Immunocytochemistry photographs of 129X1 Murine ES colonies stained for OCT3/4 (Red ) and SSEA 1 (Green). Colony A was grown on HyStem-C™, and colony B was grown on MEF feeders.
Introduction
Mitotically inactive mouse embryonic fibroblast feeder cells (iMEFs) have been widely used as a substrate to culture embryonic stem cells (ESCs) but suffer from troublesome preparation, lot-to-lot variability, and iMEF carryover during ESC passage. A synthetic substrate which mimics iMEFs would solve these problems. Since iMEFs make abundant amounts of hyaluronic acid (HA) important for hESC culture (1), we reasoned that a synthetic HA-rich matrix (HyStem-C®) would provide a suitable starting point for preparing a novel feeder-free substrate for undifferentiated ESC growth. As a first step, we hypothesized that mouse embryonic stem cells could be passaged without feeder cells on HyStem –C® for a series of five passages and maintain a pluripotent state. Cells grown in such a manner were compared to growth on iMEFs and evaluated for proper growth, morphology and expression of pluripotency markers. This evaluation was accomplished via FACs analysis, a plating efficiency assay and by scoring the colony morphology of the test conditions.
Methods and Materials
HyStem-C® Hydrogel® matrix preparation and storage: HyStem-C® Hydrogel ® (ThermoFisher Part# SV30139.01) was prepared according to the manufacturer’s instructions. We used 400µL of the prepared HyStem® in each flask (enough to completely cover the bottom). HyStem-C Hydrogel® matrix was allowed to polymerize for at least 30 minutes before use. Flasks that were not used immediately were stored for up to 1 week in a sealed humidified container at 4°C.
Serial SubcultureStudies: Primogenix‘ 129X1 mouse embryonic stem cells (mESC) p11 (ThermoFisher Part#SV30098.01) were thawed from cryogenic freeze onto iMEF feeder layers and expanded to p12 which allowed the cells to recover from the freeze and to obtain sufficient cells to seed iMEF control flasks and flasks containing only a layer of HyStem-C®. Cells were seeded into 5 mL of growth medium in 12.5 cm2 flasks at 10,000 cells per cm2. Growth medium consisted of Thermo Scientific HyClone Low Osmo DMEM (SH30870.01) supplemented with 15% Thermo Scientific HyClone ES screened FBS (SH30070.03E), 4mM Thermo Scientific HyClone L glutamine (SH30034.01), 100 U/mL Thermo Scientific HyClone Penicillin Streptomycin (SV30010), and 1000U/mL LIF (Millipore # ESG1106). This AdvanceSTEM® Low Osmo DMEM requires no additional NEAA, HEPES or Beta Mercaptoethanol.
Cells were allowed to proliferate in a humidified incubator at 37°C and 5% CO2 for 5 passages. Subculture passaging took place every 48 hours with media exchanges every 24 hours. At passages, media was removed from the flasks and cells were rinsed using Thermo Scientific HyClone PBS (SH30028.03). Cells were dissociated using Thermo Scientific HyClone 0.25% Trypsin/EDTA (SH30042.01). After dissociation, cells were counted via hemacytometer and iMEF containing cultures were reseeded onto fresh layers of MEFs and HyStem-C® cultures were seeded onto fresh HyStem-C® at 10,000 cells per cm2.
Plating Efficiency: Mitotically inactivated MEFs were plated into 6 well plates. HyStem C was used to coat 6 well tissue culture plates with approximately 200µL of the hydrogel matrix. Various media conditions were used in duplicate wells of the plating efficiency. Control media conditions consisted of Thermo Scientific HyClone DMEM (SH30022.01) supplemented with 10%, 15%, and 30%, Thermo Scientific HyClone ES screened FBS, 4mM Thermo Scientific HyClone L-glutamine, 10 mM Thermo Scientific HEPES (SH30237.01), 1X Thermo Scientific NEAA (SH30238.01), 10µL/L Beta Mercaptoethanol (Sigma M7522), 100 U/mL Thermo Scientific HyClone Penicillin/Streptomycin, and 1000U/mL LIF (Millipore # ESG1106).
Test conditions included the same medium used with the 15% FBS control and media consisted of Thermo Scientific HyClone AdvanceSTEM® Low Osmo DMEM, supplemented with 20% Thermo Scientific HyClone ES screened FBS, 4mM Thermo Scientific HyClone L glutamine, 100 U/mL Thermo Scientific HyClone Penicillin Streptomycin, and 1000U/mL LIF (Millipore # ESG1106).
129S6 p20+ cells were seeded into each replicate condition at 1000 cells/well. Cells were grown in a humidified incubator at 37°C and 5% CO2. Media was exchanged every 48 hours for 7 days, at which time colonies of cells were fixed with methanol, stained with methylene blue and basic fuchsin, counted and scored for morphology. Percent plating efficiency is derived by dividing the number of colonies counted in single well by the original seeding density of 1000 cells. Colony morphologies were scored on a scale from 0 to 4. A score of 0 reflects complete differentiation of colonies within a single culture well. A score of 4 reflects no differentiation of colonies within a single culture well. A morphology score of 3 reflects a culture with little differentiation and considered good.
Flowcytometry: Cultures were analyzed using the BD FACSAria with FACSDiva software. Cultures were harvested using Thermo Scientific HyClone 0.25% Trypsin/EDTA and fixed/stained with CytoFix/Cytoperm® fixation/permeabilization kit (BD554714). Cells were surface stained with SSEA-1-AF488 (IgM) conjugate (Santa Cruz Biotechnology # SC21702). Negative isotype control for SSEA-1 Rat IgM-AF488 conjugate (Santa Cruz Biotechn ology #SC45082). After surface staining, cells were permeablized and stained with OCT3/4-PE (IgG) conjugate (BD # 560186). Negative isotype contol for SSEA-1 Rat IgG-PE (BD# 340270). After staining, cells were rinsed and resuspended in FACS buffer for analysis.
Immunocytochemisty: At the 6th subculture p18, cells were seeded on 8 well slides on either iMEFs and HyStem –C®. After 48 hours cultures were fixed and permeablized with 2% paraformaldehyde (Sigma# 158172) and 0.1% Triton X100 (Amersco#0694). Primary antibodies SSEA-1 IgM (Developmental Studies Hybridoma Bank (MC-480) and OCT3/4-IgG2b (Santa Cruz Biotechn ology # sc5279) were added at 1:100.
The secondary antibodies Alexa Fluor 488-IgM (Invitrogen# 21042) and Alexa Fluor 568-IgG2b (Invitrogen#21144) at 1:1000. Cultures were examined and photographed using a fluorescence microscope with the appropriate filters.
Proliferation Rate of mESCs grown on HyStem-C® or MEFs is comparable.
 Figure 1: Growth curves for mESCs grown on iMEFs and HyStem-C™. This figure illustrates the growth trends of cells grown on iMEF feeder layers and HyStem-C™ over the course of 5 passages. Note that during each passage the cells were seeded at 10,000 cells/cm2. |
Figure 2: Average Population Doubling times for mESCs grown on iMEFs and HyStem-C™. This figure illustrates the average doubling time over the course of five passages for cells grown on iMEF feeder layers and HyStem-C™. |
Figure 3: Phase contrast photographs of mESC colonies over the course of the study. Photos of colonies grown on HyStem-C™ (top Row) and photos of colonies co-cultured with MEF feeders (bottom row). Morphologies bear slight differences between HyStem-C™ and MEF co-cultures. However, cultures grown on both substrates were able to maintain colonies that generally exhibited relatively regular, bright, well defined borders, characteristics of undifferentiated mESC colonies.
Colony Morphology and Plating Efficiency Analysis on HyStem-C™ or iMEFs are similar.
 Figure 4: Morphology Scores of mESC colonies grown on HyStem-C™ Compared to MEF co-cultured control. Note that HyStem-C™ produced mESC colonies with morphologies very similar to the MEF Co-Cultured controls. |
 Figure 5: Plating efficiency of mESC colonies grown on HyStem-C™ compared to MEF co-cultured control. The percentage of colonies that formed on HyStem-C™ without feeders is less than the percentage of colonies that formed on MEF feeders. However, an additional 5% FBS in Low OSMO DMEM significantly improves comparability. This is as much increase in plating efficiency as seen in mESCs grown on iMEFs even when FBS concentration is increased as much as 20%. |
Mouse ESCs grown on HyStem-C show comparable levels of pluripotency markers.
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Figure 6: Immunocytochemistry photographs of 129X1 Murine ES colonies stained for OCT3/4 (Red ) and SSEA 1 (Green). Colony A was grown on HyStem-C™, and colony B was grown on MEF feeders. |
 Figure 7: Flow Cytometry. This figure shows the percentage of cells expressing the pluripotency markers SSEA-1 and OCT3/4, as well as the percentage of cells expressing both (double positive)as assessed by flow cytometry. Overall, pluripotency expression, as measured using Oct3/4 and SSEA-1 is comparable. |
Conclusion
Similarities in growth rate, plating efficiency, and maintenance of pluripotent morphology as well as similar levels of the mESC pluripotency markers, Oct3/4 and SSEA-1 show that HyStem-C® replaced iMEF co-culture of mESCs.
Compared to iMEF co-culture, HyStem-C will likely decrease variability in cell-based high-throughput screening assays that require natural matrices because HyStem-C is consistent from lot-to-lot.
HyStem-C enables more sensitive genomic, proteomic, or metabolomic analysis of ESCs because contaminating MEFs are not required while maintaining undifferentiated phenotype.
References
- Gerecht S et al, Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells PNAS 2007 vol 104: 11298–11303
- Engler et al, Matrix Elasticity Directs Stem Cell Lineage Specification Cell 2006 vol 126: 677-689.
