Experimental Studies On The Striatal Xenotransplantation Of Microencapsulated Porcine Iris Pigment Epithelial Cells Into Parkinsonian Rats

Parkinson's disease (PD) is characterized by the degeneration of thedopaminergic neurons in the substantia nigra (SN). Cell transplantation is consideredas a promising treatment of PD. However, the effort of looking for an appropriate typeof donor cell has not been successful yet.Although RPE cells transplantation has been identified as effective for PDtreatment, the difficulty in the isolation of them has become another barricade. Irispigment epithelium (IPE) cells are anatomically continuous with ciliary epithelialcells. As they are arrayed in a simple epithelium manner, the isolation of them can beachieved in a minimal invasive fashion and is far easier than that of RPE cells. Wehereby identified that IPE cells are similar to RPE cells as for the capability ofdoparmine (DA) and neurotrophic factors secretion. Besieds, IPE cells are shown tohave iron-binding capacity, which can be helpful for PD treatment. In conclusion, ourstudy indicated that IPE cells could probably be the right donor cells fortransplantation therapies for PD.Alginate-polylysine-alginate (APA) is a kind of biologically compatiblesemipermeable membrane material, the micro-encapsulation with which can reliefimmune rejection in cell transplantation. The present study aimed to: 1. define thebiological characteristics of cultured porcine IPE cells and observe changes of themunder illumination; 2. encapsulate IPE cells with APA and observe possible changesof the biological characteristics. 3. Examine the therapeutic effects ofAPA-micro-encapsulated porcine IPE cells transplantation in the striatum of unilateralPD rats.PartⅠPrimary culture and identification of porcine IPE cells in vitroObjective: To set up a series of methods for primary culture, purification andidentification of IPE cells. Methods: Porcine IPE cells were gained by enzymedigestion, purified and transferred. Their morphologic changes and growth curveswere recorded for initial identification. Further confirmation was achieved by immuno-cyto-chemical staining with specific IPE cell markers, S-100 and cytokeratin.Purity of IPE cells was measured. Results: Positive rate of porcine IPE cells was high.The growth curve showed that the exponential growth period of IPE cells is thesecond and third day after passage, while the platform stage is the following daysafter it.PartⅡStudy on the biological characteristics of cultured porcine IPE cellsObjective: To study the biological characteristics of cultured porcine IPE cells andfind out whether they can secrete DA, neurotrophic factors, and bind with ferri ions ornot and to detect whether they can express TH on mRNA and protein levels. Methods:The DA concentration was determined by high performance liquid chromatographyelectrochemical (HPLC) assay in the supernatants collected 48 hours after IPE cellpassage. BDNF, GDNF, NGF and NT-3 concentration were figured out with ELISAand iron-binding capacity was measured with CIJI Kit. RT-PCR andimmuno-cyto-chemical staining were employed to measure expression levels of TH.Results: DA was first detected at 4 hours after passage and arrived at the peakconcentration at 48 hour after passage. The levels of BDNF, GDNF, NGF, NT-3 andiron-binding capacity were constant. Illumination increased DA secretion but did notchange secretion of the other substances. Small quantity of TH mRNA and TH proteinwere detected in porcine IPE cells.PartⅢConstruction of APA microcapsules and bioactivity changes of micro-encapsulated IPE cellsObjective: To improve microcapsule preparation conditions for porcine IPE cells andfind out changes in cell growth and substances secretion after encapsulation. Methods:APA-microencapsulated cells were constructed with a high voltage electostatic system.Optimal parameters at a fixed temperature were figured out, i.e. concentration of thealginate, distance from liquid surface and microencapsulated voltage. The shape,diameter, pressure tolerance capacity of the microcapsule and typan blue stainingdetermined cell survival rate were recorded at the 0, 3rd, 6th, 9th and 12th days undermicroscope. The concentrations of DA, BDNF, GDNE NGE NT-3 and theiron-binding capacity were recorded prior to and after microencapsulation. Results:The optimal concentration of alginate was 2.4％, corresponding distance from liquid surface 22-25mm and voltage 3.28kv. The microcapsules were near round withsmooth surface and the size is uniform. The average diameter is 275±23μm. Thenumber of microencapsulated cells remains constant and shapes remain un-affectedafter aspiration with syringe. DA secretion increased in the first 60h, after which timeno significant difference with non-microcapsule group was found. The concentrationsof BDNF, GDNF, NGF, NT-3 and iron-binding capacity showed no differencebetween the two groups.PartⅣEstablishment of rat PD model and treatment with micro-encapsulated porcine IPE cells transplantationObjective: To set up the stable 6-OHDA-lesioned PD rat model and reliableethological evaluation index and examine the therapeutic effect of micro-encapsulatedporcine IPE cells transplantation. Methods: 1, Parkinsonian rat models were madethrough unilateral 6-hydroxydopamine (6-OHDA)-lesions of medial forebrain bundle(MFB). Rotation test scores, immunohistochemistry and the other ethological indexwere used to evaluate the rat models. 2, Rats were randomly divided into three groups:microencapsulated IPE cells group, microcapsule group and physiologic saline goup,which were respectively injected with 2*10~4 IPE cells, microcapsule only and NS inunilateral striatum. 3, The apomorphine-induced rotational and the other behaviors ofrats were observed; expressions of tyrosine hydroxylase in nigral cells wereinvestigated by immunohistochemistry; contents of DA and homovanillic acid (HVA)were determined by HPLC. Changes of protein around transplantation region wereobserved through electrophoresis. Results: 1, The 6-OHDA-lesioned PD rat modelswere identified applicable considering ethological and pathological features. 2,Adjusting steps, stepping time, step length and initiation time are valuable inevaluation of this PD rat model. 3, 33％rats(10 rats) of microencapsulated IPE cellsgroup showed behavior improving 1 weeks after transplantation (compared to othergroups, P＜0.05). The improvement became more significant at the 4th week and last tothe 12th week. Rats with no behavior improvement in the microencapsulated IPE cellsgroup accepted a second injection, after which 20％rats(4 rats) showed behaviorimprovement. All in all, almost 47％rats(14 rats) showed improvement aftertransplantation. The changes of DA and HVA concentration in each group wereconsistent with changes of behavior. 4, Upon histological examination, a large number of microcapsules with porcine IPE cells were found distorted but remained intact inthe striatum. Density of TH positive fibers in the striatum near microcapsules washigher than that in the other groups. 5, Proteins around the transplantation region hadno difference with the opposite side. 6, One of the microencapsulated-IPE-cellstransplanted PD rats showed severe torsion-spasm 2 weeks after transplantation.Further investigation with histological examination showed tumor in thetransplantation site.Conclusions1. Porcine IPE cells grow actively in vitro and are easy to be purified and identified.2. Porcine IPE cells can secrete DA, BDNF, GDNF, NGF and NT-3 under basalcondition, express TH mRNA and protein and have iron binding capacity.Illumination increases DA secretion but do not influence the other secretions.3. Micro-encapsulation does not affect the shape, diameter, pressure tolerance, cellnumber, secretions of DA, BDNF, GDNF, NGF and NT-3 and iron bindingcapacity of porcine IPE cells.4. Adjusting steps, stepping time, step length and initiation time are valuable in theevaluation of 6-OHDA-lesioned of MFB PD rat model.5. The micro-encapsulated-IPE-cells transplantation has significant therapeuticeffect on PD rats, and the effect lasts for at least 12 weeks. Transplantation doesnot affect protein secretion spectrum in the corresponding site. However, how toavoid tumor evocation might be a problem needing further investigation.