™ Transfection Reagents are polymer-based and onto the column and cDNA is generated. specifically designed for in vitro DNA transfection of stem cells. 1h These transfection reagents ensure high transfection efficiencies and low cytotoxicity. Purification and release of cDNA/mRNA hybrid. Enrichment of transfected cells 15 min Enrichment of transfected cells with MACSelect™ Systems takes Elution of cDNA/mRNA hybrid. 3 hours versus several weeks for antibiotic treatment. 1 min cDNA in 1 h 30 min Figure 23: Principle of MACS Technology for mRNA isolation and cDNA synthesis. Sensitive magnetic isolation of mRNA can be performed within minutes. Subsequent in-column cDNA synthesis prevents the loss of material to further increase sensitivity and reliability.16
  • 17. Molecular applicationsFirst class genomic services for your stem cell researchTen years of microarray experienceMiltenyi Biotec has ten years of microarray experience andoffers a huge variety of genomic services. Since 2003, we are Send sample — receive resultsalso an officially certified Agilent Service provider.There is no need to establish microarray technology in your Technical support for experimentalown laboratory — simply send cells, tissue, or blood samples to design and microarray selectionour Microarray Services Department and receive reliable results • microRNA miRXplore™ Microarraysand detailed documentation in return. • Agilent Whole Genome MicroarraysFlexible expression profiling services RNA extraction and quality controlMiltenyi Biotec provides a wide range of cost-effectivemicroarray services: Optional: • microRNA expression analysis based on • Amplification and quality control miRXplore™ Microarrays • SuperAmp Service*: 1 – 10,000 cells • Agilent whole genome expression analysis• Agilent array-CGH Synthesis and purification of• Bioinformatics Services fluorescently labeled probes Microarray hybridizationSuperAmp™ Service forone-cell microarray experiments Image capture andThe SuperAmp Service is an extension of the Microarray analysis of primary dataServices and allows successful gene expression profiling from1 – 10,000 cells. Optional: Bioinformatics Services** • Pre-processingVisit www.macs-stemcells.com/molecular to see the whole • Ratio Building molecular applications portfolio. • Cluster • Discriminatory Genes • Functional Grouping • Pathway Analysis Results and report • Data on CD-ROM   * microRNAs cannot be amplified with the SuperAmp Service. ** Please inquire for microRNA Bioinformatics Services. Figure 24: Overview of genomic services workflow. 17
  • 18. Germany/Austria/ Australia China Italy SpainSwitzerland Miltenyi Biotec Miltenyi Biotec Trading Miltenyi Biotec S.r.l. Miltenyi Biotec S.L.Miltenyi Biotec GmbH Australia Pty. Ltd. (Shanghai) Co., Ltd. Via Persicetana, 2/D C/Luis Buñuel 2Friedrich-Ebert-Straße 68 Unit 16A , 2 Eden Park Drive Shanghai Office 40012 Calderara di Reno (BO) Ciudad de la Imagen51429 Bergisch Gladbach North Ryde, NSW 2113 Rm. 2309-2310, Italy 28223 Pozuelo de AlarcónGermany Australia No. 319 Xianxia Rd. Phone +39 051 6 460 411 (Madrid), SpainPhone +49 2204 8306-0 Phone +61 2 8877 7400 Shanghai 200051, P.R. China Fax +39 051 6 460 499 Phone +34 91 512 12 90Fax +49 2204 85197 Fax +61 2 9889 5044 Phone +86 21 62351005 macs@miltenyibiotec.it Fax +34 91 512 12 91macs@miltenyibiotec.de macs@miltenyibiotec.com.au Fax +86 21 62350953 macs@miltenyibiotec.es macs@miltenyibiotec.com.cn JapanUSA/Canada Benelux Miltenyi Biotec K.K. United KingdomMiltenyi Biotec Inc. Miltenyi Biotec B.V. France Nittsu-Eitai Building 5F Miltenyi Biotec Ltd.2303 Lindbergh Street Schipholweg 68 H, 2316 Leiden Miltenyi Biotec SAS 16-10 Fuyuki, Koto-ku, Tokyo Almac House, Church LaneAuburn, CA 95602, USA The Netherlands 10 rue Mercoeur 135-0041, Japan BisleyPhone 800 FOR MACS macs@miltenyibiotec.nl 75011 Paris, France Phone +81 3 5646 8910 Surrey GU24 9DR, UKPhone +1 530 888 8871 Phone +33 1 56 98 16 16 Fax +81 3 5646 8911 Phone +44 1483 799 800Fax +1 530 888 8925 Customer service Netherlands Fax +33 1 56 98 16 17 macs@miltenyibiotec.jp Fax +44 1483 799 811macs@miltenyibiotec.com Phone 0800 4020120 macs@miltenyibiotec.fr macs@miltenyibiotec.co.uk Fax 0800 4020100 Singapore Customer service Belgium Miltenyi Biotec Phone 0800 94016 Asia Pacific Pte Ltd. Fax 0800 99626 100 Beach Road Customer service Luxembourg #28-06 to 28-08 Shaw Tower Phone 800 24971 Singapore 189702 Fax 800 24984 Phone +65 6238 8183 130-096-201 Fax +65 6238 0302www.miltenyibiotec.com macs@miltenyibiotec.com.sgMiltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact.MACS, gentleMACS, autoMACS, CliniMACS, MACSQuant, MACS logo, MACSelect, VioBlue, µMACS, miRXplore, and SuperAmp are either registered trademarks or trademarksof Miltenyi Biotec GmbH. StainAlive, Stemgent, Stemfect, and NutriStem are either registered trademarks or trademarks of Stemgent Inc. All other trademarks mentionedin this document are the property of their respective owner and used for identification purposes only.Unless otherwise specifically indicated, Miltenyi Biotec products andservices are for research use only and not for therapeutic or diagnostic use. Copyright © 2011 Miltenyi Biotec GmbH. All rights reserved.