Cell Transplantation

Transplantation of human fetal central nervous system tissue has been shown to be of benefit in Parkinson's disease, and is currently being explored as a therapeutic option in Huntington's disease. The success of a neural transplant is dependent on a number of factors, including the requirement that donor cells are harvested within a given developmental window and that the cell preparation protocols take account of the biological parameters identified in animal models. Although many of the criteria necessary for a successful neural transplant have been defined in animal models, ultimately they must be validated in human studies, and some issues can only ever be addressed in human studies. Furthermore, because neural transplantation of human fetal tissue is limited to small numbers of patients in any one surgical center, largely due to practical constraints, it is crucial that tissue preparation protocols are clearly defined and reproducible, so that (i) multicenter trials are possible and are based on consistent tissue preparation parameters, and (ii) results between centers can be meaningfully analyzed. Here we describe the preparation of human fetal striatum for neural transplantation in Huntington's disease, and report on the validation of a method for estimating the developmental stage of the fetus based on direct morphometric measurements of the embryonic tissue.

Transplantation of human fetal CNS tissue is a promising therapy for neurodegenerative conditions such as Huntington's disease (HD), but its widespread adoption is limited by restricted tissue availability. One method of overcoming this problem would be to store the tissue in hibernation medium, an approach that we reported previously for human fetal striatal tissue stored for up to 24 h. We now demonstrate the feasibility of storing such tissue for up to 8 days in hibernation medium. When either fresh or 8-day hibernated striatal cells were cultured under standard conditions for 4 days, the proportion of DARPP-32-positive neurons did not differ significantly, although the total number of cells was significantly less from tissue that had been hibernated. Six weeks after transplantation into cyclosporin A-immunosuppressed unilateral quinolinic acid-lesioned rats, there was no significant difference between fresh and hibernated grafts, both in terms of graft volume and extent of striatal phenotypic markers. This study therefore clearly demonstrates that hibernation of human fetal striatal tissue for up to 8 days is not deleterious to its differentiation in culture or survival following transplantation, and is therefore an appropriate method of storage for this tissue.

Previous studies have shown that embryonic rat and human dorsal root ganglion (DRG) cells survive grafting to the cavity of extirpated adult rat DRG. Furthermore, grafted human embryonic neurons were shown to send axons peripherally and into the spinal cord, where they establish functional synaptic connections. This study analyzed the survival of orthotopically allografted rat DRG cells from embryonic stages 15 (E15) and 20 (E20), and the influence on their survival of nerve growth factor (NGF). NGF was delivered to the DRG transplants either by pump infusion or by cotransplantation of cells from Drosophila melanogaster, transgenic for human NGF. Lumbar DRGs of adult rats were removed and a collection of E15 or E20 DRGs placed in the cavity. One month after grafting the total number of DRG cells in the grafts was counted. Differentiation of subpopulations of DRG cells was estimated by counting cells immunostained for calcitonin gene-related peptide (CGRP), Griffonia simplicifolia agglutinin isolectin B4 (GSA), or heavy neurofilament protein (antibody RT97). The results show: i) similar survival of E15 and E20 grafts, with great variability in the survival of different subpopulations in E15 transplants, but a more consistent distribution of different phenotypes in E20 transplants; ii) infusion of NGF for 2 weeks increases the survival of E15 transplants, but has a negative effect on E20 transplants; iii) Drosophila cells transfected with human NGF gene survive peripheral xenografting and have a positive effect on the survival of the GSA- and CGRP-positive populations in E15 and E20 transplants; iv) Drosophila cells without the human NGF gene increase cell survival in E20 transplants. These data suggest that i) the effect of NGF is dependent on the embryonic stage of the transplants, ii) age-dependent sensitivity to NGF influences graft survival, and iii) transgenic Drosophila cells can be cotransplanted with embryonic neural tissue to the mammalian peripheral nervous system with a positive effect on the survival of neural grafts.

Replacement of damaged cells is a promising approach for treatment of age-related macular degeneration (AMD) and retinitis pigmentosa (RP); however, availability of donor tissue for transplantation remains a major obstacle. Key factors for successful engineering of a tissue include the identification of a neural cell line that is: homogeneous but can be expanded to give rise to multiple cells types; is nontumorigenic, yet capable of secreting neurotrophic factors; and is able to form three-dimensional (3D), differentiated structures. The goal of this study was to test the feasibility of tissue engineering from a multipotential human retinal cell line using a NASA-developed bioreactor. A multipotential human retinal precursor cell line was used to generate 3D structures. In addition, retinal pigment epithelium (RPE) cells were cocultured with neural cells to determine if 3D retinal structures could be generated in the bioreactor with cells grown on laminin-coated cytodex 3 beads. Cell growth, morphology, and differentiation were monitored by light and scanning electron microscopy, Western blot analysis, and analysis of glucose use and lactate production. The neuronal retinal precursor cell line cultured in a bioreactor gave rise to most retinal cell types seen in monolayer culture. They formed composite structures with cell-covered beads associated with one another in a tissue-like array. The beginning of layering and/or separation of cell types was observed. The neuronal cell types previously seen in monolayer cultures were also seen in the bioreactor. Some of the retinal cells differentiate into photoreceptors in the bioreactor with well-developed outer segment-like structures, a process that is critical for retinal function. Moreover, the neuronal cells that were generated resembled their in vivo phenotype more closely than those grown under other conditions. Outer segments were almost never seen in the monolayer cultures, even in the presence of photoreceptor-inducing growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor (TGF-α). Muller cells were occasionally seen when retinal, RPE cells were cocultured with retinal cells in the bioreactor. These have never been seen in this retinal cell line before. Cells grown in the bioreactor expressed several proteins specific for the retinal cell types: opsin, protein kinase C-α, dopamine receptor D₄, tyrosine hydroxylase, and calbindin.

Several previous studies, suggesting the potential use of embryonic xenografts in the treatment of neurological disorders, indicate that neural growth and axonal guidance factors may function across species. In this light, blocks of fetal porcine neocortex were grafted into small cortical lesion cavities made in newborn rats. Sacrifice at 3–12.5 weeks posttransplantation revealed healthy looking grafts in several animals. Apparent graft rejection evidenced by areas of necrosis and OX1 reactivity was observed in some of the older transplants. Treatment of nursing mothers or of postweaning newborns with cyclosporin A did not appear to promote graft survival. Some transplants grew to extremely large proportions and were characterized by bands of cells and bundles of axons as observed using immunohistochemical staining for pig neurofilament. Neurofilament-positive axons projected from several of the grafts to course through the corpus callosum to the contralateral cortex or to course ipsilaterally within the subcortical white matter, where labeled fibers could be traced to the midbrain crus cerebri in older transplants. Bundles of axons were also observed coursing within the ipsilateral caudate putamen where terminal branching was apparent. The normal course of transplant efferents within the host brain indicates that growing pig axons can respond to rodent axonal guidance factors.

Labeling stem cells with FDA-approved superparamagnetic iron oxide particles makes it possible to track cells in vivo with magnetic resonance imaging (MRI), but high intracellular levels of iron can cause free radical formation and cytotoxicity. We hypothesized that the use of cationic liposomes would increase labeling efficiency without toxic effects. Rabbit skeletal myoblasts were labeled with iron oxide by: 1) uptake of iron oxide incorporated into cationic transfection liposomes (group I) or 2) customary endocytosis (group II). In both groups, cell proliferation and differentiation were measured and toxicity was assayed using trypan blue and ratio fluorescence microscopy with BODIPY® 581/591 C₁₁. The effects of the intracellular iron oxide on magnetic resonance image intensities were assessed in vitro and in vivo. Both methods resulted in uptake of iron intracellularly, yielding contrast-inducing properties on MRI images. In group II, however, incubation with iron oxide at high concentrations required for endocytosis caused generation of free radicals, a decrease in proliferation, and cell death. Cytotoxic effects in the remaining cells were still visible 24 h after incubation. Conversely, in group I, sufficient intracellular uptake for detection in vivo by MRI could be achieved at 100-fold lower concentrations of iron oxide, which resulted in a high percentage of labeled cells, high retention of the label, and no cytotoxic effects even after stressing the cells with a hypoxia–reoxygenation insult. The use of cationic liposomes for iron oxide stem cell labeling increases labeling efficiency approximately 100-fold without toxic effects. This technique results in high-contrast-inducing properties on MRI images both in vitro and in vivo and could thus be a valuable tool for tracking stem cells noninvasively.

Cell engraftment is a new strategy for the repair of ischemic myocardial lesions. The hemodynamic effectiveness of this strategy, however, is not completely elucidated yet. In a rat model of cryothermia-induced myocardial dysfunction, we investigated whether syngeneic transplantation of neonatal cardiomyocytes or satellite cells is able to improve left ventricular performance. Myocardial infarction was induced in female Lewis rats by a standardized cryolesion to the obtuse margin of the left ventricle. After 4 weeks, 5 × 106 genetically male neonatal cardiomyocytes (n= 16) or satellite cells (n = 16) were engrafted into the myocardial scar. Sham-transplanted animals (n = 15) received injections with cell-free medium. Sham-operated animals (n = 15) served as controls. Left ventricular performance was analyzed 4 months after cell engraftment. Chimerism after this sex-mismatched transplantation was evaluated by detection of PCR-amplified DNA of the Y chromosome. The average heart weight of the infarcted animals significantly exceeded that of controls (p < 0.05). In sham-transplanted animals, mean aortic pressure, left ventricular systolic pressure, aortic flow (indicator of cardiac output), and left ventricular systolic reserve were significantly lower (p < 0.05) compared with sham-operated controls. This was associated with deterioration of ventricular diastolic function (maximal negative dP/dt, time constants of isovolumic relaxation; p < 0.05). Transplantation of satellite cells was found more effective than transplantation of neonatal cardiomyocytes, resulting in i) normalization of mean aortic pressure compared with sham-operated controls, and ii) significantly improved left ventricular systolic pressure and aortic flow (p < 0.05) compared with sham-transplanted animals. Left ventricular systolic reserve and diastolic function, however, were improved by neither satellite cell nor neonatal cardiomyocyte transplantation. Analysis of male genomic DNA revealed 3.98 ± 2.70 ng in hearts after neonatal cardiomyocyte engraftment and 6.16 ± 4.05 ng in hearts after satellite cell engraftment, representing approximately 103 viable engrafted cells per heart. Our study demonstrates i) long-term survival of both neonatal cardiomyocytes and satellite cells after transplantation into cryoinfarcted rat hearts, ii) slight superiority of satellite cells over neonatal cardiomyocytes in improving global left ventricular pump performance, and iii) no effect of both transplant procedures on diastolic dysfunction.

One of the major obstacles in transplanting avascular tissue or metabolically active cells for ischemic diseases is the loss of transplanted cells due to lack of oxygen and nutrients in the early posttransplantation period. Biodegradable polymeric tissue engineering scaffolds and hydrogels have a potential to incorporate cells or cellular organoids such as islets of Langerhans and growth factors. In this study, we tested the efficiency of two types of polymeric materials to carry recombinant human vascular endothelial growth factor (rhVEGF) or pancreatic tumor cell lines, namely Ins-1 and AR42J, for the induction of new vessels. Chitosan hydrogel fibers with micropores were prepared and molded into a cylinder construct (5 mm φ 8 mm height). Macroporous PLGA scaffolds with a pore size of 250–400 μm were prepared and cut into cylinders (6 mm φ 3 mm height). Both chitosan and PLGA constructs were loaded with rhVEGF (3 μg) or seeded with the cell lines (5 × 105 cells and 3 × 105 cells/construct, respectively, for AR42J and INS-1 cells), and transplanted into the fascial flaps of Wistar rats. At distinct time points up to 4 weeks postimplantation, polymers were explanted, fixed, and vessel density was counted on sections stained with anti-Factor-VIII antibody. Additionally, the kinetics of rhVEGF release from PLGA microspheres (φ of 50–80 μm) was determined using VEGF Elisa. Endogenous VEGF release from pancreatic rat cell lines was also determined. Light microscopy study was performed on H&E-stained paraffin sections of the islet-polymer samples. The vascular density of rhVEGF-loaded chitosan constructs was increased fourfold 2 weeks after subcutaneous transplantation compared with rhVEGF-unloaded controls (465 ± 144 vs. 104 ± 80 vessels per mm2, p < 0.05). Protein leakage occurred, but was not observed after 2 weeks. Higher insulin content was detected in rat islet grafts transplanted following VEGF application. More than 50% of total rhVEGF was released on the first day of in vitro culture of PLGA microspheres. rhVEGF secretion had another, but smaller, peak on the third day followed by a constant release. By comparison, endogeneous VEGF secretion of pancreatic tumor cells was measured within a 3-day culture period. Biodegradable polymer scaffolds and hydrogels may have potential use as solid supports to induce angiogenesis for pancreatic cell transplantation.

Alginate hydrogels are widely used for cell encapsulation and transplantation, and they are frequently surface reinforced with secondary polymers to enhance their mechanical rigidity and stability. We hypothesized that the molecular weight (MW) of the polymer utilized to reinforce alginate would be an important factor in their stability, particularly when the gel network was homogeneously reinforced with the polymer. This hypothesis was investigated with alginate hydrogels cross-linked with Ca2+, and reinforced throughout the bulk of the gel with poly(ethyleneimine) (PEI) having different MWs. Interactions between the two polymers became significant following gelation, leading to higher elastic moduli (E) than gels with no PEI. The decrease in E of gels incubated in isotonic salt solutions over time, utilized as an indication of gel break down, was ameliorated with an increase in the MW of the PEI. In addition, the dependencies of the moduli and visco-elasticity on the temperature also became smaller with the use of high MW PEI. This is likely due to the limited mobility of high MW PEI, leading to a higher energy for dissociation. The stable interactions between the alginate and PEI prevented alterations of the pore structure in the gels, and slowed the deterioration of gel properties even under continuous agitation in a bioreactor. The results of this study will likely be useful in designing alginate encapsulation strategies for various applications.

Autogenous cell transplantation via hydroxylapatite (HA) vehicle has been reported to have beneficial effects on the treatment of human periodontal osseous defects. The aim of this study was to explore the possibility of using gingival fibroblast-like cells in the therapy of osseous defects caused by inflammatory periodontitis by reporting long-term results of gingival fibroblast-coated hydroxylapatite (GF–HA) grafting for healing these defects. Gingival fibroblasts were cultured from healthy gingivae of treated subjects. Growth of cells on HA particles was established in vitro, and then the GF–HA complex was transplanted into the periodontal osseous defects. Clinical parameters of gingival and plaque indices, probing depth, and periapical x-ray were monitored at baseline and at various periods from 50 months to 6 years after surgery. Grafting with only HA in the osseous defects of the same patient was used for comparison. The present study shows that GF–HA-treated sites could achieve marked pocket reduction and probing attachment gain at reentry and later recalls. Good clinical bone filling of osseous defects in GF–HA-treated sites was also demonstrated in periapical radiographs (increased bone height and reappearance of the crestal cortex) and in some reentry sites. One HA-treated site was filled with connective tissue only, and the absence of new bone formation was noted during a reentry operation. Another HA-treated site exhibited a comparable increase in radiographic density, while part of HA particles were gradually lost in longer recalls. These limited observations conclude that GF–HA grafting may provide a treatment modality leading to regeneration of periodontal tissues in periodontitis-affected osseous defects. Further studies including more cases and demonstration of the deposition of differentiated periodontal tissues are necessary before further application of this therapy.

The need for transplantable beta cells with a stable phenotype has given rise to several strategies including the expansion of existing pancreatic islets and/or growth of new ones. In vitro studies of beta cell proliferation on extracellular matrices plus growth factors have highlighted a possible cell expansion technique; however, the technique was accompanied with loss of insulin secretion. Herein we showed that human islet cell proliferation was marked by a decreased expression of specific differentiation markers, particularly insulin, insulin promoting factor-1 (IPF-1), and glucokinase. After a 6-day expansion period, we tried to reexpress the beta cell differentiation markers with compounds known for their differentiation and/or insulin-secreting properties. Sodium butyrate was a potent factor of IPF-1, insulin, and glucokinase gene reexpression; it also clearly induced secretion of gastrin, a known neogenic factor. Other compounds, namely TGF-β, calcitriol, GLP-1, and activin A, efficiently enhanced the glucose sensor machinery, particularly Glut-1 and glucokinase, thus triggering glucose responsiveness. Our results indicate that specific beta cell gene expression may be induced after expansion and dedifferentiation. This rekindles interest in human beta cell expansion. The possible stabilization of specialized genes needed by beta cells to fulfill their role as nutrient sensors and metabolic regulators may also be of interest to ensure graft maintenance and efficiency.

As islet cell transplantation gains increasing interest following results published by the Edmonton group, results that have been successfully reproduced by several centers nationwide and abroad, the need of guidelines to standardize the procedure becomes highly important. We detail the key steps of the infusion procedure utilizing a closed gravity fed bag system utilized at our institution since 1990, which consists of a 600-ml transfer bag and a 150-ml rinse bag connected via sterile tubing. The use of gravity allows for a control rate of infusion as well as providing a safety mechanism through natural reduction of flow that parallels any increase in portal pressure, therefore allowing the operator to prevent precipitous pressure rises. Reports on significant rise in portal pressures during islet cell infusion as well as portal vein thrombosis have been published. Infusion at these centers was carried out using a syringe method. Using our technique, portal vein thrombosis (partial or complete) was not detected in any of the infusions performed at our institution. This method may be of assistance to minimize some of the observed complications associated with islet transplant procedures and has now been adapted by most centers performing clinical islet transplantation.