Abstract
This study characterizes dynamic and apoptotic blebbing in human embryonic stem cells (hESC), identifies dynamic blebbing as a bottleneck to successful cell attachment during passaging, and demonstrates that dynamic blebbing can be rapidly stopped by plating cells on recombinant human laminin. In freshly plated hESC, dynamic and apoptotic blebbing differed in time of occurrence, bleb retraction rate, mitochondrial membrane potential, and caspase 3&7 activation. While dynamic blebbing can be controlled with drugs that inhibit myosin II, these methods have off-target effects and are not suitable for clinical applications. Recombinant human laminin-521 or addition of laminin-111 to Matrigel provided a safe method to drastically decrease dynamic blebbing and improve cell attachment with proteins normally found in the inner cell mass. Inhibition of focal adhesion kinase, which is activated by binding of integrins to laminin, prolonged dynamic blebbing and inhibited attachment. These data show that hESC bind rapidly to laminins through an integrin, which activates focal adhesion kinase that in turn downregulates dynamic blebbing. Laminins enabled hESC to rapidly attach during passaging, improved plating efficiency, enabled passaging of single pluripotent stem cells, and avoided use of inhibitors that have non-specific off-target effects. These data provide a strategy for improving hESC culture using biologically safe recombinant human proteins.
Highlights
Human embryonic stem cells were derived in 1998 (Thomson et al, 1998), 16 years after their first mouse counterparts were reported (Martin, 1981; Evans and Kaufman, 1981). hESC are generally derived from spare blastocysts offered for research purposes by patients undergoing in vitro fertilization (Thomson et al, 1998)
We evaluated dynamic blebbing and attachment on different matrices that are used for hESC culture
We investigated the hypothesis that binding of hESC to laminin activates signaling through an integrin and focal adhesion kinase (FAK), which shuts down dynamic blebbing
Summary
Human embryonic stem cells (hESC) were derived in 1998 (Thomson et al, 1998), 16 years after their first mouse counterparts were reported (Martin, 1981; Evans and Kaufman, 1981). hESC are generally derived from spare blastocysts offered for research purposes by patients undergoing in vitro fertilization (Thomson et al, 1998). Two major improvements in hESC culture were the replacement of feeder layers with Matrigel, a hESC-qualified matrix, and the introduction of better defined, feeder-free maintenance culture media, such as mTeSR (Ludwig et al, 2006a; Ludwig et al, 2006b; McElroy and Reijo Pera, 2008; Hughes et al, 2010) In spite of these improvements, hESC do not readily attach to substrates and cannot be plated as single cells. Dynamic blebs are membrane protrusions that appear and disappear from the surface of healthy cells (Charras and Paluch, 2008). Dynamic blebbing occurs in three phases referred to as nucleation, expansion, and retraction (Charras, 2008). Blebbing provides the motive force for invasion of tissue by Entamoeba histyltica and migration of breast cancer cells during metastasis (Khajah and Luqmani, 2015)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
