Biomedical subjects

Joseph Yang

Publications and source records attributed to Joseph Yang.

11 recordsLinked to original sources

PubMed2006

Transplantable retinal pigment epithelial cell sheets for tissue engineering.

The native retinal pigment epithelium (RPE) exists as a monolayer structure and is critically involved in the maintenance of photoreceptors. Damage or destruction of the RPE due to a variety of diseases therefore often results in loss of vision. With regenerative purposes in mind, we have examined various culture conditions such as the initial cell density and the addition of various supplements in an effort to produce transplantable RPE cell sheets that can be harvested without defects. We demonstrate that the cell density in cultured RPE sheets increased linearly with the number of seeded cells and that RPE sheets were harvested without defects and limited contraction due to cytoskeletal reorganization, when TGF-beta2 was added to the growth medium. Results from histological analysis and the measurement of trans-epithelial resistance also demonstrates that these RPE cell sheets exist as monolayer structure, similar to the native RPE, with intact cell-to-cell junctions. Therefore, these methods provide significant insight into the fabrication of transplantable RPE cell sheets that can be applied to RPE regenerative therapies to restore lost vision.

Cell Count↗

PubMed2006

Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues.

Recently, the field of tissue engineering has progressed rapidly, but poor vascularization remains a major obstacle in bioengineering cell-dense tissues, limiting the viable size of constructs due to hypoxia, nutrient insufficiency, and waste accumulation. Therefore, new technologies for fabricating functional tissues with a well-organized vasculature are required. In the present study, neonatal rat cardiomyocytes were harvested as intact sheets from temperature-responsive culture dishes and stacked into cell-dense myocardial tissues. However, the thickness limit for layered cell sheets in subcutaneous tissue was approximately 80 microm (3 layers). To overcome this limitation, repeated transplantation of triple-layer grafts was performed at 1, 2, or 3 day intervals. The two overlaid grafts completely synchronized and the whole tissues survived without necrosis in the 1 or 2 day interval cases. Multistep transplantation also created approximately 1 mm thick myocardium with a well-organized microvascular network. Furthermore, functional multilayer grafts fabricated over a surgically connectable artery and vein revealed complete graft perfusion via the vessels and ectopic transplantation of the grafts was successfully performed using direct vessel anastomoses. These cultured cell sheet integration methods overcome long-standing barriers to producing thick, vascularized tissues, revealing a possible solution for the clinical repair of various damaged organs, including the impaired myocardium.

Animals↗

PubMed2006

Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze.

PURPOSE: To apply tissue-engineered cell sheet transplantation after excimer laser keratectomy as a novel approach for the reduction of postoperative corneal haze. METHODS: Limbal biopsy specimens were obtained, and epithelial cells were cultured on temperature-responsive culture inserts without the use of feeder cells. Laser keratectomy (7.0-mm ablation zone and 160-microm depth) was performed in the contralateral eye, and autologous epithelial cell sheets were transplanted to the ablated corneal stroma. Transplant and control group eyes were assessed by slit lamp biomicroscopy, and corneal haze was scored in a masked fashion, according to the Fantes grading scale. For further examination histologic and immunohistochemical analyses were performed. RESULTS: Tissue-engineered cell sheets produced stable attachment to the laser-ablated sites, resulting in epithelialization, 5 minutes after transplantation. Conversely, control corneas required 3 to 5 days for complete re-epithelialization. At both 1 and 2 months after surgery, corneal haze was significantly inhibited in the transplant group. Histologic analyses showed that the number of keratocytes undergoing apoptosis was decreased in the transplant group at 3 days after surgery. Similarly, the expression of both collagen III and alpha-smooth muscle actin, which may enhance corneal haze, were diminished in the transplant group at 2 months. CONCLUSIONS: The transplantation of tissue-engineered epithelial cell sheets can successfully prevent the development of corneal haze after excimer laser keratectomy.

Actins↗

PubMed2005

Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces.

This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.

Cell Culture Techniques↗

PubMed2005

p57Kip2 is expressed in quiescent mouse bone marrow side population cells.

Hematopoietic stem cells can be accurately identified by the side population (SP) phenotype. It has been previously shown that hematopoietic stem cells are cell cycle arrested, but the mechanisms involved are currently poorly understood. In the present study, results from quantitative real-time RT-PCR show that while SP cells have increased expression of various cyclins and cyclin-dependent kinases, the increased expression of cyclin-dependent kinase inhibitors, in particular p57(Kip2), is responsible for the observed cell cycle arrest. In addition, gene expression analysis of c-kit(+/)/Sca-1(+)/Lineage- SP (KSL-SP) cells demonstrates that only p57(Kip2) shows both higher expression compared to both SP and non-SP cells. Furthermore, immunostaining also demonstrates significantly higher protein expression in KSL-SP cells. These results demonstrate that the maintenance of bone marrow SP cells in G0/G1 may be carefully controlled by p57(Kip2).

Animals↗

PubMed2005

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea.

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2005

Limbal epithelial side-population cells have stem cell-like properties, including quiescent state.

Corneal epithelial (CE) stem cells are believed to reside in the basal layer of the limbal epithelium but remain poorly understood due to the lack of an accepted in vivo reconstitution assay as well as definitive markers for epithelial stem cells. It has been reported that side-population (SP) cells with the ability to efflux the DNA-binding dye Hoechst 33342 have stem cell-like properties and that the SP phenotype accurately represents a quiescent and immature stem cell population in the adult bone marrow. In the present study, we investigated whether SP cells isolated from the limbal epithelium have stem cell-like properties. SP cells, separated by fluorescence-activated cell sorting, comprise approximately 0.4% of all limbal epithelial cells and have markedly higher expression of the stem cell markers ABCG2, Bmi-1, and nestin but no expression of markers for differentiated CE cells compared with non-SP cells. Cell-cycle and telomerase activity analyses revealed that SP cells are growth arrested and reside in the quiescent state. Moreover, limbal epithelial SP cells did not demonstrate proliferative capabilities under typical in vitro epithelial cell culture conditions using 3T3 feeder layers. These findings present the possibility that quiescent limbal epithelial SP cells may represent an extremely immature stem cell population compared with currently defined epithelial stem or progenitor cells.

Animals↗

PubMed2005

Cell sheet engineering: recreating tissues without biodegradable scaffolds.

While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field's progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive dishes, cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds.

Animals↗

PubMed2004

Improved planar amperometric nitric oxide sensor based on platinized platinum anode. 2. Direct real-time measurement of NO generated from porcine kidney slices in the presence of l-arginine, l-arginine polymers, and protamine.

Nitric oxide generation from porcine kidney slices is assessed using a new planar NO-selective amperometric sensor. The planar shape of the sensor allows for direct NO measurements near the surface (10 microm) of renal tissue slices in real time. Renal NO production may be modulated by the addition of L-arginine, arginine homopolymers (R2, R6, R10), and protamine, all of which can potentially transport across cellular membranes and provide a substrate for nitric oxide synthase within kidney parenchyma. Real-time amperometric measurements demonstrate that most L-arginine species can translocate across the cell membrane and rapidly increase NO production. However, no increase in NO generation is observed when the dimer of L-arginine (R2) is added to the solution bathing the tissue, suggesting that this species cannot permeate cell membranes. The degree of enhancement in NO generation observed for L-arginine and the larger peptides depends on the structure and follows the following sequence: R10 (decamer) > protamine > R6 (hexamer) > L-arginine. Protamine and the R10 decamer, especially, induce the largest increases in NO generation owing to their apparent rapid translocation into cells and subsequent cleavage by proteases to create high intracellular levels of L-arginine. The effect of sensor size (for sensor dimensions of 0.15- and 1-mm outer diameters) on the measured surface NO levels is also examined. The larger sensor traps more NO but hinders access of the L-arginine species to the tissue area between the flat distal plane of the sensor and the surface of the kidney slice. The use of such NO-generating peptides may be important in numerous biological systems that depend on NO production, such as ischemia-reperfusion injury and thrombogenesis.

Animals↗

PubMed2004

Deletion of the GPI pre-anchor sequence in human p97--a general approach for generating the soluble form of GPI-linked proteins.

Melanotransferrin, also named p97, belongs to the transferrin-like group of iron-binding proteins. Unlike the other members of this family, p97 exists in two forms-one soluble form and one attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The GPI-linked form plays a role in the uptake of iron, while the soluble form of p97 has the unique ability of traversing the blood-brain barrier and may be utilized to deliver drug conjugates into the brain. To investigate these possibilities, a recombinant soluble form of p97 from the GPI-linked p97 protein is required. The approach involved sequential deletions of the p97 GPI pre-anchor sequence (PAS) up to the putative site of cleavage/attachment, releasing p97 from attachment to the GPI-anchor and rendering it soluble. Transfection of the p97 deletion constructs into both the CHO and BHK TK(-) cells was performed with the aim of optimizing the production of p97 by utilizing the cell characteristics unique to each cell line. Altering the GPI PAS resulted in the generation of a recombinant soluble form that was secreted at significantly higher rates than from the full-length expressing cell lines. Increases were from 22 x 10(-9) to 241 x 10(-9)microg/cell/h for expression in the CHO cell system and from 220 x 10(-9) to 4970 x 10(-9)microg/cell/h for the BHK system. Furthermore, there appeared to be differences in the secretion rates between the various deletions suggesting the need for closer examination of the C-terminus in achieving maximum production of the altered proteins. The results of this study are likely applicable for expressing soluble forms of other GPI-linked proteins.

Amino Acid Sequence↗

PubMed2002

The human melanoma associated protein melanotransferrin promotes endothelial cell migration and angiogenesis in vivo.

Melanotransferrin is a member of the transferrin family, which is comprised of serum transferrin, lactoferrin and ovotransferrin, and is highly expressed on melanoma cells compared to normal melanocytes. Since melanoma is an highly vascularized tumour that expresses melanotransferrin at high levels, we tested purified recombinant melanotransferrin for its capability to induce angiogenesis in the chick chorioallantoic membrane. Macroscopic and microscopic evaluation of the vascular density demonstrated that melanotransferrin exerts an angiogenic response quantitatively similar to that elicited by fibroblast growth factor-2. Overexpression of vascular endothelial growth factor-receptor-2 was observed in newly formed vessels, suggesting that the angiogenic activity of melanotransferrin may depend on activation of endogenous vascular endothelial growth factor. In addition, when antibodies against vascular endothelial growth factor were included in the assay, the angiogenic response was inhibited by 50%. In a Boyden chamber assay purified recombinant melanotransferrin induced chemotactic migration of vascular cells, which was decreased in the the presence of anti-vascular endothelial growth factor antibodies suggesting an involvement of vascular endothelial growth factor present in endothelial cells also in this assay. However, melanotransferrin was found not to directly bind to integrin alphavbeta3 or the vascular endothelial growth factor-receptor-2 as assessed in a BlAcore assay. A possible correlation between vascularization occurring during melanoma progression and the expression of melanotransferrin and vascular endothelial growth factor was established by immunolocalization of the two factors in sections of melanoma at different clinical steps of melanoma progression. These latter data strongly imply that melanotransferrin may participate in the vascularization of solid tumours and that inhibition of melanotransferrin could form the basis for intervention in tumours which use this pathway.

Adult↗