Grace Sock Leng Teo

Mesenchymal Stem Cell Homing: The Devil Is in the Details

The study of MSC trafficking is clinically relevant for minimally invasive cell therapy to promote regeneration of damaged tissue, to treat inflammation, and to promote angiogenesis. However, these studies are complicated by the diverse methods used to culture, characterize, and deliver MSCs and by the variety of methods used to assess homing events. This review provides a critical analysis of the methods used to track homing of exogenously infused MSCs and discusses strategies for enhancing their trafficking to particular tissues.

Bioinspired multivalent DNA network for capture and release of cells

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

Mesenchymal Stem Cells Transmigrate Between and Directly Through Tumor Necrosis Factor‐α‐Activated Endothelial Cells Via Both Leukocyte‐Like and Novel Mechanisms

Systemically administered adult mesenchymal stem cells (MSCs), which are being explored in clinical trials to treat inflammatory disease, exhibit the critical ability to extravasate at sites of inflammation. We aimed to characterize the basic cellular processes mediating this extravasation and compare them to those involved in leukocyte transmigration. Using high‐resolution confocal and dynamic microscopy, we show that, like leukocytes, human bone marrow‐derived MSC preferentially adhere to and migrate across tumor necrosis factor‐α‐activated endothelium in a vascular cell adhesion molecule‐1 (VCAM‐1) and G‐protein‐coupled receptor signaling‐dependent manner. As several studies have suggested, we observed that a fraction of MSC was integrated into endothelium. In addition, we observed two modes of transmigration not previously observed for MSC: Paracellular (between endothelial cells) and transcellular (directly through individual endothelial cells) diapedesis through discrete gaps and pores in the endothelial monolayer, in association with VCAM‐1‐enriched “transmigratory cups”. Contrasting leukocytes, MSC transmigration was not preceded by significant lateral migration and occurred on the time scale of hours rather than minutes. Interestingly, rather than lamellipodia and invadosomes, MSC exhibited nonapoptotic membrane blebbing activity that was similar to activities previously described for metastatic tumor and embryonic germ cells. Our studies suggest that low avidity binding between endothelium and MSC may grant a permissive environment for MSC blebbing. MSC blebbing was associated with early stages of transmigration, in which blebs could exert forces on underlying endothelial cells indicating potential functioning in breaching the endothelium. Collectively, our data suggest that MSC transmigrate actively into inflamed tissues via both leukocyte‐like and novel mechanisms. STEM CELLS2012;30:2472–2486

Chemical Engineering of Mesenchymal Stem Cells to Induce a Cell Rolling Response

Covalently conjugated sialyl Lewis X (SLeX) on the mesenchymal stem cell (MSC) surface through a biotin-streptavidin bridge imparts leukocyte-like rolling characteristics without altering the cell phenotype and the multilineage differentiation potential. We demonstrate that the conjugation of SLeX on the MSC surface is stable, versatile, and induces a robust rolling response on P-selectin coated substrates. These results indicate the potential to increase the targeting efficiency of any cell type to specific tissue.

Cellular and extracellular programming of cell fate through engineered intracrine-, paracrine-, and endocrine-like mechanisms

A cells fate is tightly controlled by its microenvironment. Key factors contributing to this microenvironment include physical contacts with the extracellular matrix and neighboring cells, in addition to soluble factors produced locally or distally. Alterations to these cues can drive homeostatic processes, such as tissue regeneration/wound healing, or may lead to pathologic tissue dysfunction. In vitro models of cell and tissue microenvironments are desirable for enhanced understanding of the biology and ultimately for improved treatment. However, mechanisms to exert specific control over cellular microenvironments remains a significant challenge. Genetic modification has been used but is limited to products that can be manufactured by cells and release kinetics of therapeutics cannot easily be controlled. Herein we describe a non-genetic approach to engineer cells with an intracellular depot of phenotype altering agent/s that can be used for altering cell fate via intracrine-, paracrine-, and endocrine-like mechanisms. Specifically, we show that human mesenchymal stem cells (MSCs) can be engineered with poly lactide-co-glycolic acid (PLGA) particles containing dexamethasone, which acts on cytoplasmic receptors. The controlled release properties of these particles allowed for sustained intracellular and extracellular delivery of agent to promote differentiation of particle-carrying cells, as well as neighboring cells and distant cells that do not contain particles.

Intravital Imaging of Mesenchymal Stem Cell Trafficking and Association With Platelets and Neutrophils

Early events of mesenchymal stem/stromal cell (MSC) adhesion to and transmigration through the vascular wall following systemic infusion are important for MSC trafficking to inflamed sites, yet are poorly characterized in vivo. Here, we used intravital confocal imaging to determine the acute extravasation kinetics and distribution of culture‐expanded MSC (2–6 hours postinfusion) in a murine model of dermal inflammation. By 2 hours postinfusion, among the MSC that arrested within the inflamed ear dermis, 47.8% ± 8.2% of MSC had either initiated or completed transmigration into the extravascular space. Arrested and transmigrating MSCs were equally distributed within both small capillaries and larger venules. This suggested existence of an active adhesion mechanism, since venule diameters were greater than those of the MSC. Heterotypic intravascular interactions between distinct blood cell types have been reported to facilitate the arrest and extravasation of leukocytes and circulating tumor cells. We found that 42.8% ± 24.8% of intravascular MSC were in contact with neutrophil‐platelet clusters. A role for platelets in MSC trafficking was confirmed by platelet depletion, which significantly reduced the preferential homing of MSC to the inflamed ear, although the total percentage of MSC in contact with neutrophils was maintained. Interestingly, although platelet depletion increased vascular permeability in the inflamed ear, there was decreased MSC accumulation. This suggests that increased vascular permeability is unnecessary for MSC trafficking to inflamed sites. These findings represent the first glimpse into MSC extravasation kinetics and microvascular distribution in vivo, and further clarify the roles of active adhesion, the intravascular cellular environment, and vascular permeability in MSC trafficking. Stem Cells 2015;33:265–277

In vivo tracking of hematopoietic cells in the retina of chimeric mice with a scanning laser ophthalmoscope

We examine the effect of bone marrow transplantation (BMT) on retinal cell turnover by performing simultaneous cell tracking of native microglia and engrafting donor bone marrow-derived cell (BMDC) populations in the retinae of live mice using a custom-built multi-color confocal scanning laser ophthalmoscope (SLO) specifically developed for murine retinal imaging. CX3CR1GFP/+ mice whose retinal microglia express the green fluorescent protein (GFP) were exposed to a lethal dose of gamma radiation and subsequently rescued with bone marrow cells from universal DsRed donor mice. Over a time course of four months after the irradiation and BMT, progressive loss of GFP+ microglia was accompanied by delayed engraftment of DsRed+ BMDC. Morphologic examination revealed that the remaining GFP+ microglia were ramified, while engrafting DsRed+ cells exhibited both ramification and dendriform shape. Leukocyte endothelial interaction, normally absent in healthy retinal vasculature, was observed even after three months, indicating sustained inflammation long after the radiation exposure. Fluorescein angiography demonstrated that the blood-retina barrier is compromised early after irradiation. In vivo imaging provides a powerful means to study dynamic cellular processes over a broad range of timescales from seconds to months that have previously not been accessible by ex vivo analysis.

Overview of Tissue Engineering Concepts and Applications

Tissue engineering strategies date back to the seventies and eighties for developing skin substitutes. Despite early approaches for replacement, repair, and regeneration of failing organs, the true emergence of tissue engineering as a medical field started in the early nineties when tissue engineering was defined as an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue function. Multiple challenges remain for translation of tissue-engineered products to the clinic. Cell type, source, and manipulation are critical parameters that need to be further studied and defined, in order to achieve the best clinical outcomes. Many approaches are too complex for scale-up to industrial level manufacture.