Norio Sakuragawa

Human Amniotic Epithelial Cells Produce Dopamine and Survive after Implantation into the Striatum of a Rat Model of Parkinson's Disease: A Potential Source of Donor for Transplantation Therapy

We have recently found that human amniotic epithelial (HAE) cells synthesize catecholamines including dopamine (DA). The present study was designed to explore the possibility of HAE cells to serve as a donor for transplantation therapy of Parkinsons disease (PD). Thus, we investigated their ability to produce DA in vitro and the survival and function of HAE cells grafted into a rat model of PD. RT-PCR and Western blotting revealed that HAE cells express tyrosine hydroxylase (TH) mRNA and protein, respectively. TH-immunohistochemistry on cultured HAE cells demonstrated that around 10% of the total cells are immunopositive for this protein. The production of DA by HAE cells was increased with time in the presence of L-tyrosine and BH(4), and was abolished with a specific TH inhibitor, alpha-methyl-rho-tyrosine. Dissociated HAE cells transduced with the Escherichia coli LacZ marker gene (beta-gal) were implanted into the previously DA-depleted striatum of immunosuppressed rats. Two weeks postgrafting HAE grafts were demonstrated to survive without overgrowth, as evidenced by the presence of beta-gal-positive cells and TH-immunoreactive cells within the grafts. The grafts also provided partial amelioration of apomorphine-induced rotational asymmetry. The results clearly indicate that HAE cells capable of producing DA can survive and function in the brain of a rat model of PD. Although DA replacement therapy of PD could possibly be achieved with implantation of HAE cells, further studies are needed to develop strategies to enhance the ability of HAE cells to produce DA as well as the graft survival.

Expression of markers for both neuronal and glial cells in human amniotic epithelial cells

Human amniotic epithelial (HAE) cells are formed from amnioblasts, separated from the epiblast at about the 8th day after fertilization. We attempted to detect various developmental antigens specific to neural cells by immunocytochemical methods. The cultured HAE cells displayed positive immunoreactivity to RC1, vimentin, A2B5, neurofilament proteins, microtubule-associated protein 2 (MAP2) and MAP2 kinase. In addition, the cells also demonstrated immunoreactivity to glial fibrillary acidic protein, CNPase, myelin basic protein and galactocerebroside. The appearance rate of positive cells was more than 50% in cells positive to RC1, A2B5, vimentin or neuronal markers, and 20-30% to glial cell markers. Double staining showed the heterogeneous appearance of oligodendrocyte lineage cells. These data indicate that HAE cells may have the putative multipotentiality of neurons, astrocytes and oligodendrocytes.

Neurotrophic function of conditioned medium from human amniotic epithelial cells.

Human amniotic epithelial cells (HAEC) may have pluripotent function because they are formed from the epiblast cells at the 8th day of fertilization. Previously, we reported that HAEC have the capacity to synthesize and release acetylcholine and catecholamine associated with the binding sites of catecholamine receptors. We show the neurotrophic function of a conditioned medium from HAEC using cultured cortical neurons of E18 rats. Extensive analyses with various techniques demonstrated that HAEC and immortalized HAEC synthesize and release brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF). Other neurotrophic factors were not detected in a cultured medium of HAEC by enzyme immunoassay. Various neurotrophic factors or growth factors did not show neurotrophic effects on E18 rat neuron except for EGF. Because EGF was not detected in the conditioned medium of HAEC, these data indicate an unidentified neurotrophic factor presently that is synthesized and released from HAEC. The amniotic membrane may have a significant role in supplying neurotrophic factors to the amniotic fluid as well as neurotransmitters, suggesting an important function to the early stages of neural development in the embryo. J. Neurosci. Res. 62:585–590, 2000.

Human amnion mesenchyme cells express phenotypes of neuroglial progenitor cells

Previous studies from our laboratory showed that human amnion epithelial cells (AECs) have multiple functions, such as synthesis and release of catecholamines, acetylcholine, neurotrophic factors, activin, and noggin. In this study, we investigated the identity of neural progenitor cells in human amnion mesenchyme cells (AMCs), which lie immediately adjacent to the AECs. Cryostat sections revealed that vimentin expression was detected in the AMCs and CK19 in AECs. Vimentin‐positive cells made up 97.5% of total cells tested in cultured AMCs. Interestingly, 3.6% of total AMCs expressed the phenotype CK19+/vimentin+, indicating coexpression of epithelial and mesenchyme cell markers. In culturing with bromodeoxyuridine (BrdU) for 24 hr, 66–82% of cells were found to be BrdU positive, suggesting that they have proliferating potency. By using RT‐PCR, AMCs express mRNA of nestin and Musashi1. With a neural cell differentiating protocol, cell bodies extended long bipolar or complex multipolar processes. Nestin (87.7% of total cells tested) and Musashi1 (93.1%) were expressed in undifferentiated cells, and their positively stained cells increased in number slightly after induction. Undifferentiated cells were stained by anti‐Tuj1 and NF‐M, and their positively stained cells increased significantly in number after induction, to 72.8% and 46.0%, respectively. Meanwhile, glial fibrillary acidic protein‐positive cells increased from 25.4% to 43.2% after induction. These studies demonstrate that AMCs have phenotypes of neuroglial progenitor cells and can be differentiated into neuroglial phenotypes by optimal differentiation protocol. Eventually, AMC‐derived stem cells may be a favorable cell vehicle in regenerative medicine.

Human amniotic epithelial cells are promising transgene carriers for allogeneic cell transplantation into liver

AbstractAs human amniotic epithelial tissue is formed on about the eighth day after fertilization, human amniotic epithelial cells (hAEC) may have multipotency to differentiate into various organs, such as brain, heart, or liver. In this study, we showed evidence of the synthesis and excretion of albumin by hAEC, by immunostaining and enzyme-linked immunoassay. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses revealed the expression of albumin mRNA and protein, respectively. In addition, hAEC also demonstrated immunoreactivity to genetic markers of liver lineage, such as human serum albumin and α-fetoprotein. Transplanted hAEC to Scid mouse liver showed positive immunoreactivity to albumin and α-fetoprotein. Genetically modified cells containing the β-galactosidase (LacZ) gene (AxCALacZ) were integrated in liver parenchyma. Human polymorphic gene analysis in Scid mouse liver after the implantation of hAEC showed that these Scid mouse livers obviously contained this human-specific gene until day 7 after the cell transplantation. As hAEC do not cause any acute rejection by allotransplantation, we conclude that hAEC may be useful as a transgene carrier to treat patients with inherited liver diseases.

Implantation of human amniotic epithelial cells prevents the degeneration of nigral dopamine neurons in rats with 6-hydroxydopamine lesions

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 Parkinsons 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 Parkinsons disease.

Evidence for active acetylcholine metabolism in human amniotic epithelial cells: applicable to intracerebral allografting for neurologic disease.

Human amniotic epithelial (HAE) cells have been used for allotransplantation in patients with lysosomal storage disease due to lack of expression of HLA antigens. Previously, we have reported the expression of differentiation markers for both neural stem cells, and neuron and glial cells. In the present study, we investigated the presence of choline acetyltransferase (ChAT) and acetylcholine (ACh) in HAE cells using different experimental approaches. Cultured HAE cells showed strong immunoreactivity against ChAT antibody. ChAT activity in primary cells was 24.9 +/- 8.5 pmol/mg protein/h. Using HPLC with electrochemical detection, ACh was detected in both cell incubation media and cell pellets indicating that these cells synthesize and release ACh in a time-dependent manner. Additional confirmation of this hypothesis was gained from the data obtained from RT-PCR and Western blot analyses which revealed the expression of ChAT mRNA and ChAT protein, respectively, in HAE cells. Results of the present study suggest that HAE cells can possibly be applied for intracerebral allografting to treat neurologic diseases in which cholinergic neurons are damaged.

Evidence for synthesis and release of catecholamines by human amniotic epithelial cells.

THE present study investigated the presence, possible synthesis and release of catecholamines (CA) by human amniotic epithelial cells (HAEC) using HPLC with electrochemical detection. The presence of CA was indicated by the detection of norepinephrine (NE), dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in extracts of cultured HAEC. Incubation of HAE cells in medium supplemented with l-tyrosine (CA precursor) and tetrahydrobiopterin (tyrosine hydroxylase cofactor) significantly increased the production of catecholamines, suggesting CA synthesis by HAEC. In contrast, pharmacological inhibition of tyrosine hydroxylase by α-methyl- p-tyrosine (MPT) significantly reduced CA production, further confirming CA synthesis by HAEC. Catecholamines were also detected in the cell incubation media, demonstrating the ability of HAEC to spontaneously secrete CA. Moreover, incubation of cells with 50 mM K+ for 10 min increased the amount of CA released into the medium. Additionally, the detection of DOPAC, a primary metabolite of DA, in HAEC strongly indicates that these cells contain DA metabolizing enzymes. The present results suggest that HAEC synthesize and release CA. These cells may be a possible candidate for transplantation therapy of neurodegenerative diseases such as Parkinsons disease and also may serve as a model to study the aspects of catecholaminergic activity.

Amniotic tissue transplantation: Clinical and biochemical evaluations for some lysosomal storage diseases

Amniotic epithelial cells has been used for transplantation in patients with lysosomal storage diseases as an enzyme replacement therapy. But its clinical effect is still the question under debate. We performed amniotic tissue transplantation on patients with different lysosomal storage diseases: one with Tay-Sachs disease, one with juvenile Gaucher disease and one with juvenile metachromatic leukodystrophy. The patient having juvenile Gaucher disease received this grafting twice. Objective clinical improvement was observed in the first trial where this patient showed an increase of soluble beta-glucosidase one week after implantation. No clinical or biochemical changes were seen in the other patients. Although there are some advantages to amniotic tissue transplantation, original methods should be modified to cell transplantation in order to avoid graft-versus-host reaction which could happen in repeated implantation.

Amniotic epithelial cells transform into neuron-like cells in the ischemic brain.

We investigated the potential use of rat amniotic epithelial (RAE) cells as donor cells for transplantation-based therapy in brain ischemia. In vitro, RAE cells were positive for both neuronal and neural stem cell markers, neurofilament microtubule-associated protein 2 and nestin. RT-PCR revealed that these cells express nestin mRNA. The RAE cells were transplanted into the hippocampus of adult gerbils that were subjected to temporal occlusion of bilateral carotid arteries. Five weeks after transplantation, grafted cells migrated into the CA1 pyramidal layer that showed selective neuronal death, and survived in a manner similar to CA1 pyramidal neurons. These results suggest that intracerebral transplantation of amniotic epithelial cells may have therapeutic potential for the treatment of ischemic damage in neuronal disorders.