of 6
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.

Differentiation of pluripotent stem cells cultured in StemFlex Medium

Category:

Entertainment

Publish on:

Views: 2 | Pages: 6

Extension: PDF | Download: 0

Share
Description
APPLICATION NOTE StemFlex Medium Differentiation of pluripotent stem cells cultured in StemFlex Medium Introduction Pluripotent stem cell (PSC) culture systems have evolved over time from feeder-dependent
Transcript
APPLICATION NOTE StemFlex Medium Differentiation of pluripotent stem cells cultured in StemFlex Medium Introduction Pluripotent stem cell (PSC) culture systems have evolved over time from feeder-dependent culture systems in which PSCs are cultured on a layer of inactivated mouse embryonic fi broblasts, to simpler, more defi ned, feeder-free culture systems in which PSCs are cultured on a variety of extracellular matrices (e.g., Gibco rhlaminin-521, Geltrex matrix, or Vitronectin Recombinant Human Protein, Truncated). The movement to these feeder-free systems has led to simpler, more effi cient methods for routine culture and expansion of PSCs, while maintaining the following hallmarks of PSCs: high nuclear-to-cytoplasmic ratio, pluripotency marker expression (e.g., OCT4, NANOG, TRA-1-6, SSEA4), normal karyotype, as well as maintenance of trilineage differentiation potential (i.e., the ability to differentiate to specifi c cell types of endoderm, mesoderm, and ectoderm). Coupling of expanded PSCs with downstream differentiation protocols provides in vitro models for understanding the fundamental basis of genetic diseases, drug discovery, as well as exploring regenerative therapy. Researchers are sometimes hesitant to change feeder-free culture systems due to concerns about performance in downstream differentiation of PSCs and that differentiation protocols will need to be updated to accommodate for changes in PSC properties. In this application note, we demonstrate that feeder-free Gibco StemFlex Medium (1) maintains trilineage differentiation potential following long-term passaging using the fl exible feeding schedule (Figure 1), (2) is compatible with downstream differentiation protocols historically used for downstream differentiation of PSCs cultured in Gibco Essential 8 Medium, and (3) is compatible with downstream Gibco differentiation kits. Suggested workflow PSCs cultured in StemFlex Medium can be differentiated using Gibco PSC Neural Induction Medium (Cat. No. A164781), Gibco PSC Cardiomyocyte Differentiation Kit (Cat. No. A292121), and Gibco PSC Defi nitive Endoderm Induction Kit (Cat. No. A36261). No changes to the currently recommended workfl ows are necessary when using StemFlex Medium. Special attention should be given to the suggested PSC seeding densities, as optimal PSC confl uency at the time of induction is crucial for successful downstream differentiation. These conditions can be cell line specifi c, and may need to be optimized for your PSC line. The recommended workfl ows for Gibco differentiation and induction kits are shown in Figure 2. Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Free day No feeding Free day No feeding Free day No feeding Passage Feed Passage Feed Figure 1. Recommended weekend-free feeding schedule. Unlike traditional PSC media, StemFlex Medium eliminates the need to manage cultures daily, enabling a truly weekend-free schedule for expansion and maintenance of PSCs. For additional feeding schedule options, visit thermofisher.com/stemflex PSC Neural Induction Medium (Cat. No. A164781, Pub. No. MAN13731) Initiate neural induction Replace PSC Neural Induction Medium Harvest and expand Plate PSCs in StemFlex Medium Observe morphology Cryopreserve, expand, differentiate Day: 1** PSCs at ~15 25% confluency* P NSCs 4 6 days P1 NSCs Seed PSCs at ~ x 1 4 cells/cm 2 * PSC Cardiomyocyte Differentiation Kit (Cat. No. A292121, Pub. No. MAN14534) Plate PSCs in StemFlex Medium Replace StemFlex Medium Cardiomyocyte Differentiation Medium A Cardiomyocyte Differentiation Medium B Cardiomyocyte Maintenance Medium, every other day Day: 4** Seed PSCs at 2 16 x 1 4 cells/cm 2 * PSCs at 3 7% confluency* Characterize, maintain, assay PSC Definitive Endoderm Induction Kit (Cat. No. A36261, Pub. No. MAN161) Plate PSCs in StemFlex Medium Definitive Endoderm Induction Medium A Definitive Endoderm Induction Medium B Characterize, differentiate, assay Day: 1** PSCs at ~15 3% confluency* 1 2 Seed PSCs at ~1 4 x 1 4 cells/cm 2 * * Seeding densities and induction confluencies for PSC differentiation may require optimization for your cell line. ** To achieve optimal confluency for induction, the number of days in culture prior to adding differentiation medium may require adjustment. With StemFlex Medium, PSCs can be plated for definitive endoderm induction on either Gibco Geltrex LDEV-Free, hesc-qualified, Reduced Growth Factor Basement Membrane Matrix (Cat. No. A14133) or Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A3184). Figure 2. Schematic workflows for differentiation and induction kits. Refer to the following manuals available at thermofisher.com for detailed protocols: Pub. No. MAN13731 for PSC Neural Induction Medium, Pub. No. MAN14534 for PSC Cardiomyocyte Differentiation Kit, and Pub. No. MAN161 for PSC Definitive Endoderm Induction Kit. Results Prior to differentiation, both Gibco Human Episomal ipscs (Cat. No. A18945) and H9 ESCs were cultured for over 5 passages in StemFlex Medium on Geltrex matrix using the flexible feeding schedule. PSCs were clump passaged with Gibco Versene Solution (Cat. No ) twice per week, skipping feeds on one weekday and both weekend days each week. A DAPI Merge Compatibility with PSC Neural Induction Medium PSC Neural Induction Medium is a serum-free medium that provides high-efficiency neural induction of human PSCs cultured in StemFlex Medium in 7 days. In preparation for neural induction, PSCs were cultured to ~7 8% confluency in StemFlex Medium, clump passaged with Versene Solution, and seeded at 25, cells/cm 2 onto a Thermo Scientific Nunc 6-well plate precoated with Geltrex matrix. Neural induction was initiated once PSCs reached 15 25% confluency. On day 7 of neural induction, newly differentiated neural stem cells (NSCs) were passaged with Gibco StemPro Accutase Cell Dissociation Reagent (Cat. No. A11151) and Gibco RevitaCell Supplement (Cat. No. A264451) and seeded at 1, cells/cm 2 onto a Thermo Scientific Nunc 96-well plate precoated with Geltrex matrix for immunocytochemistry staining and analysis. In Figure 3A, representative images are shown of NSCs derived from Human Episomal ipscs using PSC Neural Induction Medium. Cells were stained for the NSC markers NESTIN and SOX1 as well as nuclear DNA using the Invitrogen Human Neural Stem Cell Immunocytochemistry Kit (Cat. No. A24354). Figure 3B shows quantitative immunocytochemistry (ICC) data generated using the Thermo Scientific CellInsight CX5 High Content Screening Platform. NSCs derived from both Human Episomal ipscs and H9 ESCs cultured in StemFlex Medium show high expression ( 95%) of the NSC marker SOX1. B NESTIN Cells expressing SOX1 (%) SOX1 Human Episomal ipscs Human Episomal ipscs Figure 3. Induction efficiency with PSC Neural Induction Medium. PSCs cultured for 5 passages in StemFlex Medium support maintenance of ectoderm differentiation potential as shown by compatibility with PSC Neural Induction Medium. NSCs were (A) stained for the NSC markers NESTIN and SOX1 as well as nuclear DNA (DAPI) and (B) assessed for SOX1 expression by quantitative ICC. 98 H9 ESCs Compatibility with the PSC Cardiomyocyte Differentiation Kit The PSC Cardiomyocyte Differentiation Kit consists of a set of serum-free and xeno-free media that enables effi cient differentiation of human PSCs to contracting cardiomyocytes in as few as 8 days. In preparation for cardiomyocyte induction, PSCs were cultured to ~7 85% confl uency in StemFlex Medium, singularized using Gibco TrypLE Select Enzyme (Cat. No ), and seeded with RevitaCell Supplement at 2, cells/cm 2 onto a Thermo Scientifi c Nunc 12-well plate precoated with Geltrex matrix. Cardiomyocyte induction was initiated once PSCs reached ~5 6% confl uency. On day 12, newly differentiated cardiomyocytes were assessed for TNNT2 expression by fl ow cytometry using the Invitrogen Attune NxT Flow Cytometer. As shown in Figure 4, cardiomyocyte induction of both Human Episomal ipscs and H9 ESCs cultured in StemFlex Medium show high expression ( 8%) of the cardiomyocyte marker TNNT2 for optimal cell seeding densities. The data in Figure 5 show that Human Episomal ipscs cultured in StemFlex Medium can undergo successful cardiomyocyte differentiation at a wide range of induction conditions. For this ipsc line, 65% expression of the cardiomyocyte marker TNNT2 is shown for seeding densities of 15, 3, cells/cm 2 and induction confl uencies of ~35 75%. A Count B Cells expressing TNNT2 (%) Gibco Human Episomal ipscs: p51 in StemFlex Medium Unstained BL1-H Human Episomal ipscs H9 ESCs Figure 4. Induction efficiency with the PSC Cardiomyocyte Differentiation Kit. PSCs cultured for 5 passages in StemFlex Medium support maintenance of mesoderm differentiation potential as shown by compatibility with the PSC Cardiomyocyte Differentiation Kit. (A) Representative fl ow cytometry data. (B) Percentage of cells expressing the cardiomyocyte marker TNNT2 by fl ow cytometry. PSC confluency or TNNT2 expression via IncuCyte Zoom System (%) , 15, 25, 3, 35, 4, PSC confluency at time of cardiomyocyte induction (%) TNNT2 expression at day 12 by flow cytometry (%) Cell seeding density (cells/cm 2 ) using TrypLE Select and RevitaCell Supplement Figure 5. Differentiation potential at multiple seeding densities. Human Episomal ipscs cultured for 5 passages in StemFlex Medium show successful cardiomyocyte differentiation at a range of seeding densities and induction confl uencies. Compatibility with the PSC Definitive Endoderm Induction Kit The PSC Defi nitive Endoderm Induction Kit consists of two xeno-free media that enable effi cient induction of human PSCs to defi nitive endoderm. Unlike other methods that require multiple components and take fi ve or more days, the PSC Defi nitive Endoderm Induction Kit can generate 9% CXCR4 + /PDGFRα defi nitive endoderm cells with only two components in just two days. A FL2-H:: PDGFRα PE In preparation for defi nitive endoderm induction, PSCs were cultured to ~7 85% confl uency in StemFlex Medium, passaged using StemPro Accutase Cell Dissociation Reagent, and seeded with RevitaCell Supplement at 1, cells/cm 2 onto a Nunc 6-well plate precoated with Geltrex matrix. Defi nitive endoderm induction was initiated once PSCs reached ~15 3% confl uency. On day 3 of defi nitive endoderm induction, cells were assessed for expression of CXCR4 + /PDGFRα by fl ow cytometry. As shown in Figure 6, defi nitive endoderm induction of both Human Episomal ipscs and H9 ESCs cultured in StemFlex Medium show high expression ( 95%) of CXCR4. B CXCR4 + /PDGFRα cells (%) FL4-H:: CXCR4 APC Human Episomal ipscs H9 ESCs Figure 6. Induction efficiency with the PSC Definitive Endoderm Induction Kit. PSCs cultured for 5 passages in StemFlex Medium support maintenance of endoderm differentiation potential as shown by compatibility with the PSC Defi nitive Endoderm Induction Kit. (A) Representative fl ow cytometry data. (B) Percentage of CXCR4 + /PDGFRα cells by fl ow cytometry. Conclusions Together, these data confirm trilineage differentiation potential for two PSC lines cultured for over 5 passages in StemFlex Medium using the flexible feeding schedule. Additionally, the StemFlex Medium system is shown to be compatible with available Gibco differentiation products PSC Neural Induction Medium, the PSC Cardiomyocyte Differentiation Kit, and the PSC Definitive Endoderm Induction Kit. PSCs differentiated following prolonged culture in StemFlex Medium show high expression of respective lineage-specific markers, enabling success in workflows requiring downstream differentiation. Find out more at thermofisher.com/stemflex For Research Use Only. Not for use in diagnostic procedures. 217 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. IncuCyte is a trademark of Essen BioScience. Essential 8 is a trademark of Cellular Dynamics International, Inc. Versene is a trademark of Dow Chemical Co. COL
Similar documents
View more...
Search Related
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks