Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Summary

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Abstract

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation¹. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity⁴. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types^5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Protocol

1. Preparation of Mouse Embryonic Feeders

1. The MEFs used in the co-culture of hESCs should be previously treated by irradiation or Mitomycin-C to inhibit the cells from undergoing division.
2. Two hours prior to MEF seeding, coat each 60 mm plasma treated culture dish with 2 ml of 0.05% Gelatin.
3. Thaw a vial of treated MEFs and plate at ~20,000 cells/cm^2.
4. Allow the cells to adhere overnight.

2. Co-seeding hESCS onto MEFs

1. New colonies may be initiated from cryopreserved cultures or passaging current cultures onto new MEF-coated dishes, as below.
2. Wash the plate conta....

Discussion

The method presented in this manuscript offers a rapid and less costly alternative to eliminating fibroblast feeder cells from human embryonic cell cultures. The successful removal of fibroblasts is dependent on the existence of a tightly confluent monolayer of these cells that develops in longer stem cell cultures. After 7-10 days, the growing hESC colonies will push the feeder fibroblasts in an outward direction, generating an increasingly dense fibroblast monolayer between colonies. The strong cell-to-cell attachments.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DDR2	Santa Curz	7555	Fibroblast Marker
Dapi	Calbiochem	268298	Cell Nucleus Marker
SSEA-4	Millipore	MAB4304	Human ESC Marker
Oct 3/4	Santa Cruz	9081	Human ESC Marker
Flk-1	BD Pharma	555307	Early Differentiation Marker
Tra-1-81	Acris	AM20377AF4-S	Human ESC Marker
Table 1. Table of specific reagents used for immunostaining the hESC.