Nerve growth factor delivery and cell aggregation enhance choline acetyltransferase activity after neural transplantation.

Fetal cell transplantation may improve the treatment of neurodegenerative diseases, but the optimal conditions for cell transplantation have not yet been defined. In this study, we examined the influence of preaggregation of dissociated donor cells and delivery of an appropriate growth factor on cell survival following transplantation. Cell aggregates were produced by culturing dissociated cells in conventional rotation culture or in a rotating-wall bioreactor. Suspensions of dissociated septal cells from E16 embryos, or suspensions containing small aggregates (<40 microm) of these cells, were transplanted into the brains of adult rats. In some cases, transplanted cells were supplied with nerve growth factor (NGF), a neurotrophin known to promote the survival and function of septal cholinergic cells; NGF was delivered to the transplant site by controlled-release polymers or genetically engineered fibroblasts. Cell function after transplantation was determined by measuring the increase in choline acetyltransferase (ChAT) activity in brain tissue sections encompassing the transplant site. Aggregation of cells prior to transplantation enhanced ChAT activity; at 2 months posttransplantation, ChAT levels were always slightly higher in animals receiving aggregated donor cells than in those receiving single cell suspensions. NGF delivery also enhanced ChAT activity; controlled release polymers produced the largest effect 2 weeks posttransplantation, while NGF-secreting fibroblasts produced significant enhancement at 2 months posttransplantation. These different patterns of enhanced ChAT activity appear to be due to differences in the rate of NGF delivery provided by these two techniques. These studies indicate that cell aggregation and NGF delivery systems may provide important new tools for the enhancement of cell function after transplantation.

Transport and elimination of recombinant human NGF during long-term delivery to the brain.

The gene for human nerve growth factor (NGF) has been cloned into a mammalian cell line and large quantities of recombinant human NGF (rhNGF) can now be produced for clinical use, but little is known about the fate of rhNGF following delivery to the brain. In this study, we implanted polymer matrices containing 125I-labeled rhNGF into the brains of adult rats and measured spatial distributions of the released protein for 8 weeks after implantation. NGF content in the tissue was determined by counting gamma radiation in thick (1 mm) sections and by autoradiography of thin (20 microns) sections. For the first several days, the rate of NGF release from the polymer matrix was high (approximately 100 ng/day); maximal NGF concentrations, measured at the polymer-tissue interface, were correspondingly high (> 20 micrograms/ml) though day 4. At later times, the release rate decreased (2-10 ng/day) and lower maximal concentrations were observed (1-10 micrograms/ml). NGF levels were always highest in the tissue sections closest to the polymer; during the 8 weeks of the experiment, NGF levels measured in thick sections decreased 100-fold, from 30 ng/section at day 2 to 0.3 ng/section at day 54. The first 10-fold decrease occurred during the first 10 days of the study; a further 6 weeks was required to achieve the second 10-fold decrease. Throughout the experiment, the majority of NGF remained within a restricted zone around the polymer at all times; the mass of NGF decreased to 10% of the maximal level within 2-3 mm of the polymer matrix. At early times (< 1 week), radiolabel corresponding to > 20 pg of NGF was also detected in regions of the brain further removed from the polymer. Comparison of local rhNGF concentration profiles with a simple mathematical model indicated that rhNGF diffuses through the brain interstitial space and is eliminated with a half-life of approximately 45 min, although elimination appears to be substantially slower in white matter regions. This limited ability of NGF to penetrate and be retained within the brain tissue indicates that NGF will need to be delivered almost directly to the target tissue for efficacy.

Distribution of nerve growth factor following direct delivery to brain interstitium.

Several studies suggest the potential of nerve growth factor (NGF) in the treatment of patients with Alzheimer's disease. To characterize NGF transport within the brain interstitium, we implanted controlled release polymers containing NGF and [125I]NGF into the brains of adult male rats and measured spatial distributions of NGF for up to one week. NGF concentration in the brain was quantified using ELISA, radiation counting, and autoradiography. At 2 days post-implantation, quantities of NGF in excess of 50 pg per section were detected within thick (1 mm) coronal slices of the hemisphere ipsilateral to the site of implantation up to 3 mm rostral and caudal to the edge of the polymer. Lower levels of radioactivity (> 5 pg but < 50 pg NGF per section) could be detected throughout the rest of the brain. Levels were highest in the tissue sections containing the polymer, reaching 9.5 ng per section. Autoradiography of thin (20 microns) coronal sections indicated that local NGF concentrations immediately adjacent to the polymer approached 40 micrograms/ml. Analysis of sequential sections on the autoradiograph confirmed that NGF was transported only 2-3 mm from the polymer in any direction. At one week post-implantation, the pattern of NGF distribution was similar to that seen at 2 days, and concentrations remained high near the site of the implant. Comparison of local NGF concentration profiles to simple models of diffusion with first-order elimination suggests that the NGF moved through the tissue by diffusion through the interstitial space with a half-life on the order of 0.5 h. The limited range of NGF transport in brain tissue indicates that: (i) protein drug agents such as NGF will probably need to be delivered almost directly to the site of action for efficacy; and (ii) toxicities associated with delivery of NGF and other protein agents to non-target cells, as often occurs with systemic delivery of drugs, may be reduced by local, interstitial delivery since therapy can be restricted to a small volume of the brain.

PC12 cell aggregation and neurite growth in gels of collagen, laminin and fibronectin.

PC12 cells form aggregates when suspended within three-dimensional, self-assembled, type I collagen gels; these aggregates increase in size over time. In addition, when the cells are cultured in the presence of nerve growth factor, they express neurites, which extend through the three-dimensional matrix. In this report, the roles of fibronectin, laminin and nerve growth factor in PC12 cell aggregation and neurite growth following suspension in collagen matrices were evaluated. Single cells and small clusters of cells were suspended in collagen gels; the kinetics of aggregation were determined by measurement of the projected area of each aggregate, and neurite lengths were determined by measurement of end-to-end distance. Fibronectin and laminin inhibited the aggregation of PC12 cells at 50 micrograms/ml, and fibronectin, but not laminin, inhibited the growth of neurites at 100 micrograms/ml. In the absence of serum, the aggregation of cells cultured with nerve growth factor was almost completely inhibited, but the average neurite length was unaffected. In the presence of nerve growth factor, the extent of cell aggregation could not be explained simply by an increase in cell number, suggesting the presence of two separate mechanisms for aggregate growth: one dependent on cell motility and another dependent on cell proliferation.

Mathematical model of temperature-sensitive plasmid replication.

The copy number of a series of plasmids constructed at Odense University is regulated by the lambda PR/PRM promoters and the temperature-sensitive cI857 repressor. At low temperatures, these plasmids exhibit the low copy number of the parent plasmid R1 (5-6 per cell). At high temperatures, the plasmids exhibit runaway replication, reaching copy numbers of greater than 1,000 per cell. A detailed mathematical model of the temperature-sensitive replication of these plasmids has been developed incorporating three features: replication of the parent plasmid, regulation of the lambda PR/PRM promoters by the cI repressor, and thermal denaturation of the cI857 repressor. Models of the first two of these features have been described by others. We revised and extended those models, described the thermal denaturation of the cI857 repressor, and integrated these features to give a comprehensive model of temperature-sensitive plasmid replication. Model predictions were compared to experimental measurements of both steady-state copy numbers as a function of temperature and the change in copy number following temperature shifts up and down. The model accurately describes the qualitative behavior of the system and gives reasonable quantitative results. This is particularly significant since all the parameter values used in this model were determined independently: that is, there was no adjustment of parameter values to match our experimental data. The regulatory system that gives rise to the temperature-sensitive replication of these plasmids is widely used in biotechnology applications, so the elements of the model related to this regulation should be applicable to a wide variety of systems.

Cell aggregation and neurite growth in gels of extracellular matrix molecules.

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three-dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 mug/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (>/=0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four- to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. (c) 1994 John Wiley & Sons, Inc.

Stabilization of nerve growth factor in controlled release polymers and in tissue.

We have studied the release of nerve growth factor (NGF), a protein under consideration for treatment of Alzheimer's Disease, from polymer matrices and microspheres to characterize the stability of NGF, the dynamics of NGF release, and the distribution of NGF within the brain interstitium. Poly(ethylene-co-vinyl acetate) (EVAc) disks and poly(L-lactic acid) (PLA) microspheres were formed by codispersing NGF with one of a variety of molecules. The mass of mouse NGF (mNGF) detected following release from EVAc disks into buffered saline varied five-fold over the range of codispersants studied, with carboxymethyldextran providing optimal release, while the mass of recombinant human NGF (rhNGF) released varied four-fold from both EVAc disks and PLA microspheres, with albumin and carboxymethyldextran providing optimal release. Variation of the codispersant species significantly affected NGF release into buffered saline; it also had a noticeable, but small, effect of the amount of NGF found in the brain tissue following implantation of a polymer device. To improve NGF retention in tissue, NGF was conjugated to 70,000 molecular weight dextran and incorporated into a polymeric device. The distribution of NGF was enhanced by conjugation; comparison of NGF concentrations in the brain to a mathematical model of diffusion and elimination suggested that the elimination rate of NGF-dextran conjugate in the tissue was over seven times slower than the elimination rate of NGF. These results indicate that variation of the properties of the controlled release system may be useful in regulating the time course of NGF delivery to tissue, and that modification of the NGF itself can improve penetration and retention in the brain.