Thomas P. Lozito

Human mesenchymal stem cells express vascular cell phenotypes upon interaction with endothelial cell matrix

Mesenchymal stem cells (MSCs) are thought to occupy a perivascular niche where they are exposed to signals originating from vascular cells. This study focused on the effects of endothelial cell (EC)‐derived signals on MSC differentiation toward vascular cell lineages. Upon co‐culture with two types of ECs, macrovascular (macro) ECs and microvascular (micro) ECs, the former caused MSCs to increase expression of both EC and smooth muscle cell (SMC) markers, while the latter induced expression of EC markers only. These marker changes in MSCs were linked to the extracellular matrixes secreted by the ECs (EC‐matrix) rather than soluble EC‐secreted factors. Beyond enhanced marker expression, EC‐matrix also induced functional changes in MSCs indicative of development of a genuine vascular cell phenotype. These included enhanced incorporation into vessels and cytoskeletal localization of vascular SMC‐specific contractile elements. The bioactivity of EC‐matrix was sensitive to EDTA washes and required sulfated glycosaminoglycans. However, neither soluble VEGF nor substrate surfaces coated with fibronectin, collagen type IV, or laminin recreated the effects of EC‐matrix on MSC vascular differentiation. In conclusion, these results identified EC‐matrix as a critical regulator of vascular cell differentiation of MSCs. Elucidating these MSC–EC‐matrix interactions and identifying the specific EC‐matrix components involved will shed light on the perivascular signals seen by MSCs in vivo. J. Cell. Biochem. 107: 714–722, 2009. Published 2009 Wiley‐Liss, Inc.

Mesenchymal stem cells inhibit both endogenous and exogenous MMPs via secreted TIMPs.

Mesenchymal stem cells (MSCs) have been shown to be perivascular, occupying a prime location for regulating vessel stability. Here, we focused on the MSC‐contribution of key regulators of the perivascular niche, the matrix metalloproteinases (MMPs) and their inhibitors, the TIMPs. Despite secretion of active forms of MMPs by MSCs, MMP enzyme activity was not detected in MSC‐conditioned medium (MSC‐CM) due to TIMP‐mediated inhibition. By means of bifunctional‐crosslinking to probe endogenous MMP:TIMP interactions, we showed MMP‐2‐inhibition by TIMP‐2. MSCs also inhibited high levels of exogenous MMP‐2 and MMP‐9 through TIMP‐2 and TIMP‐1, respectively. Furthermore, MSC‐CM protected vascular matrix molecules and endothelial cell structures from MMP‐induced disruption. MSCs remained matrix‐protective when exposed to pro‐inflammatory cytokines and hypoxia, countering these stresses with increased TIMP‐1 expression and augmented MMP‐inhibition. Thus, MSCs are revealed as robust sources of TIMP‐mediated MMP‐inhibition, capable of protecting the perivascular niche from high levels of MMPs even under pathological conditions. J. Cell. Physiol. 226: 385–396, 2011.

Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

Wdpcp, a protein required for both planar cell polarity and ciliogenesis, regulates cell polarity and alignment via direct modulation of the actin cytoskeleton.

Mesenchymal stem cell modification of endothelial matrix regulates their vascular differentiation.

Mesenchymal stem cells (MSCs) respond to a variety of differentiation signal provided by their local environments. A large portion of these signals originate from the extracellular matrix (ECM). At the same time, MSCs secrete various matrix‐altering agents, including proteases, that alter ECM‐encoded differentiation signals. Here we investigated the interactions between MSC and ECM produced by endothelial cells (EC‐matrix), focusing not only on the differentiation signals provided by EC‐matrix, but also on MSC‐alteration of these signals and the resultant affects on MSC differentiation. MSCs were cultured on EC‐matrix modified in one of three distinct ways. First, MSCs cultured on native EC‐matrix underwent endothelial cell (EC) differentiation early during the culture period and smooth muscle cell (SMC) differentiation at later time points. Second, MSCs cultured on crosslinked EC‐matrix, which is resistant to MSC modification, differentiated towards an EC lineage only. Third, MSCs cultured on EC‐matrix pre‐modified by MSCs underwent SMC‐differentiation only. These MSC‐induced matrix alterations were found to deplete the factors responsible for EC‐differentiation, yet activate the SMC‐differentiation factors. In conclusion, our results demonstrate that the EC‐matrix contains factors that support MSC differentiation into both ECs and SMCs, and that these factors are modified by MSC‐secreted agents. By analyzing the framework by which EC‐matrix regulates differentiation in MSCs, we have uncovered evidence of a feedback system in which MSCs are able to alter the very matrix signals acting upon them. J. Cell. Biochem. 107: 706–713, 2009. Published 2009 Wiley‐Liss, Inc.

Human Bone Marrow-Derived Mesenchymal Stem Cells Display Enhanced Clonogenicity but Impaired Differentiation With Hypoxic Preconditioning

Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O2 consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis‐associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three‐dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post‐normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high‐quality differentiated tissue.

Human mesenchymal stem cells generate a distinct pericellular zone of MMP activities via binding of MMPs and secretion of high levels of TIMPs.

Mesenchymal stem cells (MSCs) are attractive candidates for inclusion in cell-based therapies by virtue of their abilities to home to wound sites. However, in-depth characterization of the specific effects of MSCs on their microenvironments is needed to realize their full therapeutic potentials. Furthermore, since MSCs of varying properties can be isolated from a diverse spectrum of tissues, a strategic and rational approach in MSC sourcing for a particular application has yet to be achieved. For example, MSCs that activate their proteolytic environments may promote tissue remodeling, while those from different tissue sources may inhibit proteases and promote tissue stabilization. This study attempts to address these issues by analyzing MSCs isolated from three adult tissue sources in terms of their effects on their proteolytic microenvironments. Human bone marrow, adipose, and traumatized muscle derived MSCs were compared in their soluble and cellular-associated MMP components and activity. For all types of MSCs, MMP activity associated with the cell surface, but activity levels and MMP profiles differed with tissue source. All MSC types bound exogenous active MMPs at their surfaces. MSCs were also able to activate exogenous proMMP-2 and proMMP-13. This is in marked contrast to the MSC soluble compartment, which strongly inhibited MMPs via endogenous TIMPs. The exact TIMP used to inhibit the exogenous MMP differed with MSC type. Thus, MSCs saturate their environment with both MMPs and TIMPs. Since they bind and activate MMPs at their surfaces, the net result is a very controlled pericellular localization of MMP activities by MSCs.

Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue

Mesenchymal stem cell (MSC) therapy is a promising approach to promote tissue regeneration by either differentiating the MSCs into the desired cell type or by using their trophic functions to promote endogenous tissue repair. These strategies of regenerative medicine are limited by the availability of MSCs at the point of clinical care. Our laboratory has recently identified multipotent mesenchymal progenitor cells (MPCs) in traumatically injured muscle tissue, and the objective of this study was to compare these cells to a typical population of bone marrow derived MSCs. Our hypothesis was that the MPCs exhibit multilineage differentiation and expression of trophic properties that make functionally them equivalent to bone marrow derived MSCs for tissue regeneration therapies. Quantitative evaluation of their proliferation, metabolic activity, expression of characteristic cell‐surface markers and baseline gene expression profile demonstrate substantial similarity between the two cell types. The MPCs were capable of differentiation into osteoblasts, adipocytes and chondrocytes, but they appeared to demonstrate limited lineage commitment compared to the bone marrow derived MSCs. The MPCs also exhibited trophic (i.e. immunoregulatory and pro‐angiogenic) properties that were comparable to those of MSCs. These results suggest that the traumatized muscle derived MPCs may not be a direct substitute for bone marrow derived MSCs. However, because of their availability and abundance, particularly following orthopaedic injuries when traumatized muscle is available to harvest autologous cells, MPCs are a promising cell source for regenerative medicine therapies designed to take advantage of their trophic properties.

Endothelial cell microparticles act as centers of matrix metalloproteinsase-2 (MMP-2) activation and vascular matrix remodeling

Endothelial cell (EC)‐derived microparticles (MPs) are small membrane vesicles associated with various vascular pathologies. Here, we investigated the role of MPs in matrix remodeling by analyzing their interactions with the extracellular matrix. MPs were shown to bind preferentially to surfaces coated with matrix molecules, and MPs bound fibronectin via integrin αV. MPs isolated from EC‐conditioned medium (Sup) were significantly enriched for matrix‐altering proteases, including matrix metalloproteinases (MMPs). MPs lacked the MMP inhibitors TIMP‐1 and TIMP‐2 found in the Sup and, while Sup strongly inhibited MMP activities but MPs did not. In fact, MPs were shown to bind and activate both endogenous and exogenous proMMP‐2. Taken together, these results indicate that MPs interact with extracellular matrices, where they localize and activate MMP‐2 to modify the surrounding matrix molecules. These findings provide insights into the cellular mechanisms of vascular matrix remodeling and identify new targets of vascular pathologies. J. Cell. Physiol. 227: 534–549, 2012.

Investigating the Neural Basis of the Auditory Continuity Illusion

In this study, we investigated one type of auditory perceptual grouping phenomenathe auditory continuity illusion (also called temporal induction). We employed a previously developed, neurobiologically realistic, large-scale neural network model of the auditory processing pathway in the cortex, ranging from the primary auditory cortex to the prefrontal cortex, and simulated temporal induction without changing any model parameters. The model processes tonal contour stimuli, composed of combinations of upward and downward FM sweeps and tones, in a delayed match-to-sample task. The local electrical activities of the neuronal units of the model simulated accurately the experimentally observed electrophysiological data, where available, and the models simulated BOLD-fMRI data were quantitatively matched with experimental fMRI data. In the present simulations, intact stimuli were matched with fragmented versions (i.e., with inserted silent gaps). The ability of the model to match fragmented stimuli declined as the duration of the gaps increased. However, when simulated broadband noise was inserted into these gaps, the matching response was restored, indicating that a continuous stimulus was perceived. The electrical activities of the neuronal units of the model agreed with electrophysiological data, and the behavioral activity of the model matched human behavioral data. In the model, the predominant mechanism implementing temporal induction is the divergence of the feedforward connections along the auditory processing pathway in the temporal cortex. These simulation results not only attest to the robustness of the model, but further predict the primary role of the anatomical connectivity of the auditory processing areas in mediating the continuity illusion.

Stem cell-based microphysiological osteochondral system to model tissue response to interleukin-1β.

Osteoarthritis (OA) is a chronic degenerative disease of the articular joint that involves both bone and cartilage degenerative changes. An engineered osteochondral tissue within physiological conditions will be of significant utility in understanding the pathogenesis of OA and testing the efficacy of potential disease-modifying OA drugs (DMOADs). In this study, a multichamber bioreactor was fabricated and fitted into a microfluidic base. When the osteochondral construct is inserted, two chambers are formed on either side of the construct (top, chondral; bottom, osseous) that is supplied by different medium streams. These medium conduits are critical to create tissue-specific microenvironments in which chondral and osseous tissues will develop and mature. Human bone marrow stem cell (hBMSCs)-derived constructs were fabricated in situ and cultured within the bioreactor and induced to undergo spatially defined chondrogenic and osteogenic differentiation for 4 weeks in tissue-specific media. We observed tissue specific gene expression and matrix production as well as a basophilic interface suggesting a developing tidemark. Introduction of interleukin-1β (IL-1β) to either the chondral or osseous medium stream induced stronger degradative responses locally as well as in the opposing tissue type. For example, IL-1β treatment of the osseous compartment resulted in a strong catabolic response in the chondral layer as indicated by increased matrix metalloproteinase (MMP) expression and activity, and tissue-specific gene expression. This induction was greater than that seen with IL-1β application to the chondral component directly, indicative of active biochemical communication between the two tissue layers and supporting the osteochondral nature of OA. The microtissue culture system developed here offers novel capabilities for investigating the physiology of osteochondral tissue and pathogenic mechanisms of OA and serving as a high-throughput platform to test potential DMOADS.