This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.
The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.
The goal of this protocol is to collect mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) from serum- and feeder-free culture conditions and to characterize its biological functions.
In general, embryonic stem cells (ESCs) have great potential for regenerative medicine and cell therapy due to their pluripotency and capacity for self-renewal1-3. However, the direct transplantation of stem cells has several limitations, such as immune rejection and tumor formation4,5. Therefore, a cell-free approach may provide an alternate therapeutic strategy for regenerative medicine and aging interventions6,7.
Senescence is viewed as a cellular counterpart to the aging of tissues and organs, characterized by a permanent state of growth arrest, altered cell physiology, and behaviors. Aging is the main risk factor for prevalent diseases including cancer, cardiovascular disease, type 2 diabetes, and neurodegeneration8. One of the obvious characteristics of aging is the decline in the regenerative potential of tissues, which is caused by stem cell aging and exhaustion9. Many significant studies have shown pharmacological molecules, such as rapamycin9, resveratrol10, and metformin11, and blood-borne systemic factors, namely GDF1112, that have the ability to consistently delay aging and extend life span.
In the present study, mESC-CM has been harvested without serum (fetal bovine serum, FBS) and feeder (mouse embryonic fibroblasts, MEFs) layers to exclude the contamination of serum factors and secretory factors from MEFs. These conditions allowed for a serum- and feeder-free CM that consequently enabled the accurate identification of mESC-specific secretory factors.
This proposed protocol is highly efficient, relatively cost effective, and easy to operate. This technique provides insights into the characterization of mESC-derived soluble factors that can mediate an anti-senescence effect, which may be used for the development of a safe and potentially advantageous cell-free therapeutic approach toward interventions for aging-associated diseases and other regenerative treatments.
NOTE: A schematic of the serum- and feeder-free CM collection protocol is shown in Figure 1.
1. Materials (Preparation of MEFs, Medium, Plates, and Solutions)
2. Culture of Mouse Embryonic Stem Cells (Figure 1A and 2A)
NOTE: Carry out all steps in a cell culture biological safety hood.
3. Collection of Serum- and Feeder-free Conditioned Medium (Figure 1B and 2B)
NOTE: Carry out all steps in a cell culture biological safety hood.
4. Effects of Mouse Embryonic Stem Cell-conditioned Medium (mESC-CM)
NOTE: The effects of mESC-CM were validated by several methods, such as SA β-gal assay, cell cycle analysis, and qRT-PCR.
Originally, mESCs are maintained on an MEF feeder in mESC medium with FBS and other supplements (Figures 1A and 2A). CM was collected from mESCs in Reduced Serum Media without a feeder layer, FBS, or other supplements (Figures 1B and 2B). This culture condition allows us to collect mESC-specific conditioned medium without potential contamination by the factors from the feeder, FBS, or other supplements. The control medium was collected under the same culture conditions, without mESCs.
mESCs show different morphologies between the two culture media: i) normal mESC culture conditions (Figure 2A) and ii) serum- and feeder-free culture conditions (Figure 2B). The mESC colonies grew on an MEF layer and demonstrated an oval and shiny appearance under the normal mESC culture conditions (Figure 2A). On the contrary, the mESCs in the serum- and feeder-free culture conditions showed a flattened and irregular morphology (Figure 2B).
The functional characterization of mESC-CM was achieved by senescence-associated methods, such as SA β-gal assay (Figure 3A), cell cycle analysis (Figure 3B), and qPCR (Figure 3C). Treatment of senescent HDFs with mESC-CM decreased the number of positive SA β-gal-positive cells, which is an indicator of cellular senescence (Figure 3A). Cell cycle analysis revealed that mESC-CM treatment dramatically increased the number of cells in the S and G2/M phase, whereas it reduced the number of cells in the G0/G1 phase (Figure 3B). In addition, mESC-CM treatment decreased the senescence-associated gene expression levels (namely, p53, p21, and p16) and the senescence-associated secretory phenotype (SASP) expression levels (IL-6).
Figure 1: Preparation and optimization of mESC-CM. Experimental strategy for the preparation and optimization of serum-free and feeder-free CM. (A) Normal mESC culture condition and (B) serum- and feeder-free mESC-CM culture condition. C: control medium without FBS and MEF; CM: conditioned medium without FBS and MEF. Modified with permission from Bae et al.15. Please click here to view a larger version of this figure.
Figure 2: Bright field images of mESCs. mESCs under (A) normal conditions and (B) serum- and feeder-free conditions. Yellow arrows indicate feeder cell (MEFs) in normal mESC culture conditions. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 3: Characterization of the anti-aging effect of mESC-CM. (A) SA β-gal activity staining and the percentage of SA β-gal-positive cells. (B) Cell cycle analysis by flow cytometry. (C) Expression levels of senescence-associated gene expression levels (p53, p21, and p16) and senescence-associated secretory phenotype (SASP) expression levels (IL-6) by qRT-PCR. Values are the mean ± SD. Figures are representative of three independent experiments. Statistically-significant differences between groups were identified by one-way ANOVA and Tukey's post-hoc test. *p <0.05, **p <0.01. Y = non-senescent cells; S: senescent cells; C: control medium without FBS and MEF; CM: conditioned medium without FBS and MEF. Scale bars = 10 µm. Modified with permission from Bae et al.15. Please click here to view a larger version of this figure.
For the successful collection of serum- and feeder-free mESC-CM, the following suggestions should be taken into consideration. The most critical factor is using early passage mESCs for the collection of mESC-CM. Previously, it has been shown that early passage mESC-CM has better anti-aging effects compared to late passage mESCs. The passage number of mESCs has been reported to affect their developmental potential16 and pluripotency17.
While additional research is needed to analyze the specific factors of the mESC secretome, which induce anti-senescence effects, we can currently conclude that mESC-CM is sufficient to decrease senescence at the cellular level.
The identification of mESC-specific secretory factors that revert senescent cells back to young cells will be critical for future studies. For high-quality analyses on the secretory molecules, such as antibody array15 and secretome analysis, the washing step during the medium collection process (step 3) is critical. If the washing step is not properly conducted, the secretory molecules will be contaminated by serum (FBS) components18,19.
The serum- and feeder-free incubation time (24 hr) is very important in the medium collection process (step 3), as the longer incubation time (over 24 hr) may increase the possibility of cell autolysis or apoptosis by starvation under the serum- and feeder- depleted conditions18,19. The normal ESC culture condition requires a feeder layer for long-term culturing of undifferentiated cells, as the feeder secretes a large number of molecules22. The gelatin-coated plate prevents the possibility of contamination from the feeder cells.
The mESC-CM, harvested from serum- and feeder-free culture conditions, has an anti-senescence ability in senescent HDFs. Anti-senescence effects of mESC-CM have been demonstrated by senescence-associated multiple readouts, such as SA β-gal activity; an enhanced proliferative potential (cell cycle analysis); and reduced p53, p21, p16, and IL-6 gene expression levels (Figure 3A–3C).
When human primary cells are treated with mESC-CM, xeno-contamination would be a critical issue for clinical application. Therefore, an investigation of the secretory factors from human ESCs or iPSCs would be an important future study for the clinical application of CM derived from human origins. The convergence of a cell-free approach based on a stem cells and an anti-senescence study is expected to expand the current understanding of senescence-associated diseases, resulting in greater insight into improvements on therapeutic approaches.
The authors have nothing to disclose.
This research was supported by the Basic Science Research Program (2013R1A1A2060930) and the Medical Research Center Program (2015R1A5A2009124) through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning. This research is also supported by a Start-up Operating Grant from The Hospital for Sick Children (H. K. Sung). We would like to thank Laura Barwell and Sarah J. S. Kim for their excellent help in editing this manuscript and Dr. Andras Nagy for providing the G4 mESC line.
DMEM | Invitrogen | #11960-044 | |
FBS | Invitrogen | #30044333 | 20%, ES cell quality |
Penicillin and streptomycin | Invitrogen | #15140 | 50units/ml penicillin and 50mg/ml strepto |
-mycin. | |||
L-glutamine | Invitrogen | #25030 | 2mM |
Nonessential amino acids (NEAA) | Invitrogen | #11140 | 100uM |
β-mercaptoethanol | Sigma | #M3148 | 100uM |
Leukemia inhibitory factor | Millipore | #ESG1107 | 100units/ml |
OPTI-MEM | Invitrogen | #22600 | |
X-gal | Sigma | #B4252 | 1mg/ml |
Paraformaldehyde (PFA) | Sigma | P6148 | 3.70% |
Dimethylformamide (DMF) | Sigma | #D4551 | |
Potassium ferricyanide | Aldrich | #455946 | 5mM |
potassium ferrocyanide | Aldrich | #455989 | 5mM |
NaCl | Sigma | #S7653 | 150mM |
MgCl2 | Sigma | #M2393 | 2mM |
Mytomycin C | Sigma | #M4287 | 10ug/ml |
Propidium iodide | Sigma | #P4170 | 50ug/ml |
TRIzol | Ambion | #15596018 | |
M-MLV reverse transcript-tase | Promega | #M170B | |
Power SYBR Green PCR master mix | Applied Biosystems | #4367659 | |
HDFs, NHDF-Ad-Der-Fibroblast | LONZA | #CC-2511 | |
Bottle top filter, | Corning | #430513 | 0.2μm |