bluuebunny/crossref_metadata_2025 huggingface dataset:
Allogeneic versus Xenogeneic Immune Reaction to Bioengineered Skin Grafts
Authors: Erdag Gulsun , Morgan Jeffrey R.
Published: April 2008

There are conflicting reports on the survival and immune reaction to allografts and xenografts of cultured skin substitutes (CSS). In this study, we investigated the allogeneic and xenogeneic responses to CSS of human keratinocytes and genetically engineered CSS expressing keratinocyte growth factor (KGF) that forms a hyperproliferative epidermis. CSS (control and KGF modified) and neonatal human foreskins were evaluated by immunohistochemistry for the expression of MHC class I and II. To study allograft rejection, grafts were transplanted to human peripheral blood mononuclear cell (huPBMC)-reconstituted SCID mice. To study xenograft rejection, grafts were transplanted to immunocompetent mice. Graft survival and immune reaction were assessed visually and microscopically. After transplantation, control CSS formed a normal differentiated epidermis, whereas KGF CSS formed a hyperproliferative epidermis. Control and KGF CSS expressed class I similar to neonatal foreskin, but did not express class II. In the allograft model, rejection of neonatal foreskins was between 5 and 9 days. In contrast, neither control nor KGF CSS was rejected by huPBMC-SCID mice. Histology showed dense mononuclear cell infiltration in human foreskins, with few, if any, mononuclear cells in control or KGF CSS. In contrast to the allogeneic reaction, CSS (control and KGF) were rejected in the xenograft model, but rejection was delayed (9–21 days) compared with neonatal skin (5–8 days). Humanized SCID mice rejected allografts of human neonatal foreskins, but did not reject control CSS or KGF CSS, even though the KGF CSS formed a hyperproliferative epidermis. Rejection of control and KGF CSS by immunocompetent mice in a xenograft model was comparable and their survival was significantly prolonged compared with neonatal skin. These results demonstrate that control CSS and hyperproliferative KGF CSS are less immunogenic than normal human skin and that sustained hyperproliferation of the epidermis does not accelerate rejection.
Methods of Human Islet Culture for Transplantation
Authors: Murdoch T. B. , McGhee-Wilson D. , Shapiro A. M. J. , Lakey J. R. T.
Published: April 2008

The ability to maintain isolated human islet preparations in tissue culture has recently been adopted by most islet transplant centers, and improves the safety as well as the practicality of islet transplantation. Maintaining islet viability and recovery, however, remains challenging in a clinical setting, due to stringent conditions required for culture. Islet culture is further complicated by the fact that islets do not form a monolayer. This review aims to clarify media, supplementation, and conditions that have been shown to be relevant to human islets, as well as to offer avenues of future research. Factors examined that may influence islet survival include base medium, glucose concentration, vitamin, inorganic ion, lipid, hormone, growth factor, amino acid, and binding protein composition and concentration, as well as culture temperature and seeding density. In addition, this article reviews novel techniques, such as coculture and matrices, that have been employed in an attempt to improve islet survival and functional viability.
Photofabricated Gelatin-Based Nerve Conduits: Nerve Tissue Regeneration Potentials
Authors: Gámez Eduardo , Goto Yoshinobu , Nagata Kengo , Iwaki Toru , Sasaki Tomio , Matsuda Takehisa
Published: April 2008

There is a strong demand for development of nerve guide conduit with prompt nerve regeneration potential for injury-induced nerve defect. Prior to study on nerve tissue engineering using Schwann cells or nerve stem cells, the effectiveness of photofabricated scaffolds based on photocurable gelatin was examined. This study describes the evaluation of in vivo nerve tissue regeneration potentials of three custom-designed and -fabricated prostheses (inner diameter, 1.2 mm; outer diameter, 2.4 mm; wall thickness, 0.60 mm; and length, 15 mm) made of photocured gelatin: a plain photocured gelatin tube (model I), a photocured gelatin tube packed with bioactive substances (laminin, fibronectin, and nerve growth factor) coimmobilized in a photocured gelatin rod (model II), and a photocured gelatin tube packed with bioactive substances coimmobilized in multifilament fibers (model III). These prostheses were implanted between the proximal and distal stumps 10 mm of the dissected right sciatic nerve of 70 adult male Lewis rats for up to 1 year. The highest regenerative potentials were found using the model III prosthesis, followed by the model II prosthesis. Markedly retarded neural regeneration was observed using the model I prosthesis. These were evaluated from the viewpoints of functional recovery, electrophysiological responses, and tissue morphological regeneration. The significance of the synergistic cooperative functions of multifilaments, which serve as a platform that provides contact guidance to direct longitudinal cell movement and tissue ingrowth and as a cell adhesive matrix with high surface area, and immobilized bioactive substances, which enhance nerve regeneration via biological stimulation, is discussed.
The Transfer of Ocular Cells Using Collagen
Authors: Yeung Chi-Kong , Chiang Sylvia Wai-Yee , Chan Kwok-Ping , Lam Dennis Shun-Chiu , Pang Chi-Pui
Published: April 2008

The present study was designed to evaluate the use of collagen gel loaded with human retinal pigment epithelium (ARPE19) in cellular transfer and to assess its viability within the gel. Collagen solution was prepared by dissolving calfskin in hydrochloric acid to make a final concentration of 2.0 mg/ml and this was mixed with 10,000 ARPE19 cells/ml. The cell viability in gel was determined using MTT assay. van Gieson stain and proliferating cell nuclear antigen (PCNA) were used to identify the location of collagen and to localize the site of cell proliferation, respectively. The ARPE19 cells in gel appeared to be healthy with a rounded morphology. The optimal collagen concentration was 1.9 mg/ml. When this concentration was used to hold cells for over 12 days, it could be seen that the growth rate was the same between day 2 and day 8 in gel and on plastic. When the cell-loaded gels were transferred onto standard tissue culture plastics, progressive cell migrations over time resembling cell migrations in organotypic explant cultures were observed. Upon intravitreal injection of cell-containing collagen suspension into a rabbit's eye, the gel became suspended within the vitreous a few hours after injection (day 0). However, it became obvious that the gel dispersed and spread around the vitreous even after just 24 h. These cells inside the vitreous were PCNA positive, indicating that the human ARPE19 cells have the capacity to proliferate even after 11 days. The present study demonstrated the potential use of collagen gel as a tool in the transfer of cellular matrix onto other substrates. The results show that the cell seeding number must be critically balanced with the concentration of gel for it to be used as transplant material.
In Vivo Evaluation of Pharmacologically Active Microcarriers Releasing Nerve Growth Factor and Conveying PC12 Cells
Authors: Tatard V. M. , Venier-Julienne M. C. , Benoit J. P. , Menei P. , Montero-Menei C. N.
Published: April 2008

Cell therapy will probably become a major therapeutic strategy in the coming years. Nevertheless, few cells survive transplantation when employed as a treatment for neuronal disorders. To address this problem, we have developed a new tool, the pharmacologically active microcarriers (PAM). PAM are biocompatible and biodegradable microparticles coated with cell adhesion molecules, conveying cells on their surface and presenting a controlled delivery of growth factor. Thus, the combined effect of growth factor and coating influences the transported cells by promoting their survival and differentiation and favoring their integration in the host tissue after their complete degradation. Furthermore, the released factor may also influence the microenvironment. In this study, we evaluated their efficacy using nerve growth factor (NGF)-releasing PAM and PC12 cells, in a Parkinson's disease paradigm. After implantation of NGF-releasing or unloaded PAM conveying PC12 cells, or PC12 cells alone, we studied cell survival, differentiation, and apoptosis, as well as behavior of the treated rats. We observed that the NGF-releasing PAM coated with two synthetic peptides (poly-D-lysine and fibronectin-like) induced PC12 cell differentiation and reduced cell death and proliferation. Moreover, the animals receiving this implant presented an improved amphetamine-induced rotational behavior. These findings indicate that PAM could be a promising strategy for cell therapy of neurological diseases and could be employed in other situations with fetal cell transplants or with stem cells.
Comparison of Fetal Porcine Aggregates of Purified β-Cells versus Islet-Like Cell Clusters as a Treatment of Diabetes
Authors: Tu Jian , Khoury Pauline , Williams Lindy , Tuch Bernard E.
Published: April 2008

Fetal pig islet-like cell clusters (ICCs) have the potential to reverse diabetes 1–5 months after transplantation. In a fetal ICC, however, β-cells constitute only 6–8% of the cells, in contrast to 65% in an adult pig islet. Attempts to purify fetal β-cells from cell clusters and compare their function to that of ICCs have not been shown previously. β-Cells were purified from ICCs isolated from the fetal pig pancreas. These were then aggregated and maintained in culture for 3 days. ICCs were isolated from fetal pig pancreas and allowed to round up in culture for 3 days. Transplantation of aggregates and ICCs (10,000 and 12,600, respectively) into diabetic immunoincompetent mice resulted in normoglycemia at 18 ± 2 and 8 ± 1 weeks, respectively (p = 0.0006). Removal of grafts after normalization of blood glucose levels resulted in rapid return of hyperglycemia in both groups. In conclusion, a purified population of immature β-cells can be produced from the fetal pig pancreas. The reason these cells take longer than ICCs to reverse diabetes when transplanted is postulated to be because of the relative lack of precursor cells from which β-cells differentiate. This finding may have implications for stem cell therapy, as other cell types, other than purified β-cells, may be necessary for appropriate function in vivo.
Studies on the Differentiation of Dopaminergic Traits in Human Neural Progenitor Cells in Vitro and in Vivo
Authors: Yang Ming , Donaldson Angela E. , Marshall Cheryl E. , Shen James , Iacovitti Lorraine
Published: April 2008

The development of cell replacement therapies for the treatment of neurodegenerative disorders such as Parkinson's disease (PD) may depend upon the successful differentiation of human neural stem/progenitor cells into dopamine (DA) neurons. We show here that primary human neural progenitors (HNPs) can be expanded and maintained in culture both as neurospheres (NSPs) and attached monolayers where they develop into neurons and glia. When transplanted into the 6-hydroxydopamine-lesioned rat striatum, undifferentiated NSPs survive longer (60% graft survival at 8–16 weeks vs. 30% graft survival at 8–13 weeks) and migrate farther than their attached counterparts. While both NSP and attached cells continue to express neuronal traits after transplantation, the spontaneous expression of differentiated transmitter-related traits is not observed in either cell type. However, following predifferentiation in culture using a previously described cocktail of reagents, approximately 25% of HNPs can permanently express the DA enzyme tyrosine hydroxylase (TH), even following replating and removal of the DA differentiation cocktail. When these predifferentiated HNPs are transplanted into the brain, however, TH staining is not observed, either because expression is lost or TH-expressing cells preferentially die. Consistent with the latter view is a decrease in total cell survival and migration, and an enhanced glial response in these grafts. In contrast, we found that the overall survival of HNPs is improved when cells engraft near blood vessels or CSF compartments or when they are placed into an intact unlesioned brain, suggesting that there are factors, as yet unidentified, that can better support the development of engrafted HNPs.
Autologous Bone Marrow Stromal Cell Transplantation for Repair of Full-Thickness Articular Cartilage Defects in Human Patellae: Two Case Reports
Authors: Wakitani Shigeyuki , Mitsuoka Tomoki , Nakamura Norimasa , Toritsuka Yukiyoshi , Nakamura Yukio , Horibe Shuji
Published: April 2008

This study assessed the effectiveness of autologous bone marrow stromal cell transplantation for the repair of full-thickness articular cartilage defects in the patellae of a 26-year-old female and a 44-year-old male. These two patients presented in our clinic because their knee pain prevented them from walking normally. After thorough examination, we concluded that the knee pain was due to the injured articular cartilage and decided to repair the defect with bone marrow stromal cell transplantation. Three weeks before transplantation, bone marrow was aspirated from the iliac crest of each patient. After erythrocytes had been removed by use of dextran, the remaining nucleated cells were placed in culture. When the attached cells reached subconfluence, they were passaged to expand in culture. Adherent cells were subsequently collected, embedded in a collagen gel, transplanted into the articular cartilage defect in the patellae, and covered with autologous periosteum. Six months after transplantation, clinical symptoms (pain and walking ability) had improved significantly and the improvement has remained in effect (5 years and 9 months posttransplantation in one case, and 4 years in the other), and both patients have been satisfied with the outcome. As early as 2 months after transplantation, the defects were covered with tissue that showed slight metachromatic staining. Two years after the first and 1 year after the second transplantation, arthroscopy was performed and the defects were repaired with fibrocartilage. Results indicate autologous bone marrow stromal cell transplantation is an effective approach in promoting the repair of articular cartilage defects.
Development of a Hydroxyapatite/Collagen Nanocomposite as a Medical Device
Authors: Itoh Soichiro , Kikuchi Masanori , Koyama Yoshihisa , Takakuda Kazuo , Shinomiya Kenichi , Tanaka Junzo
Published: April 2008

The effect of cross-linking of a hydroxyapatite/collagen (HA/Col) nanocomposite, in which HA nanocrystals and collagen fibers are aligned like natural bone by a self-organization mechanism between HA and collagen in vitro, on mechanical properties was examined. The influence of degree of cross-linking, as well as rhBMP-2 preadsorption to the composite on the substitution pattern and rate with bone, was examined. In Experiment 1, anterior fusion was carried out at the C3–C4 vertebrae on 10 dogs and they were implanted as follows: without cross-linking and without adsorbed rhBMP-2 (three dogs), with cross-linking and without adsorbed rhBMP-2 (three dogs), without cross-linking and with adsorbed rhBMP-2 (two dogs), and with cross-linking and adsorbed rhBMP-2 (two dogs). Implants were removed from each dog for histology determinations after 12, 16, and 24 weeks in the non-rhBMP-treated groups, and after 16 and 24 weeks in the rhBMP-treated groups. In Experiment 2, the HA/Col composites with cross-linking and both with and without rhBMP-2 pretreatment were implanted into a bone defect of 20 mm made in the central part of tibiae in dogs (N = 3 in each group). As a control, bone defects of 20 mm remained without implantation (N = 3). The dogs were allowed to walk using an Ilizarov extra skeletal fixator. The implants were removed after 12, 16, and 24 weeks from one dog in each group. The cross-linking of the HA/Col composite was effective in controlling both the mechanical strength and bioresorbability. A “self-organization process” on the HA/Col implant surface resulted in the formation of bone remodeling units in and around the implant. Radiographic and histological findings suggest that a combined treatment of cross-linking of the HA/Col composite with preadsorption of rhBMP-2 molecules may be a very suitable replacement of existing ceramic systems in the anterior fusion of the cervical spine, as well as inlay grafting of bone defects in weight-bearing sites.
Propagation of Adult Rat Bone Marrow-Derived Hepatocyte-Like Cells by Serial Passages in Vitro
Authors: Miyazaki Masahiro , Masaka Takuro , Akiyama Ichiro , Nakashima Emiko , Sakaguchi Masakiyo , Huh Nam-Ho
Published: April 2008

Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells. At days 34–41, 2-mm2 sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF. When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels. Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months. These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells. By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.
Embryonic Stem Cells Form Articular Cartilage, not Teratomas, in Osteochondral Defects of Rat Joints
Authors: Wakitani Shigeyuki , Aoki Hideyuki , Harada Yasuji , Sonobe Masato , Morita Yusuke , Mu Ying , Tomita Naohide , Nakamura Yukio , Takeda Satoshi , Watanabe Takeshi K. , Tanigami Akira
Published: April 2008

Embryonic stem (ES) cells are considered to be a potential tool for repairing articular cartilage defects, but so far it has been impossible to cause these cells to differentiate into chondrocytes exclusively, either in vivo or in vitro. To explore a potential new cell source of cell transplantation for articular cartilage defects, we transplanted ES cells into articular cartilage defects in immunosuppressed rats. ES cells (AB2.2 or CCE cells) were transplanted into articular cartilage defects in the patellar groove of immunosuppressed rats treated with cyclosporine. The cells were histologically observed until 8 weeks after transplantation. To determine whether the repair tissue in the defect in the AB2.2-transplanted group was derived from the transplanted cells, the neomycin-resistant gene, which had been transfected into AB2.2 cells but does not exist in rat cells, was used for detection. The cells produced cartilage, resulting in repair of the defects from 4 weeks until 8 weeks after the transplantation without forming any teratomas. The neomycin-resistant gene was detected in every sample, demonstrating that the repair tissue in the AB2.2-transplanted group was derived from the transplanted AB2.2 cells. The environment of osteochondral defects is chondrogenic for ES cells. ES cells may thus be a potential tool for repairing articular cartilage defects.
Effects of Osteogenic Induction on Mesenchymal Cells from Fetal and Maternal Parts of Human Placenta
Authors: Takahashi Kenji , Igura Koichi , Zhang Xiaohong , Mitsuru Ayako , Takahashi Tsuneo A.
Published: April 2008

To clarify whether the mesenchymal cells derived from human placenta were available for bone regeneration, we investigated the effects of osteogenic induction on mesenchymal cells of fetal and maternal parts of the placenta. The osteogenic-induced mineralization in both types of cells was measured by von Kossa staining, and the calcium concentration and the expression of osteogenic markers were assayed by RT-PCR. In the mesenchymal cells of both parts, osteopontin, osteocalcin, alkaline phosphatase, and collagen type I, which are osteogenic markers, were expressed. Moreover, the mesenchymal cells of the fetal part of the placenta were mineralized for 3 weeks, but those of the maternal part were not. These results showed that the mesenchymal cells derived from human placenta had an osteogenic phenotype and that only the mesenchymal cells of the fetal part were capable of being used as a cell source for bone reconstitution.
Engraftment Measurement in Human Liver Tissue after Liver Cell Transplantation by Short Tandem Repeats Analysis
Authors: Mas Valeria R. , Maluf Daniel G. , Thompson Melissa , Ferreira-Gonzalez Andrea , Fisher Robert A.
Published: April 2008

Hepatocyte transplantation has been proposed as a technique for bridging patients to whole-organ transplantation, for providing metabolic support during liver failure, and for replacing whole-organ transplantation in certain metabolic liver diseases. Assessment of hepatocyte engraftment has been difficult to measure, and the degree of engraftment needed to correct various liver disorders is still unknown. A sensitive, simple, and specific method of monitoring engraftment of transplanted hepatocytes for the purpose of bridging human liver failure to native regeneration using short tandem repeats (STRs) was evaluated. The analytical sensitivity of the test was evaluated using DNA mixing curves and established as 0.5% (percentage of donor DNA/recipient DNA). Sex-matched and mismatched cases were included during the validation. The clinical evaluation of the assay was performed using liver samples from two patients who underwent hepatocyte transplantation. We concluded from this study that the AmpFLSTR® Profiler Plus™ PCR Amplification Kit, a well-established technique in forensic medicine, is specific, sensitive, and a reproducible assay for measurement of engraftment after hepatocyte transplantation in both sex-matched and sex-mismatched cases.
Comparison of Embryonic Stem Cell-Derived Dopamine Neuron Grafts and Fetal Ventral Mesencephalic Tissue Grafts: Morphology and Function
Authors: Yurek David M. , Fletcher-Turner Anita
Published: April 2008

In this study we compared the function and morphology of two types of neural grafts: allografts of fetal ventral mesencephalic (VM) tissue and xenografts of embryonic stem cell (ESC)-derived dopamine neurons. Mouse embryonic stem cells were cultured and exposed to differentiation factors that induced approximately 10% of the cells to express a dopaminergic phenotype. These cells were then harvested and implanted into the denervated striatum of rats with unilateral lesions of the nigrostriatal pathway. Another group of lesioned rats received allografts of fetal ventral mesencephalic tissue. While both types of grafts yield a similar number of tyrosine hydroxylase (TH)-positive cells, amphetamine-induced rotational behavior was differentially affected by these grafts: rotational behavior was significantly reduced in lesioned rats receiving allografts of fetal VM tissue while ESC grafts had slight but insignificant effects on rotational scores. Densitometry measures of TH+ fiber outgrowth revealed a similar area of reinnervation and a comparable number of TH+ cells for ESC graft when compared with VM grafts. These data suggest there are similarities and also distinct differences in the manner in which ESC and VM grafts interact with the denervated striatum.
The Effects of Transforming Growth Factor-β2 on Dopaminergic Graft Survival
Authors: Macauley Shannon L. , Horsch Alexander D. , Oterdoom Marinus , Zheng Ming H. , Stewart Gregory R.
Published: April 2008

Dopaminergic cell transplantation is a promising therapeutic approach for the treatment of Parkinson's disease, the potential of which is limited due to poor survival and low dopamine content within engrafted tissue. In this study, the ability of transforming growth factor-β2 (TGF-β2) to influence transplant survival was evaluated. Cell suspensions containing fetal rat ventral mesencephalon (VM) cells were incubated prior to surgery with vehicle (DPBS), varying concentrations of TGF-β2 (5–1000 ng/ml), or a pan-specific antibody against TGF-β (1D11, 100 ng/ml). VM cell suspensions (200,000 cells) were unilaterally implanted into the striatum of adult Sprague-Dawley rats (n = 5–11 animals/group). Following a 3-week survival period, small but viable VM grafts containing tyrosine hydroxylase-positive (TH+) neurons and fibers were present in all animals. Addition of TGF-β2 resulted in a steep, bell-shaped dose-response curve with a significant effect on TH+/dopamine cell survival. At 50 ng/ml TGF-β2, the number of surviving dopamine neurons was increased twofold compared with controls. Addition of TGF-β2 or 1D11 did not significantly influence graft volume. Further studies, possibly in combination with other neurotrophic factors, need to be performed to obtain a greater understanding of the effects of TGF-β on dopamine neurons and fetal VM cell engraftment.
Differentiation Effects by the Combination of Spheroid Formation and Sodium Butyrate Treatment in Human Hepatoblastoma Cell Line (Hep G2): A Possible Cell Source for Hybrid Artificial Liver
Authors: Fukuda J. , Okamura K. , Ishihara K. , Mizumoto H. , Nakazawa K. , Ijima H. , Kajiwara T. , Funatsu K.
Published: April 2008

The aim of this study was to investigate the feasibility of human hepatoblastoma cell line (Hep G2), which differentiates by spheroid formation, and treatment with sodium butyrate (SB) as a cell source for hybrid artificial liver (HAL). Hep G2 spontaneously formed spheroids in polyurethane foam (PUF) within 3 days of culture and restored weak ammonia removal activity. Treatment with SB, which is a histone deacetylase inhibitor, further increased the ammonia removal activity of Hep G2 spheroids in a concentration-dependent manner. The activation of ornithine transcarbamylase—a urea cycle enzyme—was significantly related to the upregulation of ammonia removal by spheroid formation, but scarcely contributed to the further upregulation following SB treatment. In contrast with ammonia removal, treatment with SB reduced the albumin secretion of Hep G2 spheroids in a concentration-dependent manner. In the PUF-HAL module in a circulation culture, the ammonia removal rate and albumin secretion rate (per unit volume of the module) of Hep G2 spheroids treated with 5 mM SB were almost the same as those of primary porcine hepatocyte spheroids. These results suggest that simultaneous use of spheroid formation and SB treatment in Hep G2 is beneficial in enhancing the functions of human hepatocytes with potential applications in regenerative medicine and drug screening.
Large-Scale Isolation of Human Hepatocytes for Therapeutic Application
Authors: Alexandrova Krassimira , Griesel Carsten , Barthold Marc , Heuft Hans-Gert , Ott Michael , Winkler Michael , Schrem Harald , Manns Michael P. , Bredehornsp Timm , Net Marc , Vidal Martí Manyalich I , Kafert-Kasting Sabine , Arseniev Lubomir
Published: April 2008

During the last decade, hepatocyte transplantation has been suggested as a safe and potentially effective clinical option for the treatment of acute or decompensating chronic liver failure as well as for hereditary liver disease. Currently, one of the major limiting factors for clinical application is the insufficient access to suitable liver cell preparations. In cooperation with the German and Catalane organ procurement organizations, a routine procedure for the isolation of hepatocytes from donor organs rejected for transplantation (n = 117) has been established. The process is performed according to the current EC Guidelines for Good Manufacturing Practice (cGMP) and all corresponding national laws and regulations concerning donor organ and tissue procurement. In about 50% of the cases (n = 58) the three-step perfusion procedure has been completed with an average total cell yield of 5.9 × 109 cells per organ, the cell preparations displaying a mean viability of 64%. The mean specific yield was 3.6 × 106 total and 2.6 × 106 viable cells per gram liver tissue, respectively. Specific cell yields from three infantile donor livers were considerably higher. No correlation between isolation efficiency and cold ischemia time or donor age was found within the adult organ donors. In contrast, organs with a severe steatosis generally did not result in successful cell isolation. Results of sterility and endotoxin determination are also presented. In summary, a standardized and cGMP conform method of hepatocyte isolation from nontransplantable liver organs was established, which reproducibly yields large amounts of hepatocytes suitable for therapeutic application.
Protection of Photoreceptor Cells from Phototoxicity by Transplanted Retinal Pigment Epithelial Cells Expressing Different Neurotrophic Factors
Authors: Abe Toshiaki , Saigo Yoko , Hojo Masayoshi , Kano Tetsuya , Wakusawa Ryosuke , Tokita Yumi , Tamai Makoto
Published: April 2008

Transplantation of cells or tissues and the intravitreal injection of neurotrophic factors are two methods that have been used to treat retinal diseases. The purpose of this study was to examine the effects of combining both methods: the transplantation of retinal pigment epithelial (RPE) cells expressing different neurotrophic factors. The neutrophic factors were Axokine, brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The enhanced green fluorescence protein (eGFP) gene was used as a reporter gene. These genes were transduced into RPE cells by lipofection, selected by antibiotics, and transplanted into the subretinal space of 108 rats. The rats were examined at 1 week and 3 months after the transplantation to determine whether the transduced cells were present, were expressing the protein, and were able to protect photoreceptors against phototoxicity. The survival of the transplanted cells was monitored by the presence of eGFP. The degree of protection was determined by the thickness of the outer nuclear layer. Our results showed that the degree of photoreceptor protection was different for the different types of neurotrophic factors at 1 week. After 3 months, the number of surviving transplanted cell was markedly reduced, and protection was observed only with the BDNF-transduced RPE cells. A significant degree of rescue was also observed by BDNF-transduced RPE cells in the nontransplanted area of the retina at both the early and late times. Lymphocytic infiltration was not detected in the vitreous, retina, and choroid at any time. We conclude that the transplantation of BDNF-transduced RPE cells can reduce the photoreceptor damage induced by phototoxicity in the transplanted area and weakly in the nontransplanted area.
Purified Human Bone Marrow Multipotent Mesenchymal Stem Cells Regenerate Infarcted Myocardium in Experimental Rats
Authors: Zhang Shaoheng , Jia Zhuqing , Ge Junbo , Gong Lizhong , Ma Yanling , Li Tao , Guo Jingxuan , Chen Ping , Hu Qikuan , Zhang Ping , Liu Yonggang , Li Zhaoping , Ma Kangtao , Li Linsong , Zhou Chunyan
Published: April 2008

Recent findings suggest the feasibility of cardiac repair by transplantation of bone marrow mesenchymal stem cell (MSCs). However, it remains controversial regarding which cell type is the best source for transplanting into the ischemic heart because of lack of well-defined cell markers. In this study, we investigated the in vitro and in vivo effects of the novel multipotent marrow mesenchymal stem cells (MMSCs) from human bone marrow. Pluripotent markers (Oct4, Bmi1, and Abcg2) and vascular endothelial growth factor (VEGF) were detected by RT-PCR and immunofluorescence in MMSCs. Myocardial differentiation was induced in the expanded MMSC cultures by treatment with 5-azacyline. Expressions of VEGF in the animals transplanted with MMSCs were markedly increased in comparison with the animals injected with fibroblasts or saline at both mRNA and protein levels. VEGF expression was observed in both transplanted MMSCs and recipient cardiomyocytes by immunofluorescence. Confocal immunofluorescence microscopy revealed the specific markers for cardiomyocytes and endothelial cells in transplanted MMSCs 14 days after transplantation. Vessel count was increased and left ventricular function improved post-MMSC transplantation. These results indicate that transplantation of purified MMSCs from human bone marrow upregulated VEGF expression, enhanced angiogenesis, and improved the functional recovery following myocardial infarction in rats.
Minimal Focal Steatosis of Liver after Islet Transplantation in Humans: A Long-Term Study
Authors: Maffi Paola , Angeli Enzo , Bertuzzi Federico , Paties Carlo , Socci Carlo , Fedeli Carlo , De Taddeo Francesca , Nano Rita , Di Carlo Valerio , Del Maschio Alessandro , Secchi Antonio
Published: April 2008

Several reports have been published on islet transplantation in humans, but few data are available on the effect of islet infusion on the hepatic structure. Our aim was to evaluate in a longitudinal study the impact on the liver of intrahepatic islet transplantation. Clinical outcome and liver imaging were evaluated in 31 cases of islet-kidney transplantation (follow-up 38 ± 4 months, range 12–96 months). Patients were divided into three groups: full function (FF, 9 cases: established insulin independence); partial function (PF, 16 cases: transient insulin independence, prolonged C-peptide secretion); no function (NF, 6 cases: exhaustion of C-peptide secretion within the first year). Upper abdomen sonogram was regularly performed during the whole follow-up period. Percutaneous liver biopsy was performed in case of echographic abnormalities. Multiple small areas of focal hyperechogenicity were observed in nine cases after 6–12 months. These findings were observed only in FF (two) and in PF (seven) patients. Fasting C-peptide levels at the time of echography were higher in negative than in positive patients (2.42 ± 0.16 vs. 1.51 ± 0.10 ng/ml, p = 0,0001). Liver biopsies showed focal macrovesicular steatosis, surrounded by normal liver parenchyma. Normal liver function was maintained. In conclusion, our results indicate that islet transplantation can lead to structural changes of the liver parenchyma (focal steatosis). It is more often observed in patients with partial function. Sonogram can be considered a specific method to reveal liver changes after islet transplantation.
Analysis of Allogeneic and Syngeneic Bone Marrow Stromal Cell Graft Survival in the Spinal Cord
Authors: Swanger Sharon A. , Neuhuber Birgit , Himes B. Timothy , Bakshi Ajay , Fischer Itzhak
Published: April 2008

Bone marrow stromal cells (MSC) are attractive candidates for developing cell therapies for central nervous system (CNS) disorders. They can be easily obtained, expanded in culture, and promote modest functional recovery following transplantation into animal models of injured or degenerative CNS. While syngeneic MSC grafts can be used efficiently, achieving long-term survival of allogeneic MSC grafts has been a challenge. We hypothesize that improved graft survival will enhance the functional recovery promoted by MSC. To improve MSC graft survival, we tested two dosages of the immune suppressant cyclosporin A (CsA) in an allogeneic model. Syngeneic transplantation of MSC where cells survive well without immune suppression was used as a control. Sprague-Dawley rats treated with standard dose (n = 12) or high-dose (n = 12) CsA served as allogeneic hosts; Fisher 344 rats (n = 12) served as syngeneic hosts. MSC were derived from transgenic Fisher 344 rats expressing human placental alkaline phosphatase and were grafted into cervical spinal cord. Animals treated with standard dose CsA showed significant decreases in allograft size 4 weeks posttransplantation; high CsA doses yielded significantly better graft survival 4 and 8 weeks posttransplantation compared to standard CsA. As expected, syngeneic MSC transplants showed good graft survival after 4 and 8 weeks. To investigate MSC graft elimination, we analyzed immune cell infiltration and cell death. Macrophage infiltration was high after 1 week in all groups. After 4 weeks, high-dose CsA and syngeneic animals showed significant reductions in macrophages at the graft site. Few T lymphocytes were detected in any group at each time point. Cell death occurred throughout the study; however, little apoptotic activity was detected. Histochemical analysis revealed no evidence of neural differentiation. These results indicate that allogeneic transplantation with appropriate immune suppression permits long-term survival of MSC; thus, both allogeneic and syngeneic strategies could be utilized in devising novel therapies for CNS injury.
Generation of Islet-Like Hormone-Producing Cells In Vitro from Adult Human Pancreas
Authors: Atouf Fouad , Choi Yong , Fowler Michael J. , Poffenberger Greg , Vobecky Jan , Ta Malancha , Chapman George B. , Powers Alvin C. , Lumelsky Nadya L.
Published: April 2008

Transplantation of pancreatic islets can provide long-lasting insulin independence for diabetic patients, but the current islet supply is limited. Here we describe a new in vitro system that utilizes adult human pancreatic islet-enriched fractions to generate hormone-producing cells over 3–4 weeks of culture. By labeling proliferating cells with a retrovirus-expressing green fluorescent protein, we show that in this system hormone-producing cells are generated de novo. These hormone-producing cells aggregate to form islet-like cell clusters. The cell clusters, when tested in vitro, release insulin in response to glucose and other secretagogues. After transplantation into immunodeficient, nondiabetic mice, the islet-like cell clusters survive and release human insulin. We propose that this system will be useful as an experimental tool for investigating mechanisms for generating new islet cells from the postnatal pancreas, and for designing strategies to generate physiologically competent pancreatic islet cells ex vivo.
Long-Term Maintenance of Liver-Specific Functions in Cultured ES Cell-Derived Hepatocytes with Hyaluronan Sponge
Authors: Teratani Takumi , Quinn Gary , Yamamoto Yusuke , Sato Tomoya , Yamanokuchi Hiroko , Asari Akira , Ochiya Takahiro
Published: April 2008

This study investigated the three-dimensional culture of hepatocytes differentiated from mouse embryonic stem (ES) cells with a porous hyaluronan (HA) sponge support. Hepatocytes were immobilized within the pores of the support. Spheroids could be observed within the support, each containing between 20 and 50 hepatocytes. To examine the liver-specific functions of the hepatocytes in the culture, the levels of albumin secreted into the medium were analyzed. The secretion of albumin was stable over the course of 32 days, longer than that in both conventional monolayer and collagen sponge cultures. To elucidate further the liver-specific functions of hepatocytes embedded in the HA sponge, metabolic activities of the hepatocytes were examined for their ability to eliminate ammonia from culture media and the synthesis of urea nitrogen. While rates of ammonia removal and urea nitrogen synthesis were similar to those in both conventional monolayer and in collagen sponge cultures, these functions were maintained for longer duration in cells embedded in the HA sponge. These results demonstrate that the porous HA sponge is an effective support for the in vitro culture of ES-derived hepatocytes used for both basic and applied studies for cell transplantation.
Stability and Repeat Regeneration Potential of the Engineered Liver Tissues under the Kidney Capsule in Mice
Authors: Ohashi Kazuo , Kay Mark A. , Yokoyama Takashi , Kuge Hiroyuki , Kanehiro Hiromichi , Hisanaga Michiyoshi , Ko Saiho , Nakajima Yoshiyuki
Published: April 2008

Liver tissue engineering using hepatocyte transplantation has been proposed as a therapeutic alternative to liver transplantation toward several liver diseases. We have previously reported that stable liver tissue with the potential for liver regeneration can be engineered at extrahepatic sites by transplanting mature hepatocytes into an extracellular matrix. The present study was aimed at assessing the liver tissue persistence after induced regeneration by hepatectomy and repeat regeneration potential induced by repeat hepatectomy. Mouse isolated hepatocytes mixed in EHS extracellular matrix gel were transplanted under both kidney capsules of isogenic mice. The hepatocyte survival persisted for over 25 weeks. In some of the mice, we confirmed that the grafted hepatocytes developed a thin layer of liver tissues under the kidney capsule, determined by specific characteristics of differentiated hepatocytes in cord structures between the capillaries. We then assessed the regenerative potential and persistence of the exogenous liver tissue. To induce liver regeneration, we performed a two-thirds hepatectomy at 70 days after hepatocyte transplantation. Three weeks after this procedure, the engineered liver tissues showed active regeneration, reaching serum marker protein levels of 261 ± 42% of the prehepatectomy level. We found that the regenerated liver tissue was stably maintained for 100 days (length of the experiment). Repeat regeneration potential was established by performing a repeat hepatectomy (that had been two-thirds hepatectomized at day 70) 60 days after the initial hepatectomy. Again, the regenerated engineered liver tissues showed active regeneration as there was an approximately twofold increase in the serum marker protein levels. The present studies demonstrate that liver tissue, which was recognized as a part of the host naive liver in terms of the regeneration profile, could be engineered at a heterologous site that does not have access to the portal circulation.
Promotion of Neurite Outgrowth from Fetal Hippocampal Cells by TNF-α Receptor 1-Derived Peptide
Authors: Kajiwara Kazumi , Ogata Shin-Ichi , Tanihara Masao
Published: April 2008

Cytokines such as tumor necrosis factor-α (TNF-α), FasL, and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis or inflammation through binding to their specific receptors, TNFR1, Fas, and DR5, respectively. We have previously reported ligand-binding and cell death-inhibiting synthetic peptides, which were designed based on the crystal structure of a ligand–receptor complex and the homology of the amino acid sequence among the death receptor family members. Here we show that, among these death receptor-derived peptides, the TNFR1-derived peptide specifically arrested cell proliferation and promoted cell adhesion of fetal rat (E16) hippocampal cells, and promoted neurite outgrowth from hippocampus-derived neurospheres cultured with the addition of the peptide or cultured on a peptide-coated surface. Furthermore, among these death receptor-derived peptides, marked neurite outgrowth was observed only when the neurospheres were cultured on a TNFR1-derived peptide-conjugated covalently cross-linked alginate gel. The neurites from the neurospheres positively immunostained with an antibody against neurofilaments. These results suggest that the TNFR1-derived peptide promotes neuronal differentiation of the hippocampal neural stem cells and the TNFR1-derived peptide-conjugated covalently cross-linked alginate gel may be a useful material for assisting neural stem cell transplantation.
Synergistic Effects of FGF-2 with Insulin or IGF-I on the Proliferation of Human Auricular Chondrocytes
Authors: Takahashi Tsuguharu , Ogasawara Toru , Kishimoto Junji , Liu Guangyao , Asato Hirotaka , Nakatsuka Takashi , Uchinuma Eijyu , Nakamura Kozo , Kawaguchi Hiroshi , Takato Tsuyoshi , Hoshi Kazuto
Published: April 2008

Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a 1000-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor(FGF)-2, insulin-like growth factor(IGF)-I, insulin, bone morphogenetic protein-2, parathyroid hormone, growth hormone, dexamethasone, 1α25-dihydroxy vitamin D3, L-3,3′,5′-triodothyronine, interleukine-1 receptor antagonist, 17β-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with 10 ng/ml FGF-2, while the cell number synergistically gained a 10–12-fold increase with 5 μg/ml insulin or 100 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of 100 ng/ml for FGF-2, and especially for the combination of 100 ng/ml FGF-2 and 5 μg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than 10,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.
Phenotype-Specific Cells with Proliferative Potential are Produced by Polyethylene Glycol-Induced Fusion of Mouse Embryonic Stem Cells with Fetal Cardiomyocytes
Authors: Takei Shunsuke , Yamamoto Makoto , Cui Li , Yue Fengming , Johkura Kohei , Ogiwara Naoko , Iinuma Hisae , Okinaga Kota , Sasaki Katsunori
Published: April 2008

Because cardiomyocytes lose the ability to divide upon differentiation, myocardial failure is assumed to be generally irreversible. For terminal cardiac insufficiency, the potential for regenerative treatment by stem cells, especially embryonic stem (ES) cells, offers hope for the future. Recent studies showed that stem cells fuse spontaneously with cells remaining in damaged tissues, and restore tissue function. To imitate spontaneous fusion in vivo, we used polyethylene glycol (PEG) in vitro to fuse mouse ES cells and fetal cardiomyocytes and analyzed the cytochemical properties of the fused cells. Confocal laser scanning microscopy coupled with lipophilic dye labeling of the living cell membranes showed that there were fused cells of ES cells and cardiomyocytes after PEG treatment. By flow cytometry, the fusion efficiency between ES cells and cardiomyocytes was estimated to be about 45% of the total resulting cells. When green fluorescent protein (GFP)-expressing ES cells were fused with cardiomyocytes, the fused cells had immunoreactivity for GFP in their cytoplasm and cardiac troponin I in their myofibrils. Some of these cells also expressed proliferating cell nuclear antigen up to 11 days after fusion, the last time point examined. This study shows that PEG-induced fusions of mouse ES cells and cardiomyocytes have the cardiomyocyte phenotype and proliferation potential.