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.
Subject(s)
Human Embryonic Stem Cells/metabolism , Integrins/metabolism , Laminin/metabolism , Humans , Signal TransductionABSTRACT
New methods are needed to reduce dynamic blebbing which inhibits cell attachment and survival during passaging of pluripotent stem cells. We tested the hypothesis that activation of the P2X7 receptor by extracellular ATP during passaging initiates dynamic blebbing. The P2X7 receptor was present in human embryonic stem cells (hESC), but not in differentiating cells. Extracellular ATP concentrations were 14× higher in medium during passaging. Addition of ATP to culture medium prolonged dynamic blebbing and inhibited attachment. Inhibition of P2X7 by specific drugs or by siRNA significantly reduced dynamic blebbing and improved cell attachment. When cells were incubated in calcium chelators (EGTA or BAPTA), blebbing decreased and attachment improved. Calcium influx was observed using Fura-4 when ATP was added to culture medium and inhibited in the presence of the P2X7 inhibitor. Over-expressing activated Rac in hESC reduced blebbing and promoted cell attachment, while a Rac inhibitor prolonged blebbing and reduced attachment. These data identify a pathway involving P2X7 that initiates and prolongs dynamic blebbing during hESC passaging. This pathway provides new insight into factors that increase dynamic blebbing and identifies new targets, such as P2X7, that can be used to improve the culture of cells with therapeutic potential.
Subject(s)
Cell Surface Extensions/metabolism , Human Embryonic Stem Cells/metabolism , Receptors, Purinergic P2X7/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Adenosine Triphosphate/metabolism , Biomarkers/metabolism , Calcium/metabolism , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Extracellular Space/metabolism , Gene Knockdown Techniques , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/drug effects , Humans , Ion Channel Gating/drug effects , Models, Biological , Purinergic P2X Receptor Antagonists/pharmacology , Thiazoles/pharmacology , Time-Lapse Imaging , rac GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
Blebbing is an important biological indicator in determining the health of human embryonic stem cells (hESC). Especially, areas of a bleb sequence in a video are often used to distinguish two cell blebbing behaviors in hESC: dynamic and apoptotic blebbings. This paper analyzes various segmentation methods for bleb extraction in hESC videos and introduces a bio-inspired score function to improve the performance in bleb extraction. Full bleb formation consists of bleb expansion and retraction. Blebs change their size and image properties dynamically in both processes and between frames. Therefore, adaptive parameters are needed for each segmentation method. A score function derived from the change of bleb area and orientation between consecutive frames is proposed which provides adaptive parameters for bleb extraction in videos. In comparison to manual analysis, the proposed method provides an automated fast and accurate approach for bleb sequence extraction.
