Pedro Lei

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.

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

Serum‐free spheroid suspension culture maintains mesenchymal stem cell proliferation and differentiation potential

There have been many clinical trials recently using ex vivo‐expanded human mesenchymal stem cells (MSCs) to treat several disease states such as graft‐versus‐host disease, acute myocardial infarction, Crohns disease, and multiple sclerosis. The use of MSCs for therapy is expected to become more prevalent as clinical progress is demonstrated. However, the conventional 2‐dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the MSC‐based indications further exacerbates this manufacturing challenge. In contrast, expanding MSCs as spheroids does not require a cell attachment surface and is amenable to large‐scale suspension cell culture techniques, such as stirred‐tank bioreactors. In the present study, we developed and optimized serum‐free media for culturing MSC spheroids. We used Design of Experiment (DoE)‐based strategies to systematically evaluate media mixtures and a panel of different components for effects on cell proliferation. The optimization yielded two prototype serum‐free media that enabled MSCs to form aggregates and proliferate in both static and dynamic cultures. MSCs from spheroid cultures exhibited the expected immunophenotype (CD73, CD90, and CD105) and demonstrated similar or enhanced differentiation potential toward all three lineages (osteogenic, chondrogenic, adipogenic) as compared with serum‐containing adherent MSC cultures. Our results suggest that serum‐free media for MSC spheroids may pave the way for scale‐up production of MSCs in clinically relevant manufacturing platforms such as stirred tank bioreactors.

Retrovirus-Associated Heparan Sulfate Mediates Immobilization and Gene Transfer on Recombinant Fibronectin

ABSTRACT Recombinant retroviruses have been shown to bind to fibronectin (FN) and increase the efficiency of gene transfer to a variety of cell types. Despite recent work to optimize gene transfer on recombinant FN, the mechanism of retrovirus binding to FN and the interactions of target cells with the bound virus remain elusive. We investigated the roles of virus surface glycoprotein (gp70), cell-conditioned medium, and proteoglycans in mediating retrovirus binding to FN. We also examined the role of Polybrene (PB) in these interactions. We found that gp70 is not involved in retrovirus binding to FN. Immobilization of the virus, however, does not overcome its receptor requirement, and gp70 is still needed for successful gene transfer. Our results clearly show that retrovirus binds FN through virus-associated heparan sulfate (HS) and that binding is necessary for transduction without PB. Two distinct modes of gene transfer occur depending on PB: (i) in the presence of PB, retrovirus interacts directly with the target cells; and (ii) in the absence of PB, retrovirus binds to FN and target cells interact with the immobilized virus. PB may promote the former mode by interacting with the virus HS and reducing the negative charge of the viral particles. Interestingly, the latter mode is more efficient, leading to significantly enhanced gene transfer. A better understanding of these interactions may provide insight into virus-cell interactions and lead to a more rational design of transduction protocols.

Fibrin-mediated lentivirus gene transfer: implications for lentivirus microarrays.

We employed fibrin hydrogel as a bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 and 7.5mg/ml. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner suggesting that fibrin degradation by target cells may be necessary for successful gene delivery. Under these conditions transduction may be limited only to cells interacting with the matrix thereby providing a method for spatially-localized gene delivery. Indeed, when lentivirus-containing fibrin microgels were spotted in an array format gene transfer was confined to virus-containing fibrin spots with minimal cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may provide an effective matrix for spatially-localized gene delivery with potential applications in high-throughput lentiviral microarrays and in regenerative medicine.

JNK is a novel regulator of intercellular adhesion

c-Jun N-terminal Kinase (JNK) is a family of protein kinases, which are activated by stress stimuli such as inflammation, heat stress and osmotic stress, and regulate diverse cellular processes including proliferation, survival and apoptosis. In this review, we focus on a recently discovered function of JNK as a regulator of intercellular adhesion. We summarize the existing knowledge regarding the role of JNK during the formation of cell-cell junctions. The potential mechanisms and implications for processes requiring dynamic formation and dissolution of cell-cell junctions including wound healing, migration, cancer metastasis and stem cell differentiation are also discussed.

Regulated Insulin Delivery From Human Epidermal Cells Reverses Hyperglycemia

Alternative insulin therapies are being sought that will provide euglycemic control for people with diabetes mellitus. The epidermis is a self-renewing tissue that is easily accessible and can provide large numbers of autologous cells that can be used for generating insulin-secreting skin substitutes. Lentiviral vectors have been engineered to produce a fusion protein between the furin-cleavable proinsulin and the self-dimerization mutant of FK506-binding protein to yield bioactive insulin in keratinocytes; this insulin is released as a response to exogenous administration of a small organic molecule, rapamycin. The engineered keratinocytes retained normal morphology and grew in a manner similar to lentiviral-treated control cells. Epidermal keratinocytes in culture and in stratified bioengineered epidermis released insulin within 30 minutes after addition of rapamycin, and secretion slowed or stopped within 2-3 hours after removal of the inducing agent. When the cells were implanted into athymic mice that had been rendered diabetic with streptozotocin (STZ), insulin was detected in the plasma within 1 hour after addition of rapamycin. Concomitantly, serum glucose decreased to normal levels even in diabetic animals with severe hyperglycemia. Repeated rapamycin administration yielded similar results. These experiments provide proof-of-concept that insulin released from the skin in a regulatable manner can reverse hyperglycemia.

PKC-δ binds to E-cadherin and mediates EGF-induced cell scattering

EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinomas but the underlying mechanisms are not completely understood. Using human tumor epithelial cells overexpressing EGFR we demonstrated that EGF-induced cell scattering was mediated by protein kinase C-delta (PKC-delta). PKC-delta knockdown by siRNA significantly inhibited EGF-induced internalization of E-cadherin into the cytoplasm and blocked cell scattering. EGF phosphorylated PKC-delta at Y311 and ectopic expression of the mutant Y311F prevented PKC-delta binding to E-cadherin and EGF-induced cell scattering. Moreover, depletion of Src using siRNA decreased EGF-induced phosphorylation of PKC-delta at Y311 and blocked scattering. Finally, EGF reduced expression of the tight junction protein, occludin, and this effect was also mediated by PKC-delta through Src. In summary, PKC-delta mediated the effects of EGF on adherens and tight junctions thereby playing an important role in cell-cell adhesion with possible wider implications in tumor metastasis or epithelial-to-mesenchymal transition.

JNK regulates compliance-induced adherens junctions formation in epithelial cells and tissues

Summary We demonstrate that c-Jun N-terminal kinase (JNK) responds to substrate stiffness and regulates adherens junction (AJ) formation in epithelial cells in 2D cultures and in 3D tissues in vitro and in vivo. Rigid substrates led to JNK activation and AJ disassembly, whereas soft matrices suppressed JNK activity leading to AJ formation. Expression of constitutively active JNK (MKK7-JNK1) induced AJ dissolution even on soft substrates, whereas JNK knockdown (using shJNK) induced AJ formation even on hard substrates. In human epidermis, basal cells expressed phosphorylated JNK but lacked AJ, whereas suprabasal keratinocytes contained strong AJ but lacked phosphorylated JNK. AJ formation was significantly impaired even in the upper suprabasal layers of bioengineered epidermis when prepared with stiffer scaffold or keratinocytes expressing MKK7-JNK1. By contrast, shJNK1 or shJNK2 epidermis exhibited strong AJ even in the basal layer. The results with bioengineered epidermis were in full agreement with the epidermis of jnk1−/− or jnk2−/− mice. In conclusion, we propose that JNK mediates the effects of substrate stiffness on AJ formation in 2D and 3D contexts in vitro as well as in vivo.