Stelios T. Andreadis

Fibrin promotes migration in a three-dimensional in vitro model of wound regeneration

We developed an in vitro model of wound reepithelialization based on engineered composite skin equivalents of human keratinocytes. Such organotypic cultures are unique in that regulatory mechanisms of cell growth and differentiation can be investigated under conditions mimicking those in vivo. We employed this model system to evaluate fibrin as a substrate for keratinocyte growth and migration after incisional wounding. Our results show that fibrin decreases the length of the lag phase of keratinocyte activation and increases the consistency of the healing response. In addition, the response of these skin equivalents to wounding mimicks that of animal models in terms of the kinetics of reepithelialization, the spatiotemporal distribution of proliferating cells in and around the wound, the unique phenotype exhibited by the cells in the newly formed epidermis, the upregulation of key molecular anchors that initiate cell migration, and the formation of basement membrane during wound closure. Our results suggest that this model can be used to study molecular mechanisms of reepithelialization and evaluate biomaterials as vehicles for controlled delivery of genes and proteins to promote wound healing.

Biomimetic Delivery of Keratinocyte Growth Factor upon Cellular Demand for Accelerated Wound Healing in Vitro and in Vivo

Exogenous keratinocyte growth factor (KGF) significantly enhances wound healing, but its use is hampered by a short biological half-life and lack of tissue selectivity. We used a biomimetic approach to achieve cell-controlled delivery of KGF by covalently attaching a fluorescent matrix-binding peptide that contained two domains: one recognized by factor XIII and the other by plasmin. Modified KGF was incorporated into the fibrin matrix at high concentration in a factor XIII-dependent manner. Cell-mediated activation of plasminogen to plasmin degraded the fibrin matrix and cleaved the peptides, releasing active KGF to the local microenvironment and enhancing epithelial cell proliferation and migration. To demonstrate in vivo effectiveness, we used a hybrid model of wound healing that involved transplanting human bioengineered skin onto athymic mice. At 6 weeks after grafting, the transplanted tissues underwent full thickness wounding and treatment with fibrin gels containing bound KGF. In contrast to topical KGF, fibrin-bound KGF persisted in the wounds for several days and was released gradually, resulting in significantly enhanced wound closure. A fibrinolytic inhibitor prevented this healing, indicating the requirement for cell-mediated fibrin degradation to release KGF. In conclusion, this biomimetic approach of localized, cell-controlled delivery of growth factors may accelerate healing of large full-thickness wounds and chronic wounds that are notoriously difficult to heal.

Cell-controlled and spatially arrayed gene delivery from fibrin hydrogels.

We investigated fibrin-mediated gene transfer by embedding pDNA within the hydrogel during polymerization and using two modes of gene transfection with cells placed either on the surface (2D transfection) or within the hydrogel (3D transfection). Using this model, we found that cell transfection depended strongly on the local cell-pDNA microenvironment as defined by the 2D vs. 3D context, target cell type and density, as well as fibrinogen and pDNA concentrations. When cells were embedded within the fibrin matrix lipofectamine-induced cell death decreased significantly, especially at low target cell density. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner, suggesting that fibrin degradation may be necessary for efficient gene transfer. We also provided proof-of-concept that fibrin-mediated gene transfer can be used for spatially localized gene delivery, which is required in cell-transfection microarrays. When lipoplex-containing hydrogels were spotted in an array format gene transfer was strictly confined to pDNA-containing fibrin spots with no cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may be used as a biomaterial to deliver genes in an efficient, cell-controlled and spatially localized manner for potential applications in vitro or in vivo.

JNK phosphorylates β-catenin and regulates adherens junctions

The c‐Jun amino‐terminal kinase (JNK) is an important player in inflammation, proliferation, and apoptosis. More recently, JNK was found to regulate cell migration by phosphorylating paxillin. Here, we report a novel role of JNK in cell adhesion. Specifically, we provide evidence that JNK binds to E‐cadherin/β‐catenin complex and phosphorylates β‐catenin at serine 37 and threonine 41, the sites also phosphorylated by GSK‐3β. Inhibition of JNK kinase activity using dominant‐negative constructs reduces phosphorylation of β‐catenin and promotes localization of E‐cadherin/β‐catenin complex to cell‐cell contact sites. Conversely, activation of JNK induces β‐catenin phosphorylation and disruption of cell contacts, which are prevented by JNK siRNA. We propose that JNK binds to β‐catenin and regulates formation of adherens junctions, ultimately controlling cell‐to‐cell adhesion.—Lee, M.‐H., Koria, P., Qu, J., Andreadis, S. T. JNK phosphorylates β‐catenin and regulates adherens junctions. FASEBJ. 23, 3874‐3883 (2009). www.fasebj.org

Contractile smooth muscle cells derived from hair-follicle stem cells

AIMS We hypothesized that hair-follicle stem cells can differentiate toward smooth contractile muscle cells, providing an autologous cell source for cardiovascular tissue regeneration. METHODS AND RESULTS Smooth muscle progenitor cells (SMPCs) were obtained from ovine hair follicles using a tissue-specific promoter and fluorescence-activated cell sorting. Hair-follicle smooth muscle progenitor cells (HF-SMPCs) expressed several markers of vascular smooth muscle including alpha-actin, calponin, myosin heavy chain (MHC), caldesmon, smoothelin, and SM22. HF-SMPCs were highly proliferative and showed high clonogenic potential without any signs of chromosomal abnormalities as evidenced by karyotype analysis. HF-SMPCs compacted fibrin hydrogels to a similar extent as vascular smooth muscle cells from ovine umbilical veins (V-SMCs), indicating the development of the force-generating machinery. In addition, cylindrical tissue equivalents prepared with HF-SMPCs displayed significant contractility in response to vasoactive agonists including KCl and the thromboxane A2 mimetic U46619, suggesting that these cells had developed receptor and non-receptor-mediated pathways of contractility. Finally, transforming growth factor-beta1 promoted differentiation of HF-SMPCs toward a mature SMC phenotype as suggested by increased expression of MHC and enhanced matrix compaction. CONCLUSION Our results suggest that hair follicles may be an easily accessible, autologous, and rich source of functional SMPC for cardiovascular tissue engineering and regenerative medicine.

BMP2 Genetically Engineered MSCs and EPCs Promote Vascularized Bone Regeneration in Rat Critical-Sized Calvarial Bone Defects

Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.

High Efficiencies of Gene Transfer with Immobilized Recombinant Retrovirus: Kinetics and Optimization

We used a combination of mathematical modeling and experiments to investigate the rate‐limiting steps of retroviral transduction on surface‐bound fibronectin (FN) and identify the conditions that maximize the efficiency of gene transfer. Our results show that fibronectin‐assisted gene transfer (FAGT) is a strong function of the time and temperature of virus incubation in FN‐coated plates. Gene transfer increases sharply at short times, reaches a maximum at intermediate times, and eventually declines as a result of loss of retroviral activity. The maximum transduction efficiency and the time at which this is attained increase with decreasing temperature of virus incubation. Depending on the temperature and the type of target cells, the initial rate of gene transfer increases by 3‐ to 10‐fold and the maximum transduction efficiency increases by 2‐ to 4‐fold as compared to traditional transduction (TT). Interestingly, Polybrene (PB) inhibits FAGT in a dose‐dependent manner by inhibiting binding of retrovirus to FN. In contrast to traditional transduction, FAGT yields higher than 10‐fold transduction efficiencies with concentrated retrovirus stocks. Gene transfer is directly proportional to the concentration of the virus‐containing medium with no sign of saturation for the range of concentrations tested. These results suggest that immobilization of recombinant retrovirus can be rationally optimized to yield high efficiency of gene transfer to primary cells and improve the prospect of gene therapy for the treatment of human disease.

Nanog Reverses the Effects of Organismal Aging on Mesenchymal Stem Cell Proliferation and Myogenic Differentiation Potential

Although the therapeutic potential of mesenchymal stem cells (MSCs) is widely accepted, loss of cell function due to donor aging or culture senescence are major limiting factors hampering their clinical application. Our laboratory recently showed that MSCs originating from older donors suffer from limited proliferative capacity and significantly reduced myogenic differentiation potential. This is a major concern, as the patients most likely to suffer from cardiovascular disease are elderly. Here we tested the hypothesis that a single pluripotency‐associated transcription factor, namely Nanog, may reverse the proliferation and differentiation potential of bone marrow‐derived MSC (BM‐MSC) from adult donors. Microarray analysis showed that adult (a)BM‐MSC expressing Nanog clustered close to Nanog‐expressing neonatal cells. Nanog markedly upregulated genes involved in cell cycle, DNA replication, and DNA damage repair and enhanced the proliferation rate and clonogenic capacity of aBM‐MSC. Notably, Nanog reversed the myogenic differentiation potential and restored the contractile function of aBM‐MSC to a similar level as that of neonatal (n)BM‐MSC. The effect of Nanog on contractility was mediated—at least in part—through activation of the TGF‐β pathway by diffusible factors secreted in the conditioned medium of Nanog‐expressing BM‐MSC. Overall, our results suggest that Nanog may be used to overcome the effects of organismal aging on aBM‐MSC, thereby increasing the potential of MSC from aged donors for cellular therapy and tissue regeneration. STEM CELLS 2012;30:2746–2759

Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

AIMS Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. METHODS AND RESULTS We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. CONCLUSION We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology.

Clonal multipotency and effect of long-term in vitro expansion on differentiation potential of human hair follicle derived mesenchymal stem cells.

Hair follicle harbors a rich stem cell pool with mesenchymal lineage differentiation potential. Although previous studies with rodent cells demonstrated that hair follicle sheath and papilla cells possess multi-lineage differentiation potential, human hair follicle derived mesenchymal stem cells (hHF-MSCs) have not been characterized in detail in terms of their multipotency. In addition, it is not clear whether these cells are true stem cells that can differentiate along multiple lineages or whether they represent a collection of progenitor cells with restricted differentiation potential. Here we report that hHF-MSCs are highly proliferative cells that can be maintained in culture for ~45 population doublings before they start to show signs of cellular senescence. Under appropriate culture conditions, hHF-MSCs differentiated along the myogenic, osteogenic, adipogenic and chondrogenic lineages, as demonstrated by kinetic gene expression profiling and functional assays. Interestingly, the differentiation potential decreased with time in culture in a lineage-specific manner. Specifically, myogenesis and chondrogenesis showed a moderate decrease over time; osteogenesis was maximum at intermediate passages and adipogenesis was highly sensitive to long-term culture and was diminished at late passages. Finally, hHF-MSCs were clonally multipotent as the majority of hHF-MSCs clones (73%) demonstrated bi- or tri-lineage differentiation potential. These results suggest that hHF-MSCs may present as an alternative source of easily accessible, autologous stem cells for tissue engineering and regenerative medicine.