PubMed · 14257969
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R MAHON, J SOUMIREU, G BRUN. 1964. [AMNIOTIC EMBOLISM].. https://pubmed.ncbi.nlm.nih.gov/14257969/
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PURPOSE: To examine the feasibility of using sterilized, freeze-dried amniotic membrane (FD-AM) as a substrate for cultivating autologous corneal epithelial cells for ocular surface reconstruction. METHODS: Human AM deprived of amniotic epithelial cells by incubation with EDTA was freeze dried, vacuum packed, and sterilized with gamma-irradiation. The resultant FD-AM was characterized for its physical, biological, and morphologic properties by stretch stress tests, immunohistochemistry, electron microscopy, and cell culture. In addition, 3 weeks after an ocular surface injury, the conjunctivalized corneal surfaces of eyes in eight rabbits were surgically reconstructed by transplantation of autologous cultivated corneal epithelial cells on FD-AM. RESULTS: A stretch stress test revealed no significant differences between sterilized FD-AM and cryopreserved AM. Immunohistochemistry for several extracellular matrix molecules and electron microscopic analysis of FD-AM revealed that the process of drying and irradiation did not affect its biological and morphologic properties. The corneal epithelial cells cultivated on FD-AM had four to five stratified, well-differentiated cell layers. Corneas that were grafted with the cultivated corneal epithelial cells on FD-AM were clear and were all epithelialized at 10 days after surgery. CONCLUSIONS: The sterilized, freeze-dried AM retained most of the physical, biological, and morphologic characteristics of cryopreserved AM; consequently, it is a useful biomaterial for ocular surface reconstruction.
PURPOSE: To investigate the phenotype of fetal and adult human limbal cells cultured on human amniotic membrane and the ability of cultured adult human limbal cells to repair limbal stem cell deficiency in a rabbit model. METHODS: Human adult and fetal limbal cells were isolated and cultured either on plastic plates or on human amniotic membrane. Connexin43, p63, and keratins 3 and 12 (K3 and K12) were detected by immunofluorescence and RT-PCR. Limbal stem cell deficiency was established in rabbits using chemical ablation and mechanical debridement. Cultured adult human limbal cells were transplanted onto rabbit corneas one month after injury, then fixed and imbedded in paraffin forty days later. Immunofluorescent staining of human-nuclear antigen, p63, K3, and connexin43 identified human-specific cells, progenitor cells, and differentiated corneal epithelial cells, respectively. RESULTS: Adult and fetal cultured limbal cells appeared similar in morphology. RT-PCR results showed that cells cultured from the human adult and fetal limbal area expressed both p63 and K12, whereas cells from central adult epithelium expressed K12 only. Immunofluorescent staining showed that more cells were p63 positive when cultured on human amniotic membrane than on plastic. Double staining for p63 and connexin43 showed some p63-positive cells co-expressing connexin43. After transplantation of adult human limbal cells cultured on human amniotic membrane, injured rabbit corneas were completely reconstructed exhibiting epithelial integrity, improved corneal clarity, and little or no neovascularization. The majority of repopulated epithelial cells expressed anti-human nuclear antibody. Cells expressing p63 occurred throughout the new epithelium. CONCLUSIONS: During healing, expression of p63 is not limited to epithelial stem cells but may also mark transient amplifying progenitor cells. Culture on human amniotic membrane suppresses differentiation of limbal epithelial cells and promotes the proliferation of p63 expressing cells. Amniotic membrane-cultured human limbal cells fully reconstructed rabbit corneas having limbal stem cell deficiency, with human cells providing most of the cells of the new epithelium. Expression p63 is distributed throughout the reconstructed tissue.
We recently found that human amniotic epithelial (HAE) cells secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on dopamine (DA) neurons. The present study explored whether implantation of HAE cells can be a possible means to deliver trophic factors into the brain to prevent the death of DA neurons in a rat model of Parkinson's disease. We first investigated the ability of HAE cells to produce factors capable of promoting DA cell survival in vitro, and then tested whether HAE cell grafts survive and prevent the death of nigral DA neurons in rats with 6-hydroxydopamine lesions. A treatment with conditioned medium derived from HAE cell cultures enhanced the survival of tyrosine hydroxylase (TH)-immunopositive DA cells in serum-free cultures. The conditioned medium also protected the morphological integrity of TH-positive neurons against toxic insult with 6-hydroxydopamine. HAE cells were grafted into the midbrain of immunosuppressed rats. The rats were then subjected to a unilateral nigrostriatal lesion induced by intrastriatal infusions of 6-hydroxydopamine. HAE cell transplants were found to survive without evidence for overgrowth 2 weeks postgrafting. The number of nigral DA cells, detected with either TH-immunohistochemistry or retrograde labelling with fluorogold, was significantly increased in rats given the grafts as compared to that in control animals without the grafts. The results indicate that HAE cells produce diffusible molecules that can enhance the survival of DA neurons. Although the factors that contribute to the currently observed effects remain to be fully determined, implantation of HAE cells could be a viable strategy to counteract the loss of DA neurons in Parkinson's disease.