Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

Summary

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.

Abstract

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.

Introduction

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-renewal^1-3. However, the direct transplantation of stem cells has several limitations, such as immune rejection and tumor formation^4,5. Therefore, a cell-free approach may provide an alternate therapeutic strategy for regenerative medicine and aging interventions^6,7.<....

Protocol

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)

1. Prepare 500 ml of medium to culture the MEFs. Supplement Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS (ESC quality), 50 units/ml penicillin, and 50 mg/ml streptomycin.
2. Isolate MEFs from embryos following an established routine protocol¹³ and maintain them in MEF medium.
3. Prepare 500 ml of medium to culture the mESCs. DMEM is supplemented with 15% FBS and 2 mM L-glutamine, 100 µM non-essential amino acids (NEAA), 100 µM ....

Results

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.......

Discussion

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 t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	#11960-044
FBS	Invitrogen	#30044333	20%, ES cell quality
Penicillin and streptomycin	Invitrogen	#15140	50 units/ml penicillin and 50 mg/ml streptomycin
L-glutamine	Invitrogen	#25030	2 mM
Nonessential amino acids (NEAA)	Invitrogen	#11140	100 µM
β-mercaptoethanol	Sigma	#M3148	100 µM
Leukemia inhibitory factor	Millipore	#ESG1107	100 units/ml
OPTI-MEM	Invitrogen	#22600
X-gal	Sigma	#B4252	1 mg/ml
Paraformaldehyde (PFA)	Sigma	P6148	3.70%
Dimethylformamide (DMF)	Sigma	#D4551
Potassium ferricyanide	Aldrich	#455946	5 mM
potassium ferrocyanide	Aldrich	#455989	5 mM
NaCl	Sigma	#S7653	150 mM
MgCl2	Sigma	#M2393	2 mM
Mitomycin C	Sigma	#M4287	10 µg/ml
Propidium iodide	Sigma	#P4170	50 µg/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	0.2 μm