Prevention of complement-mediated cytotoxicity against rat islets by encapsulation in a cellulose sulphate membrane

The immuno-isolation by semipermeable membranes should prevent the immunological destruction of the transplanted tissue. The polyelectrolyte complex membrane from cellulose sulphate and poly(dimethyldiallylammoniumchloride), which does not alter the viability of the encapsulated rat islets, can prevent complement-mediated cytotoxicity. The 51Cr release from prelabelled islets, which was induced by either unspecific cytotoxicity of rabbit sera against the rat islets or by an anti-MHC rat serum in presence of complement, was prevented. The diffusion of immunoglobulins as well as of factors of the complement system was inhibited by encapsulation. The capsule membrane also prevents the beta-cell destruction in cytotoxic sera from newly diagnosed type-I diabetics measured as insulin leakage after blocking the active hormone secretion. In conclusion, the cellulose sulphate membrane seems to be useful for the transplantation of encapsulated islets.     

Live encapsulated Lactobacillus acidophilus cells in yogurt for therapeutic oral delivery: preparation and in vitro analysis of alginate-chitosan microcapsules

Targeted delivery of live microencapsulated bacterial cells has strong potential for application in treating various diseases, including diarrhea, kidney failure, liver failure, and high cholesterol, among others. This study investigates the potential of microcapsules composed of two natural polymers, alginate and chitosan (AC), and the use of these artificial cells in yogurt for delivery of probiotic Lactobacillus acidophilus bacterial live cells. Results show that the integrity of AC microcapsules was preserved after 76 h of mechanical shaking in MRS broth and after 12 h and 24 h in simulated gastric and intestinal fluids. Using an in vitro computer-controlled simulated human gastrointestinal (GI) model, we found 8.37 log CFU/mL of viable bacterial cells were present after 120 min of gastric exposure and 7.96 log CFU/mL after 360 min of intestinal exposure. In addition, AC microcapsules composed of chitosan 10 and 100 at various concentrations were subjected to 4-week storage in 2% milk fat yogurt or 0.85% physiological solution. It was found that 9.37 log CFU/mL of cells encapsulated with chitosan 10 and 8.24 log CFU/mL of cells encapsulated with chitosan 100 were alive after 4 weeks. The AC capsule composed of 0.5% chitosan 10 provided the highest bacterial survival of 9.11 log CFU/mL after 4 weeks. Finally, an investigation of bacterial viability over 72 h in different pH buffers yielded highest survival of 6.34 log CFU/mL and 10.34 log CFU/mL at pH 8 for free and AC-encapsulated cells, respectively. We conclude from these findings that encapsulation allows delivery of a higher number of bacteria to desired targets in the GI tract and that microcapsules containing bacterial cells are good candidates for oral artificial cells for bacterial cell therapy.     

菲立磁标记食蟹猴骨髓间充质干细胞的脑内移植示踪研究

中枢神经系统损伤后的再生修复问题一直是神经科学领域关注的重点之一,骨髓间充质干细胞移植治疗拓宽了人类中枢神经系统损伤的治疗前景,而非侵入性的磁共振成像能活体追踪移植细胞,评价移植效果。应用菲立磁标记食蟹猴骨髓来源的间充质干细胞,在脑立体定位仪引导下,自体脑内移植。结果显示,菲立磁标记间充质干细胞的有效率高达90%以上,移植区磁共振影像呈明显的低信号改变。标记的间充质干细胞移植后在脑内存活,并向周围的脑实质内迁移。移植8周后,发现移植细胞通过血管向对侧脑部迁移,但并未发现移植细胞向神经细胞分化。这些结果提示,菲立磁可用于标记、追踪脑内移植的食蟹猴骨髓间充质干细胞,标记的移植细胞可在脑内存活、迁移。     

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.     

Differentiation and migration of long term expanded human neural progenitors in a partial lesion model of Parkinson's disease

Human neural progenitor cells (HNPCs) can be expanded in large numbers for significant periods of time to provide a reliable source of neural cells for transplantation in neurodegenerative disorders such as Parkinson's disease (PD). In the present study, HNPCs isolated from embryonic cortex were expanded as neurospheres in cell culture for 10 months. Just prior to transplantation, a proportion of the HNPCs were treated in a "predifferentiation" protocol in combination with the neurotropic factor NT4, in order to yield significant numbers of neurons. For transplantation, either undifferentiated HNPCs, or predifferentiated HNPCs were transplanted into the substantia nigra of a rat model of Parkinson's disease. At 12 weeks, there was good survival with proliferation of transplanted HNPCs occurring after transplantation but ceasing before the animals were sacrificed. Transplants of predifferentiated cells contained a higher proportion of neurons. The presence of a lesion in the striatum had a significant influence on the migration of transplanted cells from the substantia nigra into the striatum. There was no significant behavioural recovery or effect of transplanted HNPCs on the loss of dopaminergic cells from the host brain. In conclusion, HNPCs may provide a source of cells for use in the treatment of Parkinson's disease.     

Complex Coacervates Formed between Whey Protein Isolate and Carboxymethylcellulose for Encapsulation of β-Carotene from Sacha Inchi Oil: Stability,In Vitro Digestion and Release Kinetics

The present work aimed to study the influence of the pH and protein ratio on the formation of complex coacervates of carboxymethylcellulose (CMC) and whey protein isolated nanoparticles (WPIN). These biopolymers and transglutaminase, as a cross-linking agent, were used to encapsulate sacha inchi oil (SIO) containing β-carotene (β-C). The stability of β-C from SIO microcapsules (β-SIO microcapsules) was evaluated under in vitro digestion using an INFOGEST 2.0 in vitro digestion protocol. The release of β-C in a simulated food model was studied, and mathematical models were used to determine the mechanism. A ratio of 1:6 (CMC/WPIN) at pH 3.5 was used for the formation of the complex. Chemical and morphological analyses suggested that SIO was microencapsulated and that a high encapsulation efficiency was obtained. The β-C from β-SIO microcapsules was preserved in vegetable oil (food model), and Fickian diffusion occurred. The β-C from β-SIO microcapsules was preserved under oral and gastric conditions, and higher release occurred during intestinal digestion when samples were subjected to in vitro digestion simulation. After in vitro digestion, the β-C from β-SIO microcapsules presented higher stability (83.37%) and acceptable bioaccessibility (31.16%). There are few studies in the literature of encapsulated SIO using the CMC/WPIN complex or studies of the release of β-carotene from SIO during in vitro digestion and in food simulants. The knowledge obtained in this study will facilitate the use and applications of β-C-loaded microcapsule delivery systems.

     

Encapsulation of parathyroid cells in hollow fibers: a preliminary report

The purpose of experiments was to evaluate the survival and functioning of human parathyroid cells after encapsulation in hollow fibers (HFs). The polypropylene HFs K600(PP Accurel (Akzo-Nobel, Germany) of inner diameter 0.6 mm, wall thickness 0.2 mm, original or surface modified were used for encapsulation. Production of parathormone (PTH) by encapsulated cells was measured in vitro. HF were filled with parathyroid cell suspension and tightly closed. Encapsulated cells were cultured for 9 or 33 days in RPMI 1640 containing 10% FCS or in Chang's medium. The level of PTH, produced by encapsulated cells was evaluated in the culture medium with radioimmunoassay test (RIA). The assays were performed every 2-4 days. The result of PTH assay was similar in both types of tested media as well as with unmodified and modified HFs, being 2-4 pg/ml of culture medium per 10(3) encapsulated cells. In conclusion, encapsulation in original or modified HFs ensures diffusion of nutrients from culture medium to encapsulated cells and allows for functioning of cells for at least 33 days in vitro.     

Yeast Cell Microcapsules as a Novel Carrier for Cholecalciferol Encapsulation: Development,Characterization and Release Properties

In this study Saccharomyces cerevisiae yeast cells was used as a novel vehicle for encapsulation of vitamin D₃. The effects of initial cholecalciferol concentration (100,000 and 500,000 IU/g yeast), yeast cell pretreatment (plasmolysis with NaCl) and drying method (spray or freeze drying) on microcapsules properties were investigated. It was found that the vitamin concentration and drying method had significant influence on encapsulation efficiency (EE) and size of yeast microcapsules. Furthermore, EE values were more increased by the plasmolysis treatment. The highest EE was obtained for plasmolysed and spray dried yeast cells prepared using initial cholecalciferol concentration of 2.5 mg per gram of yeast cells (76.10?±?6.92%). The values of mean particle size were 3.43–7.91 μm. The presence of cholecalciferol in yeast microcapsules was confirmed by X-ray diffraction (XRD) and Fourier transform-infrared (FT-IR) analyses. The in vitro cholecalciferol release from yeast microcapsules in phosphate buffer saline solution (PBS) followed a controlled release manner consistent with a Fickian diffusion mechanism. In addition, the release studies in simulated gastrointestinal tract showed sustained release of cholecalciferol in the stomach condition and significant release in intestinal medium.     

Partial recovery of dopaminergic pathway after graft of adult mesenchymal stem cells in a rat model of Parkinson's disease

Cellular therapy with adult stem cells appears as an opportunity for treatment of Parkinson's disease. To validate this approach, we studied the effects of transplantation of rat adult bone-marrow mesenchymal stem cells in a rat model of Parkinson's disease. Animals were unilaterally lesioned in the striatum with 6-hydroxydopamine. Two weeks later, group I did not undergo grafting, group II underwent sham grafting, group III was intra-striatal grafted with cells cultured in an enriched medium and group IV was intra-striatal grafted with cells cultured in a standard medium. Rotational amphetamine-induced behavior was measured weekly until animals were killed 6 weeks later. One week after graft, the number of rotations/min was stably decreased by 50% in groups III and IV as compared with groups I and II. At 8 weeks post-lesion, the density of dopaminergic markers in the nerve terminals and cell bodies, i.e. immunoreactive tyrosine hydroxylase, membrane dopamine transporter and vesicular monoamine transporter-2 was significantly higher in group III as compared with group I. Moreover, using microdialysis studies, we observed that while the rate of pharmacologically induced release of dopamine was significantly reduced in lesioned versus intact striatum in no grafted rats, it was similar in both sides in animals transplanted with mesemchymal stem cells. These data demonstrate that graft of adult mesemchymal stem cells reduces behavioral effects induced by 6-hydroxydopamine lesion and partially restores the dopaminergic markers and vesicular striatal pool of dopamine. This cellular approach might be a restorative therapy in Parkinson's disease.     

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche^1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment⁴ such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures². These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded⁵. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions² to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes¹. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.     

Differentiation of Apical Bud Cells in a Newly Developed Apical Bud Transplantation Model Using GFP Transgenic Mice as Donor

Rodent mandibular incisors have a unique anatomical structure that allows teeth to grow throughout the lifetime of the rodent. This report presents a novel transplantation technique for studying the apical bud differentiation of rodent mandibular incisors. Incisal apical end tissue with green fluorescent protein from transgenic mouse was transplanted to wild type mice, and the development of the transplanted cells were immunohistologically observed for 12 weeks after the transplantation. Results indicate that the green fluorescent apical end tissue replaced the original tissue, and cells from the apical bud differentiated and extended toward the incisal edge direction. The immunostaining with podoplanin also showed that the characteristics of the green fluorescent tissue were identical to those of the original. The green fluorescent cells were only found in the labial side of the incisor up to 4 weeks. After 12 weeks, however, they were also found in the lingual side. Here the green fluorescent cementocyte-like cells were only present in the cementum close to the dentin surface. This study suggests that some of the cells that form the cellular cementum come from the apical tissue including the apical bud in rodent incisors.     

In vivo characterization of somatodendritic dopamine release in the substantia nigra of 6-hydroxydopamine-lesioned rats

We investigated the effect of an injection of 6-hydroxydopamine (6-OHDA) into the rat medial forebrain bundle (MFB) on the degeneration and the function of the dopaminergic cell bodies in the substantia nigra (SN) 3 and 5 weeks after lesioning. After injection of 6-OHDA into the MFB a complete loss of dopamine content was apparent in the striatum 3 weeks after lesioning. In the SN the amount of tyrosine hydroxylase-immunoreactive dopamine cells decreased gradually, with a near-complete lesion (> 90%) obtained only after 5 weeks, indicating that neurodegeneration of the nigral cells was still ongoing when total dopamine denervation of the striatum had already been achieved. Baseline dialysate and extracellular dopamine levels in the SN, as determined by in vivo microdialysis, were not altered by the lesion. A combination of compensatory changes of the remaining neurones and dopamine originating from the ventral tegmental area may maintain extracellular dopamine at near-normal levels. In both intact and lesioned rats, the somatodendritic release was about 60% tetrodotoxin (TTX) dependent. Possibly two pools contribute to the basal dopamine levels in the SN: a fast sodium channel-dependent portion and a TTX-insensitive one originating from diffusion of dopamine. Amphetamine-evoked dopamine release and release after injection of the selective dopamine reuptake blocker GBR 12909 were attenuated after a near-complete denervation of the SN (5 weeks after lesioning). So, despite a 90% dopamine cell loss in the SN 5 weeks after an MFB lesion, extracellular dopamine levels in the SN are kept at near-normal levels. However, the response to a pharmacological challenge is severely disrupted.     

骨髓间充质干细胞黑质内移植对帕金森病大鼠的治疗作用

目的探索骨髓间充质干细胞（BMSCs）移植到帕金森病（Parkinson’s disease,PD）大鼠毁损侧黑质内,PD模型大鼠的姿势不对称性和黑质及纹状体内酪氨酸羟化酶（tyrosinehy droxylase,TH）表达的改变,以及BM—SCs在大鼠脑内的存活、分化情况。方法黑质、前脑内侧束两点法注射6一羟多巴胺（6-OHDH）并行为学分析筛选PD模型大鼠。将PD模型大鼠随机分为移植组和对照组。BMSCs移植术后4周和8周,观察大鼠姿势不对称性,免疫组织化学及免疫荧光显色方法检测黑质和纹状体酪氨酸羟化酶（tyrosine hydroxylase,TH）的表达变化以及BMSCs在大鼠体内的存活、迁移及分化情况。结果BMSCs黑质内移植可使PD模型大鼠的转动频率由（10．62±2．97）r／min降至（4．65±1．08）r／min（P〈0．01）,显著增加毁损侧黑质TH阳性细胞数量和纹状体内TH阳性纤维密度。BMSCs在大鼠黑质内可以存活至少8周,部分细胞分化为神经干细胞、神经元和神经胶质细胞。结论黑质内移植BMSCs对PD模型大鼠有一定的治疗作用。     

Transplantation of Melanocytes Obtained from the Skin Ameliorates Apomorphine-Induced Abnormal Behavior in Rodent Hemi-Parkinsonian Models

Tyrosinase, which catalyzes both the hydroxylation of tyrosine and consequent oxidation of L-DOPA to form melanin in melanocytes, is also expressed in the brain, and oxidizes L-DOPA and dopamine. Replacement of dopamine synthesis by tyrosinase was reported in tyrosine hydroxylase null mice. To examine the potential benefits of autograft cell transplantation for patients with Parkinson’s disease, tyrosinase-producing cells including melanocytes, were transplanted into the striatum of hemi-parkinsonian model rats or mice lesioned with 6-hydroxydopamine. Marked improvement in apomorphine-induced rotation was noted at day 40 after intrastriatal melanoma cell transplantation. Transplantation of tyrosinase cDNA-transfected hepatoma cells, which constitutively produce L-DOPA, resulted in marked amelioration of the asymmetric apomorphine-induced rotation in hemi-parkinsonian mice and the effect was present up to 2 months. Moreover, parkinsonian mice transplanted with melanocytes from the back skin of black newborn mice, but not from albino mice, showed marked improvement in the apomorphine-induced rotation behavior up to 3 months after the transplantation. Dopamine-positive signals were seen around the surviving transplants in these experiments. Taken together with previous studies showing dopamine synthesis and metabolism by tyrosinase, these results highlight therapeutic potential of intrastriatal autograft cell transplantation of melanocytes in patients with Parkinson’s disease.     

Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules

We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (p<0.05). The APA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.     

Viability and protein secretion from human Hepatoma (HepG2) cells encapsulated in 400-mum polyacrylate microcapsules by submerged nozzle-liquid jet extrusion

An interfacial precipitation process to encapsulate mammalian cells in hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) microcapsules of approximately 750 in approximately m diameter was previously described. It was not possible to produce smaller capsules due to low shearing force. A new droplet generation scheme was developed by suspending the cell and polymer co-extrusion nozzle in a uniform co-axial fluid jet which enabled the production of 300 to 600-microm diameter capsules. HepG2 hepatoma cells in 400-microm-diameter HEMA-MMA capsules were able to retain their metabolic activity during and after the encapsulation process. The in vitro secretion of plasma proteins alpha(1)-acid glycoprotein, alpha(1)-antitrypsin, and fibrinogen by the encapsulated cells was retained. The encapsulated cells secreted less fibrinogen (340 kD) relative to alpha(1)-acid glycoprotein (42kD), indicating the sieving effect (but not absolute cut-off) of the HEMA-MMA membrane. (c) 1994 John Wiley & Sons, Inc.     

Immobilization of mesenchymal stem cells and monocytes in biocompatible microcapsules to cell therapy

The aim of cell therapy is to replace, repair, or enhance the function of damaged tissues or organs. Several factors complicate the development of cellular therapies. Of primary importance is protection of the implanted cells from the host's immune system. Cells are encapsulated in selectively semipermeable and biocompatible membranes that block entry of immune mediators but allow outward diffusion of active molecules produced by the cells. The immobilization of mesenchymal stem cells and monocytes, in micrometric (30-60 microm) alginate-barium microcapsules based on atomization processes, has been achieved successfully. This size is necessary to the administration of microcapsules via injection (Hamilton syringe with a needle size of 100 microm) and aerosol. Microencapsulated cells survive at least 2 weeks after preparation in vitro.     

Behavioural effects of genetically engineered cells releasing dopa and dopamine after intracerebral grafting in a rat model of Parkinson's disease.

The relative importance of synaptic versus paracrine dopamine transmission for the occurrence of functional effects following intrastriatal grafting is not fully established. In the present study we grafted cell lines, expressing the form I of human tyrosine hydroxylase after infection with a recombinant retrovirus and selection in tyrosine-free-medium, to the denervated striatum in order to analyse the extent to which extracellular dopamine levels can be restored and the effect of a diffuse release of dopamine on motor impairement in a rat model of Parkinson's disease. In petri dish, the modified fibroblast cells (NIH.3T3) release DOPA constitutively whereas the modified endocrine cells (RIN) store and release dopamine in a regulated way. Interestingly, in denervated striatum, grafts of modified fibroblast cells produce DOPA which was efficiently converted into dopamine by the host striatal tissue. In the grafted striatum, both fibroblast and endocrine cells restore subnormal levels of diffuse release of dopamine which is notably unaffected and stimulated, respectively, by high concentration of potassium, in connection with the in vitro properties of the grafted cells. The intrastriatal grafts of modified cells partially reversed the apomorphine-induced but not the amphetamine-induced motor asymmetry. We discuss the implications of these results in the context of Parkinson disease.     

Factors Influencing the Differentiation of Dopaminergic Traits in Transplanted Neural Stem Cells

1. Our previous studies demonstrated that when neural stem cells (NSCs) of the C17.2 clonal line are transplanted into the intact or 6-hydroxydopamine (6-OHDA) lesioned rat striatum, in most, but not all grafts, cells spontaneously express the dopamine (DA) biosynthetic enzymes, tyrosine hydroxylase (TH), and aromatic L-amino acid decarboxylase (Yang, M., Stull, N. D., Snyder, E. Y., Berk, M. A., and Iacovitti, L. (2002). Exp. Neurol.).2. These results suggested that there were certain conditions which were more conducive to the development of DA traits in NSCs and possibly other neurotransmitter phenotypes.3. In the present study, we modified a number of variables in vitro (i.e. passage number, confluence) and/or in vivo (degree, type, and site of injury) before assessing the survival, migration, and differentiation of engrafted NSCs.4. We found that low confluence cultures were comprised exclusively of flattened polygonal cells, which when transplanted, migrated widely in the brain but did not express TH.5. In contrast, high confluence cultures contained both polygonal cells and an overlying bed of fusiform cells.6. When these NSCs were maintained for 12–20 passages and then transplanted, virtually all engrafted cells in 65% of the grafts expressed TH but not markers of other neurotransmitter systems.7. Importantly, all TH⁺ grafts were accompanied by significant physical damage to the brain while TH^– grafts were not, suggesting that local injury-related factors were also important.8. Of no apparent influence on TH expression, regardless of how cells were grown prior to implantation, was the site of transplantation (cortex or striatum) or the degree of chemical lesion (intact, partial or full).9. We conclude that transplanted NSCs can express traits specifically associated with DA neurons but only when cells are grown under certain conditions in vitro and then transplanted in proximity to injury-induced factors present in vivo.     

Characterisation of the Xenogeneic Immune Response to Microencapsulated Fetal Pig Islet-Like Cell Clusters Transplanted into Immunocompetent C57BL/6 Mice

Xenotransplantation of microencapsulated fetal pig islet-like cell clusters (FP ICCs) offers a potential cellular therapy for type 1 diabetes. Although microcapsules prevent direct contact of the host immune system with the xenografted tissue, poor graft survival is still an issue. This study aimed to characterise the nature of the host immune cells present on the engrafted microcapsules and effects on encapsulated FP ICCs that were transplanted into immunocompetent mice. Encapsulated FP ICCs were transplanted into the peritoneal cavity of C57BL/6 mice. Grafts retrieved at days 1, 3, 7, 14 and 21 post-transplantation were analysed for pericapsular fibrotic overgrowth (PFO), cell viability, intragraft porcine gene expression, macrophages, myofibroblasts and intraperitoneal murine cytokines. Graft function was assessed ex vivo by insulin secretion studies. Xenogeneic immune response to encapsulated FP ICCs was associated with enhanced intragraft mRNA expression of porcine antigens MIP-1α, IL-8, HMGB1 and HSP90 seen within the first two weeks post-transplantation. This was associated with the recruitment of host macrophages, infiltration of myofibroblasts and collagen deposition leading to PFO which was evident from day 7 post-transplantation. This was accompanied by a decrease in cell viability and loss of FP ICC architecture. The only pro-inflammatory cytokine detected in the murine peritoneal flushing was TNF-α with levels peaking at day 7 post transplantation. This correlated with the onset of PFO at day 7 implying activated macrophages as its source. The anti-inflammatory cytokines detected were IL-5 and IL-4 with levels peaking at days 1 and 7, respectively. Porcine C-peptide was undetectable at all time points post-transplantation. PFO was absent and murine intraperitoneal cytokines were undetectable when empty microcapsules were transplanted. In conclusion, this study demonstrated that the macrophages are direct effectors of the xenogeneic immune response to encapsulated FP ICCs leading to PFO mediated by a combination of both pro- and anti-inflammatory cytokines.