Fibroblast Grafts Research Articles

CD47 prevents the elimination of diseased fibroblasts in scleroderma

Scleroderma is a devastating fibrotic autoimmune disease. Current treatments are partly effective in preventing disease progression but do not remove fibrotic tissue. Here, we evaluated whether scleroderma fibroblasts take advantage of the “don’t-eat-me-signal” CD47 and whether blocking CD47 enables the body’s immune system to get rid of diseased fibroblasts. To test this approach, we used a Jun-inducible scleroderma model. We first demonstrated in patient samples that scleroderma upregulated transcription factor JUN and increased promoter accessibilities of both JUN and CD47. Next, we established our scleroderma model, demonstrating that Jun mediated skin fibrosis through the hedgehog-dependent expansion of CD26+Sca1– fibroblasts in mice. In a niche-independent adaptive transfer model, JUN steered graft survival and conferred increased self-renewal to fibroblasts. In vivo, JUN enhanced the expression of CD47, and inhibiting CD47 eliminated an ectopic fibroblast graft and increased in vitro phagocytosis. In the syngeneic mouse, depleting macrophages ameliorated skin fibrosis. Therapeutically, combined CD47 and IL-6 blockade reversed skin fibrosis in mice and led to the rapid elimination of ectopically transplanted scleroderma cells. Altogether, our study demonstrates the efficiency of combining different immunotherapies in treating scleroderma and provides a rationale for combining CD47 and IL-6 inhibition in clinical trials.

Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration.

The epicardium and its derivatives provide trophic and structural support for the developing and adult heart. Here we tested the ability of human embryonic stem cell (hESC)-derived epicardium to augment the structure and function of engineered heart tissue (EHT) in vitro and to improve efficacy of hESC-cardiomyocyte grafts in infarcted athymic rat hearts. Epicardial cells markedly enhanced the contractility, myofibril structure and calcium handling of human EHTs, while reducing passive stiffness compared to mesenchymal stromal cells. Transplanted epicardial cells formed persistent fibroblast grafts in infarcted hearts. Co-transplantation of hESC-derived epicardial cells and cardiomyocytes doubled graft cardiomyocyte proliferation rates in vivo, resulting in 2.6-fold greater cardiac graft size and simultaneously augmenting graft and host vascularization. Notably, co-transplantation improved systolic function compared with hearts receiving either cardiomyocytes alone, epicardial cells alone or vehicle. The ability of epicardial cells to enhance cardiac graft size and function make them a promising adjuvant therapeutic for cardiac repair.

Autologous Platelet-Rich Plasma (CuteCell PRP) Safely Boosts In Vitro Human Fibroblast Expansion.

Nowadays autologous fibroblast application for skin repair presents an important clinical interest. In most cases, in vitro skin cell culture is mandatory. However, cell expansion using xenogeneic or allogenic culture media presents some disadvantages, such as the risk of infection transmission or slow cell expansion. In this study, we investigated an autologous culture system to expand human skin fibroblast cells in vitro with the patient's own platelet-rich plasma (PRP). Human dermal fibroblasts were isolated from patients undergoing abdominoplasty, and blood was collected to prepare nonactivated PRP using the CuteCell™ PRP medical device. Cultures were followed up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Fibroblasts cultured in medium supplemented with PRP showed dose-dependently significantly higher proliferation rates (up to 7.7 times with 20% of PRP) and initiated a faster migration in the in vitro wound healing assay compared with FBS, while chromosomal stability was maintained. At high concentrations, PRP changed fibroblast morphology, inducing cytoskeleton rearrangement and an increase of alpha-smooth muscle actin and vimentin expression. Our findings show that autologous PRP is an efficient and cost-effective supplement for fibroblast culture, and should be considered as a safe alternative to xenogeneic/allogenic blood derivatives for in vitro cell expansion. Impact Statement Autologous dermal fibroblast graft is an important therapy in skin defect repair, but in vitro skin cell culture is mandatory in most cases. However, cell expansion using xenogeneic/allogenic culture media presents some disadvantages, such as the risk of infection transmission. We demonstrated that an autologous culture system with the patient's own platelet-rich plasma is an efficient, cost-effective, and safe supplement for fibroblast culture. As it respects the good manufacturing practices and regulatory agencies standards, it should be considered as a potent alternative and substitute to xenogeneic or allogenic blood derivatives for the validation of future clinical protocols using in vitro cell expansion.

Amniotic cells share clusters of differentiation of fibroblasts and keratinocytes, influencing their ability to proliferate and aid in wound healing while impairing their angiogenesis capability

An alternative to cultured skin cell grafts usage in burn treatment is the graft of allogenic stem cells. We verified whether amniotic stem cells are better than the present therapeutic standard: grafts of autologous keratinocytes and fibroblasts along with autologous adipose-derived stem cells, and whether amniotic stem cells can support the growth of autologous keratinocytes and fibroblasts in the culture.The study was performed on the material from 18 amnia. Skin cells were obtained from 3 patients. In order to assess the influence of stem cells on keratinocytes and fibroblasts, the following experiments were performed: impact on viability and cell cycle, wound healing capability, angiogenesis capability, influence on the proliferation speed and capability to differentiate into skin cells.We demonstrated that human amniotic membrane-derived mesenchymal stem cells (hAMMSCs) share amniotic proteins with skin cells. Amniotic stem cells may replace skin fibroblasts in grafts due to the close similarity in their surface antigens, with significantly larger proliferative potential and ability to stimulate wound healing. It was shown that adding amniotic cells to both keratinocytes and fibroblast cultures accelerates directional migration by ≥ 40%. We confirmed in this study the influence of amniotic cells on the proliferation and cell cycle of fibroblasts and keratinocytes.Amniotic stem cells can be successfully used not only as a first choice graft but also to replace 3T3 line cells, supporting the proliferation of the cells during the culturing, as well as a supplementary graft supporting an autologous graft of keratinocytes and fibroblasts.

Culture of pyramidal neural precursors, neural stem cells, and fibroblasts on various biomaterials

A combinatory approach using biomaterials together with cells may improve the efficacy of cell therapy for treatment of various diseases/indications. In the current study, we cultured pyramidal neural precursors (PNPs), neural stem cells (NSCs), and fibroblasts on different materials that included fibrin, collagen, hyaluronic acid (HA), sciatic nerves, and matrigel, to search for the most suitable biomaterial for culture of each cell type. Collagen was fabricated in both an aligned collagen-poly (lactic-co-glycolic acid) (PLGA) composite and an alveolate form; fibrin and hyaluronic acid were made in an aligned form only. Pyramidal neurons have strong projection ability and have potentials in neural circuit reconstruction. However, PNPs showed difficulty in attaching to and growing neurites on most of the materials tested, except for matrigel, in which neurite growth was observed in a three dimentional culture. NSCs and derivatives hold promise in treating neurological diseases. On aligned fibrin, NSCs could differentiate and grow neurites in a directional manner before fibrin was degraded in 2 days. On aligned collagen-PLGA, induced neural stem cells (iNSCs) could survive and differentiate for at least 2 weeks, but the neurites failed to extend in an aligned way. Fibroblast graft are useful in many indications, such as in skin burns. Fibroblasts generally grew better on the tested materials than did the neural cells, and fibroblasts could grow directionally on the aligned fibrin and scattered around on the alveolate collagen. The study provided information which may be used to further optimize the materials to support culture of each type of cells.

Rodent Neural Progenitor Cells Support Functional Recovery after Cervical Spinal Cord Contusion.

Previously, we and others have shown that rodent neural progenitor cells (NPCs) can support functional recovery after cervical and thoracic transection injuries. To extend these observations to a more clinically relevant model of spinal cord injury, we performed unilateral midcervical contusion injuries in Fischer 344 rats. Two-weeks later, E14-derived syngeneic spinal cord-derived multi-potent NPCs were implanted into the lesion cavity. Control animals received either no grafts or fibroblast grafts. The NPCs differentiated into all three neural lineages (neurons, astrocytes, oligodendrocytes) and robustly extended axons into the host spinal cord caudal and rostral to the lesion. Graft-derived axons grew into host gray matter and expressed synaptic proteins in juxtaposition with host neurons. Animals that received NPC grafts exhibited significant recovery of forelimb motor function compared with the two control groups (analysis of variance p < 0.05). Thus, NPC grafts improve forelimb motor outcomes after clinically relevant cervical contusion injury. These benefits are observed when grafts are placed two weeks after injury, a time point that is more clinically practical than acute interventions, allowing time for patients to stabilize medically, simplifying enrollment in clinical trials, and enhancing predictability of spontaneous improvement in control groups.

Early Inflammatory Responses Following Cell Grafting in the CNS Trigger Activation of the Subventricular Zone: A Proposed Model of Sequential Cellular Events.

While multiple rodent preclinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety, and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP(+) mouse embryonic fibroblasts (mEFs) in the CNS of immunocompetent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neoangiogenesis, microglia/macrophage recruitment, astrogliosis, and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the subventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses and will eventually allow for successful manipulation of this intervention.

Injection of embryonic median ganglionic eminence cells or fibroblasts within the amygdala in rats kindled from the piriform cortex

Intracerebral infusion of anticonvulsant agent secreting cells has proven to raise the threshold for seizure generation in epileptogenic areas. Median ganglionic eminence (MGE) is the main embryonic region where future GABAergic cells originate. Here we report the results of intraamygdaline grafting of MGE cells versus fibroblasts in a piriform cortex kindling model of epilepsy in the rat. Rats were implanted with an electrode in the left piriform cortex and subjected to infusion at the left basolateral amygdala of cells obtained from the MGE of embryos or fibroblasts. Some of the donor cells were obtained from transgenic rats expressing the green fluorescent protein (GFP). Seizure and neurologic behavior were recorded, and inmunohistochemical and ultrastructural studies were carried out. Cells obtained from the embryonic MGE elevated both the afterdischarge and the seizure threshold progressively, being significant 3 weeks after their injection. On the contrary, fibroblasts injected into the amygdala raised the seizure thresholds the first week, the effect weaning during the following weeks. Fibroblasts and MGE cells were shown at the injected amygdala. No behavioral side effects were recorded in either experimental group. MGE cells implanted at the amygdala may control the focal component of temporolimbic seizures. This effect may be mediated by local release of GABA.

Annual Meeting of the Society for Cutaneous Ultrastructure Research (33rd Annual Meeting of SCUR) 8–10 June 2006 WARSZAWA, POLAND

Unsupported keratinocyte grafts, while fragile, provide excellent clinical benefits and wound coverage. We aim to overcome the fragile nature of these grafts by using keratinocytes and fibroblast grafts grown on biocompatible, biodegradable, hexagonalpacked porous poly-calprolactone membranes. To confirm the feasibility of this proposal, human epidermal keratinocytes and dermal fibroblasts were cultured on membranes with controlled-sized pores (ranging from 3–20 microns). We examined the effects of growing monocultures on these porous membranes and on flat (pore less) polymer films. Cell adhesion, growth and migration rates and basic morphology were examined using confocal laser scanning microscopy. Cell ultrastructure was subsequently examined using scanning and transmission electron microscopy. Human keratinocytes and fibroblasts attached to all porous membranes. Furthermore, small pore (3 and 5 micron) membranes provided the best surfaces, on which fibroblasts could attach, adhere, divide and grow. However, keratinocytes, the most discerning of the two cell types adhered and grew best on 3 micron pore membranes. In addition, cells were prevented from undergoing transmembrane migration (to the opposing membrane surface) by the 3 micron porous membrane. Larger pores (45microns) allowed migration of both fibroblasts (and to a lesser extent keratinocytes) on to the opposite side of the membrane. Our data show that keratinocytes and fibroblasts attached and grew optimally on small pore membranes due to greater pores numbers and a larger surface area over which cells adhere. The small pore size also inhibits cells from traversing the membrane while, at the same time allowing cell adhesion, matrix communication and diffusion of soluble nutrients/factors from the environment. These characteristics are important in development of graft technology, in the treatment of human skin wounds and in the study of cell-substrate adhesion on novel patterned structures

Failure of Schwann cells as supporting cells for adult neural progenitor cell grafts in the acutely injured spinal cord

Adult neural progenitor cells (NPC) co-grafted with fibroblasts replace cystic lesion defects and promote cell-contact-mediated axonal regeneration in the acutely injured spinal cord. Fibroblasts are required as a platform to maintain NPC within the lesion; however, they are suspected to create an inhospitable milieu for regenerating central nervous system (CNS) axons. Therefore, we thought to replace fibroblasts by primary Schwann cells, which might serve as a superior scaffold to maintain NPC within the lesion and might further enhance axon regrowth and remyelination following spinal cord injury. Adult rats underwent a cervical dorsal column transection immediately followed by transplantation of either NPC/Schwann cell or NPC/Schwann cell/fibroblast co-grafts. Animals receiving Schwann cell or fibroblast grafts alone, or Schwann cell/fibroblast co-grafts served as controls. At 3 weeks after injury/transplantation, histological analysis revealed that only fibroblast-containing grafts were able to replace the cystic lesion defect. In both co-cultures and co-grafts, Schwann cells and NPC were segregated. Almost all NPC migrated out of the graft into the adjacent host spinal cord. As a consequence, only peripheral-type myelin, but no CNS-type myelin, was detected within co-grafts containing NPC/Schwann cells. Corticospinal axon regeneration into Schwann-cell-containing co-grafts was reduced. Taken together, Schwann cells within NPC grafts contribute to remyelination. However, Schwann cells fail as a supporting platform to maintain NPC within the graft and impair CNS axon regeneration; this makes them an unfavorable candidate to support/augment NPC grafts following spinal cord injury.

Fibroblasts Show More Potential as Target Cells than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal-epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.

Autologous Adult Rodent Neural Progenitor Cell Transplantation Represents a Feasible Strategy to Promote Structural Repair in the Chronically Injured Spinal Cord

Adult neural progenitor cells (NPCs) represent an attractive source for cell-based regenerative strategies in CNS disease. In animal models of spinal cord injury, syngenic adult NPCs, which were isolated from pooled post-mortem CNS tissue and co-transplanted together with fibroblasts, have been shown to promote substantial structural repair. The autologous transplantation of adult NPCs represents a major advantage compared with other sources of neural stem/progenitor cells. However, the feasibility of autologous NPC generation from a single biopsy in a relevant preclinical CNS disease model has yet to be demonstrated. To investigate this matter, adult Wistar rats underwent a cervical spinal cord lesion, which was followed by a minimal subventricular zone aspiration biopsy 2 days later. NPCs were isolated and propagated separately for each animal for the following 8 weeks. Thereafter, they were co-transplanted with simultaneously harvested skin fibroblasts in an autologous fashion into the cervical spinal cord lesion site. A total of 4 weeks later, graft survival, tissue replacement and axonal regeneration were assessed histologically. Animals receiving either allogenic NPCs combined with fibroblasts or autologous pure fibroblast grafts served as control groups. Within 8 weeks after the biopsy more than 3 million NPCs could be generated from a single aspiration biopsy, which displayed a differentiation pattern indistinguishable from syngenic NPC grafts. NPCs within autologous co-grafts readily survived, replaced cystic lesion defects completely and differentiated exclusively into glial phenotypes, thus paralleling previous findings with syngenic NPCs. The delayed transplantation 8 weeks after the spinal cord lesion elicited substantial axonal regeneration. These findings demonstrate that the therapeutic strategy to induce structural repair by transplanting adult autologous NPCs, after the successful propagation from a small brain biopsy into an acute CNS disease model, such as spinal cord injury, is feasible at the preclinical level.

Alginate Encapsulated BDNF-Producing Fibroblast Grafts Permit Recovery of Function after Spinal Cord Injury in the Absence of Immune Suppression

Encapsulation of cells has the potential to provide a protective barrier against host immune cell interactions after grafting. Previously we have shown that alginate encapsulated BDNF-producing fibroblasts (Fb/BDNF) survived for one month in culture, made bioactive neurotrophins, survived transplantation into the injured spinal cord in the absence of immune suppression, and provided a permissive environment for host axon growth. We extend these studies by examining the effects of grafting encapsulated Fb/BDNF into a subtotal cervical hemisection on recovery of forelimb and hindlimb function and axonal growth in the absence of immune suppression. Grafting of encapsulated Fb/BDNF resulted in partial recovery of forelimb usage in a test of vertical exploration and of hindlimb function while crossing a horizontal rope. Recovery was significantly greater compared to animals that received unencapsulated Fb/BDNF without immune suppression, but similar to that of immune suppressed animals receiving unencapsulated Fb/BDNF. Immunocytochemical examination revealed neurofilament (RT-97), 5-HT, CGRP and GAP-43 containing axons surrounding encapsulated Fb/BDNF within the injury site, indicating axonal growth. BDA labeling however showed no evidence of regeneration of rubrospinal axons in recipients of encapsulated Fb/BDNF, presumably because the amounts of BDNF available from the encapsulated grafts are substantially less than those provided by the much larger numbers of Fb/BDNF grafted in a gelfoam matrix in the presence of immune suppression. These results suggest that plasticity elicited by the BDNF released from the encapsulated cells contributed to reorganization that led to behavioral recovery in these animals and that the behavioral recovery could proceed in the absence of rubrospinal tract regeneration. Alginate encapsulation is therefore a feasible strategy for delivery of therapeutic products produced by non-autologous engineered fibroblasts and provides an environment suitable for recovery of lost function in the injured spinal cord.

Long-term restoration of nigrostriatal system function by implanting GDNF genetically modified fibroblasts in a rat model of Parkinson's disease.

The motor behavior and levels of dopamine and its metabolites in the striatum were studied in rats that received a unilateral injection of 6-OHDA and underwent grafting of rat-derived primary fibroblasts that had been genetically modified to express lacZ and human glial cell line-derived neurotrophic factor (GDNF). Rotation behavior tests were performed each week and striatal levels of DA and its metabolites were measured every 4 weeks after grafting of fibroblasts that expressed lacZ, with or without additional transfection of the GDNF transgene. Rats grafted with GDNF-producing fibroblasts showed a significant improvement in motor behavior as determined by the rotation test, with a less pronounced reduction in the levels of dopamine and its metabolites in the striatum as compared with those in the control animals or brain parts. In addition, there was a lower decrease in the number of TH immunoreactive neurons in the substantia nigra ipsilateral to the lesion in rats with GDNF-producing fibroblasts than in rats with lacZ-expressing fibroblasts. These results support the notion that intracerebral grafting of fibroblasts that express GDNF is a potentially useful therapeutic strategy for treating Parkinson's disease.

Axonal Regeneration through Regions of Chondroitin Sulfate Proteoglycan Deposition after Spinal Cord Injury: A Balance of Permissiveness and Inhibition

Increased expression of certain extracellular matrix (ECM) molecules after CNS injury is believed to restrict axonal regeneration. The chondroitin sulfate proteoglycans (CSPGs) are one such class of ECM molecules that inhibit neurite outgrowth in vitro and are upregulated after CNS injury. We examined growth responses of several classes of axons to this inhibitory environment in the presence of a cellular fibroblast bridge in a spinal cord lesion site and after a growth factor stimulus at the lesion site (fibroblasts genetically modified to secrete NGF). Immunohistochemical analysis showed dense labeling of the CSPGs NG2, brevican, neurocan, versican, and phosphacan at the host-lesion interface after spinal cord injury (SCI). Furthermore, robust expression of NG2, and to a lesser extent versican, was also observed throughout grafts of control and NGF-secreting fibroblasts. Despite this inhibitory milieu, several axonal classes penetrated control fibroblast grafts, including dorsal column sensory, rubrospinal, and nociceptive axons. Axon growth was amplified more in the presence of NGF-secreting grafts. Confocal microscopy demonstrated that axon growth was, paradoxically, preferentially associated with NG2-rich substrates in both graft types. NG2 expression also increased after sciatic nerve injury, wherein axons successfully regenerate. Cellular sources of NG2 in SCI and peripheral nerve lesion sites included Schwann cells and endothelial cells. Notably, these same cellular sources in lesion sites produced the cell adhesion molecules L1 and laminin, and these molecules all colocalized. Thus, axons grow along substrates coexpressing both inhibitory and permissive molecules, suggesting that regeneration is successful when local permissive signals balance and exceed inhibitory signals.

Transplantation of syngeneic Schwann cells to the retina of the rhodopsin knockout (rho(-/-)) mouse.

To determine whether subretinal Schwann cell transplantation can prolong the survival of photoreceptors in the rhodopsin knockout (rho(-/-)) mouse. Schwann cells were prepared from postnatal day (PN) 5 to 7 mouse pups and grafted subretinally into the eyes of PN35 rho(-/-) mice. RT-PCR was performed on similarly prepared cells to determine growth factor production in vitro. Eyes were retrieved at PN70 for anatomic and statistical analysis. Control animals received grafts of fibroblasts or sham surgery. RT-PCR demonstrated the presence of message for ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic factor (GDNF) in the cultured Schwann cells. Schwann cell grafts produced a statistically significant rescue of photoreceptors in a restricted area of retina at PN70, but the effect was lost by PN140. Preserved inner segments could be identified, but outer segments were never present. Sham surgery also resulted in photoreceptor rescue but at a reduced level. Fibroblast grafts appeared to produce little or no rescue effect. Grafts of Schwann cells or fibroblasts and sham surgery induced a reactive Müller glial response. Schwann cells can prolong photoreceptor survival in the rhodopsin knockout mouse until at least PN70.

Grafts of BDNF-Producing Fibroblasts Rescue Axotomized Rubrospinal Neurons and Prevent Their Atrophy

We have reported that intraspinal transplants of fibroblasts genetically modified to express brain-derived neurotrophic factor (BDNF) promote rubrospinal axon regeneration and functional recovery following subtotal cervical hemisection that completely ablated the rubrospinal tract. In the present study we examined whether these transplants could prevent cell loss and/or atrophy of axotomized Red nucleus neurons. Adult rats received a subtotal spinal cord cervical hemisection followed by a graft of unmodified fibroblasts or fibroblasts producing BDNF into the lesion cavity. One or 2 months later, fluorogold was injected several segments caudal to the lesion-transplant site to retrogradely label those Red nucleus neurons whose axons have regenerated. Unmodified fibroblasts failed to protect against either cell loss or atrophy. Neuron counts and soma-size measurements in Nissl-stained preparations showed a 45% loss of recognizable neurons and 40% atrophy of the surviving neurons in the injured Red nucleus. Grafts of BDNF-producing fibroblasts reduced neuron loss to less than 15% and surviving neurons showed only a 20% decrease in mean soma size. Soma size analysis of fluorogold-labeled Red nucleus neurons indicated that the Red nucleus neurons whose axons regenerated caudal to the graft did not atrophy. We conclude that fibroblasts engineered ex vivo to secrete BDNF and grafted into a partial cervical hemisection promote axon regeneration while reducing cell loss and atrophy of neurons in the Red nucleus. These results suggest that transplants of genetically engineered cells could be an important tool for delivery of therapeutic factors that contribute to the repair of spinal cord injury.

Delivery of hyper-interleukin-6 to the injured spinal cord increases neutrophil and macrophage infiltration and inhibits axonal growth.

Cytokine growth factors of the interleukin (IL)-6 family have recently been shown to play an important role in central nervous system (CNS) development, repair, and inflammation. These cytokines, which interact via specific membrane receptors, share a signal-transducing receptor subunit, glycoprotein 130 (gp130). Gp130 is expressed by motoneurons in the gray matter of the rat spinal cord and by several brainstem nuclei that project to the spinal cord including the red, reticular, and vestibular nuclei. In this study, we examined whether stimulation of gp130 signaling, with the use of grafts of fibroblasts genetically modified to deliver the fusion protein, hyper-IL-6 (H-IL-6), which consists of the cytokine growth factor, IL-6, and its alpha receptor, would elicit growth of injured spinal cord axons. Particular emphasis was placed on examining the potentially competing effects of growth factor versus proinflammatory influences of H-IL-6 in the context of spinal cord injury. Our results demonstrated that grafts delivering H-IL-6 induce a sixfold increase in the number of neutrophils (P < 0.05) and a twofold increase in the areas of spinal tissue occupied by macrophages and activated microglia (P < 0.01) at the site of the spinal cord injury when compared with control grafts. Of note, this augmentation in inflammatory cell infiltration correlated with a significant twofold increase in lesion size (P < 0.05) and a fourfold reduction in axonal growth (P < 0.01) at the lesion site. Thus, potential neurotrophic properties of this cytokine family of growth factors must be balanced against their inflammatory properties when considering therapeutic application to CNS injury.

Erratum: Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme

Erratum: Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme

Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme.

Down-regulation of retroviral vector expression occurs in a number of cell types after transplantation. Although a number of vector elements have been shown to affect expression in specific experimental situations, the results can vary depending on the specific cDNA being expressed, the individual retroviral elements included in vectors, the promoter, or the inclusion of selectable markers. In previous experiments with the lysosomal enzyme beta-glucuronidase, silencing has occurred in more than 95% of transduced cells regardless of the position of the expression unit within the vector, whether a eukaryotic or viral promoter was used, whether a bacterial selectable marker gene was present or not, the target cell type, or the species of the host. It has been a consistent finding that a small number of continuously expressing cells persist for long periods after transplantation. In this study we found that deletion of all the transcriptional regulatory elements from the vector LTR, inclusion of a permissive primer binding site sequence, and use of a eukaryotic housekeeping promoter could greatly increase the number of expressing cells in fibroblast grafts in subcutaneous neo-organs and in the brain. Furthermore, the level of enzyme expression was increased five-fold on a per positive cell basis, indicating that the deleted regulatory elements were exerting a negative effect on expression in the few cells that were positive before modification of the vector. This resulted in more than a 50-fold increase in total activity compared with the previous highest expressing vector.