Regulation of osteogenic and chondrogenic differentiation of mesenchymal stem cells in PEG-ECM hydrogels

Bone-marrow-derived mesenchymal stem cells (MSCs) are candidates for regeneration applications in musculoskeletal tissue such as cartilage and bone. Various soluble factors in the form of growth factors and cytokines have been widely studied for directing the chondrogenic and osteogenic differentiation of MSCs, but little is known about the way that the composition of extracellular matrix (ECM) components in three-dimensional microenvironments plays a role in regulating the differentiation of MSCs. To define whether ECM components influence the regulation of osteogenic and chondrogenic differentiation by MSCs, we encapsulated MSCs in poly-(ethylene glycol)-based (PEG-based) hydrogels containing exogenous type I collagen, type II collagen, or hyaluronic acids (HA) and cultured them for up to 6 weeks in chondrogenic medium containing transforming growth factor-β1 (10 ng/ml) or osteogenic medium. Actin cytoskeleton organization and cellular morphology were strongly dependent on which ECM components were added to the PEG-based hydrogels. Additionally, chondrogenic differentiation of MSCs was marginally enhanced in collagen-matrix-based hydrogels, whereas osteogenic differentiation, as measured by calcium accumulation, was induced in HA-containing hydrogels. Thus, the microenvironments created by exogenous ECM components seem to modulate the fate of MSC differentiation.     

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. © 2010 Wiley‐Liss, Inc.     

Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors

Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell‐derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non‐MP secreted factors (Sup) were isolated from serum‐free medium conditioned by human microvascular ECs (HMEC‐1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP‐containing MPs were isolated from cells transduced with CMV‐GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP‐MPs, but not free GFP. Thus, only MP‐associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP‐1, MMP‐3, CCL‐2/MCP‐1 and IL‐6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF‐κB signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms.     

Recreating the perivascular niche ex vivo using a microfluidic approach

Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow‐derived MSCs) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well‐defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their α6β1 integrin receptor and EC‐deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency. Biotechnol. Bioeng. 2010;107: 1020–1028. © 2010 Wiley Periodicals, Inc.     

Extracellular matrix provides an optimal niche for the maintenance and propagation of mesenchymal stem cells

Relatively little is known about the cellular and molecular mechanisms underlying the control of mesenchymal stem cell (MSC) proliferation, differentiation, and survival. This presents difficulties in following and characterizing cells along the lineage because of our inability to isolate and obtain a sufficient number of homogeneous MSCs using current culture systems for in vitro expansion. Adjusting the cellular machinery to allow greater proliferation can lead to other unwanted outcomes, such as unmanageable precancerous changes, or differentiation down an undesired pathway. Recently, it has become increasingly evident that the extracellular matrix (ECM) is an important component of the cellular niche in a tissue, supplying critical biochemical and physical signals to initiate and sustain cellular functions. Indeed, it is very doubtful that the intricate and highly ordered nature of the ECM could be reproduced with synthetic or purified components. This review cites evidence that supports an alternative approach for maintenance of MSCs by simulating in vitro the bone marrow ECM, where MSCs reside in vivo, and discusses the potential mechanisms whereby the ECM regulates the exposure of cells to growth factors that subsequently control MSC replication and differentiation, and also how the ECM provides unique cues that govern the lineage specification and differentiation of MSCs. Birth Defects Research (Part C) 90:45–54, 2010. © 2010 Wiley‐Liss, Inc.     

Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration

Model systems mimicking the extracellular matrix (ECM) have greatly helped in quantifying cell migration in three dimensions and elucidated the molecular determinants of cellular motility in morphogenesis, regeneration, and disease progression. Here we tested the suitability of proteolytically degradable synthetic poly(ethylene glycol) (PEG)-based hydrogels as an ECM model system for cell migration research and compared this designer matrix with the two well-established ECM mimetics fibrin and collagen. Three-dimensional migration of dermal fibroblasts was quantified by time-lapse microscopy and automated single-cell tracking. A broadband matrix metalloproteinase (MMP) inhibitor and tumor necrosis factor-alpha, a potent MMP-inducer in fibroblasts, were used to alter MMP regulation. We demonstrate a high sensitivity of migration in synthetic networks to both MMP modulators: inhibition led to an almost complete suppression of migration in PEG hydrogels, whereas MMP upregulation increased the fraction of migrating cells significantly. Conversely, migration in collagen and fibrin proved to be less sensitive to the above MMP modulators, as their fibrillar architecture allowed for MMP-independent migration through preexisting pores. The possibility of molecularly recapitulating key functions of the natural extracellular microenvironment and the improved protease sensitivity makes PEG hydrogels an interesting model system that allows correlation between protease activity and cell migration.     

Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling

The effects of mesenchymal stem cells (MSCs) on proliferation and cell fate determination of neural stem cells (NSCs) have been investigated. NSCs were co-cultured with MSCs or NIH3T3 cells using an in vitro transwell system. After 4 days, immunofluorescence staining showed that the number of cells positive for the cell proliferation antigen, ki-67, in neurospheres in MSCs was greater than in NIH3T3 cells. In some experiments, the top-layers of MSCs and NIH3T3 cells were removed to induce NSCs differentiation. Seven days after initiating differentiation, the levels of the neuronal marker, NSE, were higher in NSCs in MSCs co-culture group, and those of glial fibrillary acidic protein (GFAP) were lower, compared with NIH3T3 cells co-culture group. These were confirmed by immunofluorescence. The role of the Notch signaling pathway analyzed with the specific inhibitor, DAPT, and by examining the expression of Notch-related genes using RT-PCR showed that after co-culturing with MSCs for 24 h, NSCs expressed much higher levels of ki-67, Notch1, and Hes1 than did NSCs co-cultured with NIH3T3 cells. Treatment with DAPT decreased ki-67, Notch1 and Hes1 expression in NCSs, and increased Mash1 expression. The data indicate that the interactions between MSCs and NSCs promote NSCs proliferation and are involved in specifying neuronal fate, mediated in part by Notch signaling.     

Hydrogel‐based three‐dimensional cell culture for organ‐on‐a‐chip applications

Recent studies have reported that three‐dimensionally cultured cells have more physiologically relevant functions than two‐dimensionally cultured cells. Cells are three‐dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ‐on‐a‐chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation‐based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ‐on‐a‐chip. Last, we also discuss the challenges of hydrogel‐based three‐dimensional cell culture techniques and propose future directions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:580–589, 2017     

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.     

Regulation of stemness and stem cell niche of mesenchymal stem cells: Implications in tumorigenesis and metastasis

Human mesenchymal stem cells (MSCs) derived from adult tissues have been considered a candidate cell type for cell‐based tissue engineering and regenerative medicine. These multipotent cells have the ability to differentiate along several mesenchymal lineages and possibly along non‐mesenchymal lineages. MSCs possess considerable immunosuppressive properties that can influence the surrounding tissue positively during regeneration, but perhaps negatively towards the pathogenesis of cancer and metastasis. The balance between the naïve stem state and differentiation is highly dependent on the stem cell niche. Identification of stem cell niche components has helped to elucidate the mechanisms of stem cell maintenance and differentiation. Ultimately, the fate of stem cells is dictated by their microenvironment. In this review, we describe the identification and characterization of bone marrow‐derived MSCs, the properties of the bone marrow stem cell niche, and the possibility and likelihood of MSC involvement in cancer progression and metastasis. J. Cell. Physiol. 222: 268–277, 2010. © 2009 Wiley‐Liss, Inc.     

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.     

Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment

Enhancing differentiation of mesenchymal stem cells (MSCs) to endothelial cells may improve their ability to vascularize tissue and promote wound healing. This study describes a novel role for nitric oxide (NO) in reprogramming MSCs towards an endothelial lineage and highlights the role of Wnt signaling and epigenetic modification by NO. Rat MSCs were transduced with lentiviral vectors expressing endothelial nitric oxide synthase (pLV-eNOS) and a mutated caveolin gene (pLV-CAV-1^F92A) to enhance NO generation resulting in increased in vitro capillary tubule formation and endothelial marker gene expression. An exogenous source of NO could also stimulate CD31 expression in MSCs. NO was associated with an arterial-specific endothelial gene expression profile of Notch1, Dll4, and Hey2 and significantly reduced expression of venous markers. Wnt signaling associated with NO was evident through increased gene expression of Wnt3a and β-catenin protein, and expression of the endothelial marker Pecam-1 could be significantly reduced by treatment with the Wnt signaling inhibitor Dkk-1. The role of NO as an epigenetic modifier was evident with reduced gene expression of the methyltransferase, DNMT1, and bisulfite sequencing of the endothelial Flt1 promoter region in NO-producing MSCs showed significant demethylation compared to control cells. Finally, subcutaneous implantation of NO-producing MSCs seeded in a biomaterial scaffold (NovoSorb®) resulted in survival of transplanted cells and the formation of blood vessels. In summary, this study describes, NO as a potent endothelial programming factor which acts as an epigenetic modifier in MSCs and may provide a novel platform for vascular regenerative therapy.     

Impaired Endothelium-Mesenchymal Stem Cells Cross-talk in Systemic Sclerosis: a Link Between Vascular and Fibrotic Features

Introduction

To assess if an impaired cross-talk between endothelial cells (ECs) and perivascular/multipotent mesenchymal stem cells (MSCs) might induce a perturbation of vascular repair and leading to a phenotypic switch of MSC toward myofibroblast in Systemic Sclerosis (SSc).

Methods

We investigated different angiogenic and profibrotic molecules in a tridimentional matrigel assay, performing co-cultures with endothelial cells (ECs) and bone marrow derived MSCs from patients and healthy controls (HC). After 48 hours of co-culture, cells were sorted and analyzed for mRNA and protein expression.

Results

ECs-SSc showed a decreased tube formation ability which is not improved by co-cultures with different MSCs. After sorting, we showed: i. an increased production of vascular endothelial growth factor A (VEGF-A) in SSc-MSCs when co-cultured with SSc-ECs; ii. an increased level of transforming growth factor beta (TGF-β) and platelet growth factor BB (PDGF-BB) in SSc-ECs when co-cultured with both HC- and SSc-MSCs; iii. an increase of TGF-β, PDGF-R, alpha smooth muscle actin (α-SMA) and collagen 1 (Col1) in both HC- and SSc-MSCs when co-cultured with SSc-ECs.

Conclusion

We showed that during SSc, the ECs-MSCs crosstalk resulted in an altered expression of different molecules involved in the angiogenic processes, and mainly SSc-ECs seem to modulate the phenotypic switch of perivascular MSCs toward a myofibroblast population, thus supporting the fibrotic process.     

Fabrication of viable and functional pre‐vascularized modular bone tissues by coculturing MSCs and HUVECs on microcarriers in spinner flasks

Slow vascularization often impedes the viability and function of engineered bone replacements. Prevascularization is a promising way to solve this problem. In this study, a new process was developed by integrating microcarrier culture and coculture to fabricate pre‐vascularized bone microtissues with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs). Initially, coculture medium and cell ratio between MSCs and HUVECs were optimized in tissue culture plates concerning cell proliferation, osteogenesis and angiogenesis. Subsequently, cells were seeded onto CultiSpher S microcarriers in spinner flasks and subjected to a two‐stage (proliferative‐osteogenic) culture process for four weeks. Both cells proliferated and functioned well in chosen medium and a 1 : 1 ratio between MSCs and HUVECs was chosen for better angiogenesis. After four weeks of culture in spinner flasks, the microtissues were formed with high cellularity, evenly distributed cells and tube formation ability. While coculture with HUVECs exerted an inhibitory effect on osteogenic differentiation of MSCs, with downregulated alkaline phosphatase activity, mineralization and gene expression of COLI, RUNX2 and OCN, this could be attenuated by employing a delayed seeding strategy of HUVECs against MSCs during the microtissue fabrication process. Conclusion: Collectively, this work established an effective method to fabricate pre‐vascularized bone microtissues, which would lay a solid foundation for subsequent development of vascularized tissue grafts for bone regeneration.     

Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) have strong potential in regeneration of musculoskeletal tissues including cartilage and bone. The microenvironment, comprising of scaffold and soluble factors, plays a pivotal role in determining the efficacy of cartilage tissue regeneration from MSCs. In this study, we investigated the effect of a three-dimensional synthetic-biological composite hydrogel scaffold comprised of poly (ethylene glycol) (PEG) and chondroitin sulfate (CS) on chondrogenesis of MSCs. The cells in CS-based bioactive hydrogels aggregated in a fashion which mimicked the mesenchymal condensation and produced cartilaginous tissues with characteristic morphology and basophilic extracellular matrix production. The aggregation of cells resulted in an enhancement of both chondrogenic gene expressions and cartilage specific matrix production compared to control PEG hydrogels containing no CS-moieties. Moreover, a significant down-regulation of type X collagen expression was observed in PEG/CS hydrogels, indicating that CS inhibits the further differentiation of MSCs into hypertrophic chondrocytes. Overall, this study demonstrates the morphogenetic role of bioactive scaffold-mediated microenvironment on temporal pattern of cartilage specific gene expressions and subsequent matrix production during MSC chondrogenesis.     

Biomimetic poly(ethylene glycol)-based hydrogels as scaffolds for inducing endothelial adhesion and capillary-like network formation

The extracellular matrix (ECM) is an attractive model for designing synthetic scaffolds with a desirable environment for tissue engineering. Here, we report on the synthesis of ECM-mimetic poly(ethylene glycol) (PEG) hydrogels for inducing endothelial cell (EC) adhesion and capillary-like network formation. A collagen type I-derived peptide GPQGIAGQ (GIA)-containing PEGDA (GIA-PEGDA) was synthesized with the collagenase-sensitive GIA sequence attached in the middle of the PEGDA chain, which was then copolymerized with RGD capped-PEG monoacrylate (RGD-PEGMA) to form biomimetic hydrogels. The hydrogels degraded in vitro with the rate dependent on the concentration of collagenase and also supported the adhesion of human umbilical vein ECs (HUVECs). Biomimetic RGD/GIA-PEGDA hydrogels with incorporation of 1% RGD-PEGDA into GIA-PEGDA hydrogels induced capillary-like organization when HUVECs were seeded on the hydrogel surface, while RGD/PEGDA and GIA-PEGDA hydrogels did not. These results indicate that both cell adhesion and biodegradability of scaffolds play important roles in the formation of capillary-like networks.     

Aggregation of bovine anterior cruciate ligament fibroblasts or marrow stromal cells promotes aggrecan production

The development of a tissue‐engineered alternative for current ligament grafts requires the creation of a fibrocartilaginous interface between the engineered ligament midsubstance and bone tissue. Therefore, the focus of this study was to examine the potential for cartilaginous extracellular matrix (ECM) formation by altering culture parameters for bovine anterior cruciate ligament (ACL) fibroblasts and marrow stromal cells (MSCs). Specifically, cells were cultured without chondrogenic media supplements on aggrecan‐coated surfaces, tissue culture‐treated control surfaces, and nonadhesive surfaces that promoted cell aggregation, and examined over 14 days. Aggrecan‐coated surfaces promoted the aggregation of ACL fibroblasts and MSCs within 24 h after seeding. Aggrecan gene expression was significantly upregulated in cell aggregates, regardless of how cell clustering was induced, with as much as 10.9 ± 1.2‐fold upregulation in ACL fibroblasts and 9.7 ± 1.1‐fold in MSCs after 3 days, compared to control surfaces. Dimethylmethylene blue (DMMB) results and immunostaining verified the presence of aggrecan in ACL fibroblast and MSC aggregates throughout the culture period. Results indicate that ACL fibroblasts retained the ability to alter their gene expression and produce aggrecan, though MSCs, in general, had a more consistent response to aggregation. These findings support the use of aggregate‐inducing materials to encourage production of aggrecan and suggest that altering the degree of clustering could produce a range of phenotypes from a single cell source. As such, this represents a first step which may inform future approaches to producing tissue‐engineered ligament grafts. Biotechnol. Bioeng. 2011; 108:151–162. © 2010 Wiley Periodicals, Inc.