bluuebunny/crossref_metadata_2025 huggingface dataset:
The Fetal Dermal but Not Loose Fascial Mesenchymal Cells Possess Regenerative Activity of Dermal Structure
Authors: Kishi Kazuo , Katsube Kenichi , Satoh Hiroko , Imanishi Nobuaki , Nakajima Hideo , Nakajima Tatsuo
Published: April 2008

Fetal skin possesses a regenerative activity until certain developmental stages. However, the origin of cells that regenerate dermis after wounding has not been clarified yet. In the present study we located the origin of cells that reconstruct fetal dermal structure by histological examination and by marking cells in the loose fascia. Next we evaluated the regenerative activity of fetal dermal mesenchymal cells by cotransplanting with fetal epidermal cells onto the skin defect of scid mice. We conclude that fetal dermal mesenchymal cells but not loose fascial cells possess regenerative activity even on the environment in scid mice.
The Effects of Cryopreservation on Human Hepatocytes Obtained from Different Sources of Liver Tissue
Authors: Terry Claire , Mitry Ragai R. , Lehec Sharon C. , Muiesan Paulo , Rela Mohamed , Heaton Nigel D. , Hughes Robin D. , Dhawan Anil
Published: April 2008

Successful cryopreservation of human hepatocytes is important to establish hepatocyte banks for clinical use or in vitro research. The availability of donor tissue from unused liver segments/lobes and non-heart-beating donors (NHBD) has provided newer sources of hepatocytes. The quality of hepatocytes at the time of cryopreservation is important as cells isolated from liver tissue of borderline quality may not withstand the stresses associated with cryopreservation and subsequent thawing. Human hepatocytes were cryopreserved after isolation from mainly donor tissues (n = 40). In vitro assessment of the viability and function of the fresh and thawed cryopreserved hepatocytes was performed. Viability, attachment efficiency, enzyme activity, and albumin production of hepatocytes were all significantly decreased, and LDH leakage significantly increased, on thawing after cryopreservation. The viability of cryopreserved hepatocytes isolated from tissue rejected for orthotopic liver transplantation (36 ± 15%) was significantly lower than those isolated from tissue where part was used for liver transplantation (47 ± 14%, p = 0.002), but there were no significant differences in functional parameters. The viability of cryopreserved hepatocytes isolated from NHBD tissue (29 ± 9%, p = 0.001) and from steatotic donor tissue (35 ± 11%, p = 0.019) was significantly lower than those isolated from normal donor tissue (49 ± 14%). There was no difference in functional parameters, except for albumin production of hepatocytes from NHBD tissue (2.9 ±1.0 μg/h/mg protein) being significantly lower than those from normal donor tissue (4.8 ± 2.8 μg/h/mg protein, p = 0.03). The viability and attachment efficiency of cryopreserved hepatocytes isolated from liver tissue from resections for tumors was significantly higher, and the LDH leakage significantly lower, than those isolated from all donor tissue. Hepatocytes isolated from NHBD and steatotic tissue were more vulnerable to the effects of cryopreservation. Further research is required to improve hepatocyte isolation and cryopreservation protocols for different types of liver tissue.
Selection of Cell Source for Ligament Tissue Engineering
Authors: Ge Zigang , Goh James Cho Hong , Lee Eng Hin
Published: April 2008

Use of appropriate types of cells could potentially improve the functionality and structure of tissue engineered constructs, but little is known about the optimal cell source for ligament tissue engineering. The object of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Fibroblasts isolated from anterior cruciate ligament, medial collateral ligament (MCL), as well as bone marrow mesenchymal stem cells (MSC) were compared using the following parameters: proliferation rate, collagen excretion, expression of collagen type I, II, and III, as well as α-smooth muscle actin. Green fluorescent protein (GFP) transfected MSCs were used to trace their fate in the knee joints. MSC, ACL, and MCL fibroblasts were all highly stained with antibodies for collagen types I and III and α-smooth muscle actin while negatively stained with collagen type II. Proliferation rate and collagen excretion of MSCs were higher than ACL and MCL fibroblasts (p < 0.05), and MSCs could survive for at least 6 weeks in knee joints. In summary, MSC is potentially a better cell source than ACL and MCL fibroblasts for anterior cruciate ligament tissue engineering.
A Simple and Highly Effective Method for the Stable Transduction of Uncultured Porcine Hepatocytes Using Lentiviral Vector
Authors: Nguyen Tuan Huy , Khakhoulina Tatiana , Simmons Andrew , Morel Philippe , Trono Didier
Published: April 2008

Gene therapy is an attractive approach for the treatment of a wide spectrum of liver diseases. Lentiviral vectors allow the stable integration of transgenes into the genome of nondividing differentiated cells including hepatocytes and could provide long-lasting expression of a therapeutic gene. To develop such approaches, preclinical studies in large animal models such as pigs are necessary to evaluate the feasibility and safety of stable lentiviral integration and long-term vector expression. In addition, effective lentivector-mediated gene transfer onto porcine hepatocytes could advance in cell-based therapies for acute liver failure. To investigate this issue, porcine hepatocytes were transduced in suspension immediately after their isolation in University of Wisconsin (UW) solution containing vitamin E. Up to 80% of hepatocytes stably expressed a GFP transgene after a single exposure to lentiviral vector coding for GFP under the control of either liver-specific or ubiquitous promoters. Moreover, porcine hepatocytes cryopreserved in UW solution containing fetal bovine serum, dimethyl sulfoxide, and vitamin E remained highly transducible with lentiviral vector after thawing. When thawed, transduced in suspension, and immediately transplanted into the spleen of immunodeficient mice, ex vivo lentivirally transgene marked xenogeneic hepatocytes were detected in murine liver. We demonstrated that porcine hepatocytes are highly susceptible to lentiviral vector and describe an easy methodology to efficiently, rapidly, and stably introduce transgenes into uncultured porcine hepatocytes.
Comparative Analysis of Genetically Engineered Immunodeficient Mouse Strains as Recipients for Human Myoblast Transplantation
Authors: Silva-Barbosa Suse D. , Butler-Browne Gillian S. , Di Santo James P. , Mouly Vincent
Published: April 2008

The development of an optimized animal model for the in vivo analysis of human muscle cells remains an important goal in the search of therapy for muscular dystrophy. Here we examined the efficiency of human myoblast xenografts in three distinct immunodeficient mouse models. We found that different conditioning regimes used to provoke host muscle regeneration (i.e., cardiotoxin versus cryodamage) had a marked impact on xenograft success. Tibialis anterior muscle of Rag2-, Rag-/γc-, and Rag-/γc-/C5- mice was treated by cardiotoxin or cryodamage, submitted to enzymatic digestion, and analyzed by cytofluorometry to quantitate inflammatory cells. Human myoblasts were injected into pretreated muscles from immunodeficient recipients and the cell engraftment evaluated by immunocytochemistry, 4—8 weeks after transplantation. Donor cell differentiation and dispersion within the host muscles was also investigated. Host regeneration in cardiotoxin-treated mice was accompanied by a higher inflammatory cell infiltration when compared to that induced by cryodamage. Accordingly, when compared to the cardiotoxin group, more human myogenic cells were found after cryodamage. When the distinct immunodeficient mice were compared, we found that the alymphoid strain lacking the complement component C5 (Rag-/γc-/C5- mice) was the most efficient host for human muscle xenografts, when compared with C5+Rag-/γc- mice or Rag- mice. Our results demonstrate that cryolesion-conditioned muscles of Rag-/γc-/C5- mice provide the best environment for long-term in vivo human myoblast differentiation, opening the way for a novel approach to study the pathophysiology of human muscle disorders.
The Escape of Temperature-Sensitive T Antigen Immortalized Rat Hepatocytes from Conditional Immortalization
Authors: Kim Byung-Ho , Han Yo Seb , Choe Bong-Keun , Cho Hyeseong , Nam Gi Deog , Lee Jin Woo , Kim Young Il , Park Jai Kyung , Dong Seok Ho , Kim Hyo Jong , Chang Young Woon , Lee Joung Il , Chang Rin
Published: April 2008

Conditionally immortalized hepatocytes (CIH) established with a gene for the temperature-sensitive mutant of the T antigen (tsT) have characteristics to stop proliferating and to differentiate at nonpermissive temperatures (37—39°C) due to inactivation of the T antigen. Therefore, they may be a good alternative to primary hepatocytes for experimental investigations or clinical applications. Deinduction of the T antigen results in a transient increase of p53 in these cells, leading to reexpression of normal senescence because of the telomere attrition occurring during the early stages of immortalization. To determine this T antigen dependency for the maintenance of immortality, a type of rat CIH was cultured continuously at 39°C. The frequency of occurrence of T-antigen-independent clones ranged from 0.053% to 0.093%. These clones maintained the temperature-sensitive property of the T antigen; nevertheless, they were able to progress to the S phase and proliferate without undergoing apoptosis at 39°C as at 33°C, a permissive temperature. The temperature-sensitive point mutation of tsT was not affected in these clones and the T antigen was functioning properly. The integrity of the p53 pathway was also maintained from the point of Western blot analysis of p21. Although the telomerase continued to be expressed and the telomere length was maintained, marked chromosomal damage could not be avoided in these cells. It is a plausible explanation that this escape phenomenon from conditional immortalization may be related to the change of other genes involved in cell cycles, which have yet to be elucidated. In conclusion, CIH could lose their temperature-sensitive characteristics without the change of tsT, itself, and the T antigen is not always necessary to maintain their immortality. Therefore, the results obtained from experimental investigations using these cells should be interpreted carefully, and unpredictable phenotypic changes should also be taken into consideration when using them in clinical applications.
Effect of Oxygenated Perfluorocarbons on Isolated Rat Pancreatic Islets in Culture
Authors: Bergert Hendrik , Knoch Klaus-Peter , Meisterfeld Ronny , Jäger Melanie , Ouwendijk Joke , Kersting Stephan , Saeger Hans Detlev , Solimena Michele
Published: April 2008

One impediment for a wider application of islet transplantation is the limited number of donor pancreata for islet isolation. A more efficient utilization of available organs could in part alleviate this problem. Perfluorocarbons (PFCs) have a high oxygen solubility coefficient and maintain high oxygen partial pressures for extended time. They serve also as oxygen “reservoirs” for harvested organs in pancreas organ transplantation. The aim of this study was to test whether the use of PFCs could also be beneficial for the secretory activity and overall viability of cultured purified islets before transplantation. Purified rat islets were cultured in static conditions with or without oxygen-saturated PFCs for 1 or 7 days. Cell death and apoptosis were assessed by trypan blue staining, DNA strand breaks, and caspase 3/7 activity. mRNA levels of insulin and ICA512/IA-2, a membrane marker of secretory granules (SGs), were quantitated by real-time PCR, whereas insulin content and secretion were measured by RIA. Polypyrimidine tract binding protein (PTB), which promotes SG biogenesis, was assessed by Western blotting. The number of SGs and the ultrastructural appearance of β-cells were analyzed by cryoimmunoelectronmicroscopy for insulin. Various parameters, including caspase activity, insulin and ICA512/IA-2 mRNA levels, PTB expression, number of secretory granules, and ultrastructural appearance did not significantly differ between control and PFC-cultured islets. On the other hand, PFC culture islets showed significantly increased DNA fragmentation and a reduced insulin stimulation index at both time points compared to control islets. While advantageous for the transport of human harvested organs, the use of PFH in the culture may be comparable to and/or not provide advantage over conventional protocols for culture of islets for transplantation.
Effects of Cryopreservation on Cell Viability and Insulin Secretion in a Model Tissue-Engineered Pancreatic Substitute (TEPS)
Authors: Mukherjee Neil , Chen Zhenzhen , Sambanis Athanassios , Song Ying
Published: April 2008

The use of encapsulated insulin-secreting cells constitutes a promising approach towards the treatment of insulin-dependent diabetes. However, long- term storage for off-the-shelf availability still remains an issue, which can be addressed by cryopreservation. This study investigated cryopreservation of a model tissue-engineered pancreatic substitute by two ice-free cryopreservation (vitrification) solutions (designated VS55 and PEG400) in comparison to a conventional freezing protocol. The model substitute consisted of insulin-secreting mouse insulinoma βTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads. Cell viability and static insulin secretion from the thawed cryopreserved groups were characterized and compared against fresh controls. Cell viability tests using alamarBlue® showed that, compared to the fresh groups, the VS55 had the highest viability (p < 0.05), followed by both the PEG400 (p < 0.001) and the frozen groups (p < 0.001). In response to a square wave of glucose, the static insulin secretion data showed that the VS55 and PEG400 groups had similar induction levels against the fresh group, whereas the frozen group had the poorest secretion rate. Cryosubstitution of capsules showed ice formation in the frozen group but no ice in the vitrified groups. Microscopic observations revealed holes and/or tears within beads subjected to freezing, whereas no such abnormalities were detected in the vitrified samples. Overall, vitrification was found to be a promising preservation procedure for this encapsulated cell system.
Human Serum for Culture of Articular Chondrocytes
Authors: Tallheden Tommi , Van Der Lee Josefine , Brantsing Camilla , Månsson Jan-Eric , Sjögren-Jansson Eva , Lindahl Anders
Published: April 2008

In the field of cell and tissue engineering, culture expansion of human cells in monolayer plays an important part. Traditionally, cell cultures have been supplemented with serum to support attachment and proliferation, but serum is a potential source of foreign protein contamination and viral protein transmission. In this study, we evaluated the use of human serum for experimental human articular chondrocyte expansion and to develop a method for preparation of large volumes of high-quality human serum from healthy blood donors. Human autologous serum contained high levels of epidermal-derived growth factor and platelet-derived growth factor-AB and supported proliferation up to 7 times higher than FCS in primary chondrocyte cultures. By letting the coagulation take place in a commercially available transfusion bag overnight, up to 250 ml of growth factor-rich human serum could be obtained from one donor. The allogenic human serum supported high proliferation rate without loosing expression of cartilage-specific genes. The expanded chondrocytes were able to redifferentiate and form cartilage matrix in comparable amounts to autologous serums. In conclusion, the transfusion bags allow preparation of large volumes of growth factor-rich human serum with the capacity to support in vitro cell expansion. The data further indicate that by controlling the coagulation process there are possibilities of optimizing the release of growth factors for other emerging cell therapies.
Age Effects on Vascular Smooth Muscle: An Engineered Tissue Approach
Authors: Solan Amy , Niklason Laura
Published: April 2008

Tissue engineering of blood vessels offers a potential new therapy for patients with vascular occlusive disease. In addition, tissue engineering technologies offer the opportunity to study the biology of vascular cells in a biomimetic, three-dimensional environment. A model for vascular tissue engineering was used to study the effects of vascular cell age on extracellular matrix (ECM) deposition, cellular mitosis, and protein synthesis under controlled conditions in vitro. Blood vessels were grown using a three-dimensional polyglycolic acid (PGA) mesh that was seeded with either infant or adult porcine vascular smooth muscle cells. Mechanical forces in the form of pulsatile radial distension were applied for the duration of the 7-week growth period. Overall, infant cells exhibited higher levels of cellular proliferation, ECM deposition, and remodeling activity than cells derived from adult animals. In addition, vessels cultured from infant cells had enhanced physical properties compared to vessels cultured from adult cells. The differentiation state of the smooth muscle cells in the infant and adult constructs was unchanged from the native state. However, the levels of immature pro-collagen, although undetectable in the vessels grown from adult cells, were similar in native vessels and in vessels grown with infant cells. These studies have important implications for the study of aging and vascular disease and remodeling, as well as for the field of tissue engineering.
Intrasplenic Transplantation of Encapsulated Genetically Engineered Mouse Insulinoma Cells Reverses Streptozotocin-Induced Diabetes in Rats
Authors: Aoki Takeshi , Hui Hongxiang , Umehara Yutaka , Licalzi Sergio , Demetriou Achilles A. , Rozga Jacek , Perfetti Riccardo
Published: April 2008

Pancreatic islet transplantation is limited by shortage of donor organs. Although β-cell lines could be used, their secretion of insulin is characteristically glucose independent and immunoisolation is required. Here we show that intrasplenic transplantation of encapsulated glucose-responsive mouse insulinoma cells reversed streptozotocin (STZ)-induced diabetes in rats. MIN-6 cells derived from a transgenic mouse expressing SV 40 large T antigen in pancreatic β-cells were transfected with minigene encoding for human glucagon-like-peptide-1 under the control of rat insulin promoter. The cells were encapsulated in alginate/poly-L-lysine and used for cell transplantation in STZ-diabetic rats. Rats with nonfasting blood glucose (n-FBG) greater than 350 mg/dl were used. In group I rats (n = 6) 20 million encapsulated cells were injected into the spleen. Group II rats (n = 6) received empty capsules. n-FBG was measured biweekly. After 4 and 8 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed in group I; normal rats served as controls. Plasma insulin level was measured every other week (RIA). After 8 weeks, spleens were removed 1 day before sacrifice. In rats transplanted with cells the n-FBG was 100—150 mg/dl until the end of the study. After splenectomy, all cell recipients became diabetic (glucose 400 ± 20 mg/dl). Transplanted rats showed increase in body weight and insulin production (3.3 ± 1.0 ng/ml versus 0.92 ± 0.3 ng/ml; p < 0.01) and had normal IPGTT. Spleens contained capsules with insulin-positive cells. Overall, data from this work indicate that intrasplenic transplantation of xenogeneic encapsulated insulin-producing cells without immunosuppression reversed diabetes in rats. Excellent survival and function of the transplanted cells was due to the fact that the cells were separated from the bloodstream by the immunoisolatory membrane only and insulin was delivered directly to the liver (i.e., in a physiological manner).
Delivery of TAT/PTD-Fused Proteins/Peptides to Islets via Pancreatic Duct
Authors: Klein Dagmar , Mendoza Valeska , Pileggi Antonello , Molano R. Damaris , Barbé-Tuana Florencia M. , Inverardi Luca , Ricordi Camillo , Pastori Ricardo L.
Published: April 2008

Delivering cytoprotective proteins/peptides into pancreata prior to islet isolation through protein transduction (PT) is a novel strategy to enhance the yield of viable transplantable islets. Previous work has shown that the protein transduction domain PTD-5 efficiently transduced islets via the pancreatic duct. TAT/PTD is a well-characterized PTD with the capability to cross even the hemato–encephalic barrier. In this study, we investigated the utilization of the 11-aa TAT protein transduction domain (TAT/PTD) to deliver peptides or proteins of different sizes ranging from 1.2 to 120 kDa, as the TAT/PTD and TAT/PTD-BH4 peptide, or the TAT/PTD–β-galactosidase fusion protein, into islets through the pancreatic duct. Using flow cytometry analysis we found that TAT/PTD derivatives transduced practically 100% of the islet cell population. Moreover, confocal laser scanning microscopy in live, nonfixed islets confirmed these results assessing transduction of TAT/PTD molecules into intact nondisaggregated islets. TAT–β-galactosidase peptide conjugated to FITC was not compartment selective, as both cytoplasmic and nucleic cellular compartments were positively stained. Furthermore, TAT–β-galactosidase peptide delivery was highly effective, as even cells located in the inner core region of the islets were transduced. Finally, transduced TAT–β-galactosidase fusion protein was biologically active after islet isolation and manipulation, and islet insulin secretion capability was not compromised by peptide transduction. These findings suggest that the transduction of chimeric TAT/PTD proteins can represent an efficient tool of molecular delivery independent of the size, to enhance or modify a specific phenotype at the nuclei or cytoplasmic level.
Adult-Fetal Fibroblast Interactions: Effects on Cell Migration and Implications for Cell Transplantation
Authors: Sandulache Vlad C. , Dohar Joseph E. , Hebda Patricia A.
Published: April 2008

Wound healing is a complex process involving close cooperation between multiple cell types. During wound healing, fibroblasts are primarily responsible for synthesis of the replacement extracellular matrix. Fibroblast therapy is under investigation in this and other laboratories for its potential use to modulate the final outcome of the wound-healing process. This study addresses the potential interactions between transplanted and host fibroblasts, using a two-dimensional mixed culture model. Our results show that fibroblasts of two different phenotypes, fetal and adult, exhibit different speeds of in vitro migration. These migration speeds are conserved in mixed cocultures, suggesting that the migratory response is an intrinsic property of the fibroblast rather than a response to juxtacrine or paracrine signals. These results have relevance for cell-based therapies in that they demonstrate that donor fibroblasts of a different phenotype may at least partially retain that phenotype in the host environment and in the presence of endogenous fibroblasts.
Neural Stem Cells Implanted into MPTP-Treated Monkeys Increase the Size of Endogenous Tyrosine Hydroxylase-Positive Cells Found in the Striatum: A Return to Control Measures
Authors: Bjugstad Kimberly B. , Redmond D. Eugene , Teng Yang D. , Elsworth J. D. , Roth R. H. , Blanchard B. C. , Snyder Evan Y. , Sladek John R.
Published: April 2008

Neural stem cells (NSC) have been shown to migrate towards damaged areas, produce trophic factors, and replace lost cells in ways that might be therapeutic for Parkinson's disease (PD). However, there is very little information on the effects of NSC on endogenous cell populations. In the current study, effects of implanted human NSC (hNSC) on endogenous tyrosine hydroxylase-positive cells (TH+ cells) after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were explored in nonhuman primates. After MPTP damage and in PD, the primate brain is characterized by decreased numbers of dopamine neurons in the substantia nigra (SN) and an increase in neurons expressing TH in the caudate nucleus. To determine how implanted NSC might affect these cell populations, 11 St. Kitts African green monkeys were treated with the selective dopaminergic neurotoxin, MPTP. Human NSC were implanted into the left and right caudate nucleus and the right SN of eight of the MPTP-treated monkeys. At either 4 or 7 months after NSC implants, the brains were removed and the size and number of TH+ cells in the target areas were assessed. The results were compared to data obtained from normal untreated control monkeys and to the three unimplanted MPTP-treated monkeys. The majority of hNSC were found bilaterally along the nigrostriatal pathway and in the substantia nigra, while relatively few were found in the caudate. In the presence of NSC, the number and size of caudate TH+ cells returned to non-MPTP-treated control levels. MPTP-induced and hNSC-induced changes in the putamen were less apparent. We conclude that after MPTP treatment in the primate, hNSC prevent the MPTP-induced upregulation of TH+ cells in the caudate and putamen, indicating that hNSC may be beneficial to maintaining a normal striatal environment.
Inhibition of Chromatin Condensation Prevents Transgene Silencing in a Neural Progenitor Cell Line Transplanted to the Rat Brain
Authors: Rosenqvist Nina , Jakobsson Johan , Lundberg Cecilia
Published: April 2008

The use of ex vivo gene therapy in the central nervous system has so far suffered from transgene downregulation. Condensation of the transgenic sequences has been proposed to be a mechanism involved in this silencing. In this study we inhibited either histone deacetylation or DNA methylation in neural progenitor cell lines, transduced with a lentiviral vector carrying green fluorescent protein (GFP), prior to grafting them into the rat striatum. The expression of GFP was significantly higher in grafts pretreated with either of the inhibitors. After 1 week in vivo we detected an 11-fold increase in the number of GFP-expressing cells due to the inhibition of DNA methylation in vitro with azadeoxycytidine and a ninefold increase when inhibiting histone deacetylation with trichostatin A. This suggests that a pretreatment paradigm could be used to increase efficacy of ex vivo delivery of a therapeutic protein locally in the brain.
In Vivo Noninvasive Monitoring of a Tissue Engineered Construct Using 1H NMR Spectroscopy
Authors: Stabler C. L. , Long R. C. , Constantinidis I. , Sambanis A.
Published: April 2008

Direct, noninvasive monitoring of tissue engineered substitutes containing live, functional cells would provide valuable information on dynamic changes that occur postimplantation. Such changes include remodeling both within the construct and at the interface of the implant with the surrounding host tissue, and may result in changes in the number of viable cells in the construct. This study investigated the use of 1H NMR spectroscopy in noninvasively monitoring the viable cell number within a tissue engineered construct in vivo. The construct consisted of mouse βTC3 insulinomas in a disk-shaped agarose gel, surrounded by a cell-free agarose gel layer. Localized 1H NMR spectra were acquired from within implanted constructs, and the total choline resonance was measured. Critical issues that had to be addressed in accurately quantifying total choline from the implanted cells included avoiding signal from host tissue and correcting for interfering signal from diffusing solutes. In vivo NMR measurements were correlated with MTT assays and NMR measurements performed in vitro on explanted constructs. Total choline measurements accurately and noninvasively quantified viable βTC3 cell numbers in vivo, in the range of 1 × 106 to more than 14 × 106 cells, and monitored changes in viable cell number that occurred in the same construct over time. This is the first study using NMR techniques to monitor viable cell numbers in an implanted tissue substitute. It established architectural characteristics that a construct should have to be amenable to NMR monitoring, and it set the foundation for future in vivo investigations with other tissue engineered implants.
Effect of Microcapsule Composition and Short-Term Immunosuppression on Intraportal Biocompatibility
Authors: Toso Christian , Mathe Zoltan , Morel Philippe , Oberholzer Jose , Bosco Domenico , Sainz-Vidal Dianelys , Hunkeler David , Buhler Leo H. , Wandrey Christine , Berney Thierry
Published: April 2008

With higher nutrient and oxygen supply and close contact to blood, the portal vein is a possible alternative to the peritoneal cavity for transplantation of encapsulated cells. Data regarding intraportal biocompatibility of microcapsules are lacking. Microcapsules were built from five alginate types differing in their molar mass and mannuronic/guluronic acid ratios by complex formation with divalent cations (barium or calcium) or mixtures of divalent cations and polycations. They were injected in the portal vein of rats, and cellular and fibrotic pericapsular infiltration thickness was measured 3 and 7 days after implantation. Overgrowth was characterized using various stainings or immunohistochemistry (hematoxylin and eosin, Giemsa, ED-1 for monocyte/macrophage, α-actin for myofibroblasts, CD31 for endothelial cells). The impact of short-term immunosuppression (gadolinium-chloride IV 20 mg/kg/day on days −1 and 4 as well as 10 days of rapamycin PO 1 mg/kg/day, tacrolimus PO 3 mg/kg/day, or combinations of rapamycin/tacrolimus or gadolinium/tacrolimus) was further assessed 3, 7, and 42 days after implantation. Overall, overgrowth increased from day 3 to day 7 (p < 0.05). Three and 7 days after implantation, polycation-containing microcapsules induced more reaction than microbeads (p < 0.0001 and p < 0.01). Considering polycation-free beads, barium-alginate induced the weakest reaction. Biocompatibility of microbeads was independent of mannuronic/guluronic acid ratio and molar mass of the alginate. Infiltration was mainly a monocyte/macrophage-rich foreign body reaction, but an eosinophil-containing immunoallergic reaction was also observed. Short-term immunosuppression significantly reduced infiltration in all conditions and up to 42 days after implantation. Biocompatibility after intraportal infusion was best for barium-alginate microbeads and poorest for polycation-containing microcapsules. Short- and long-term overgrowth could be significantly reduced by short-term immunosuppression.
Short-Term Culture with the Caspase Inhibitor z-VAD.fmk Reduces Beta Cell Apoptosis in Transplanted Islets and Improves the Metabolic Outcome of the Graft
Authors: Montolio Marta , Téllez Noèlia , Biarnés Montserrat , Soler Joan , Montanya Eduard
Published: April 2008

In the initial days after transplantation islets are particularly vulnerable and show increased apoptosis and necrosis. We have studied the effects of caspase inhibition on this early beta cell death in syngeneically transplanted islets. Streptozotocin-diabetic C57BL/6 mice were transplanted with 150 syngeneic islets, an insufficient mass to restore normoglycemia, preincubated with or without the pan-caspase inhibitor z-VAD. fmk 2 h before transplantation. Beta cell apoptosis was increased in control islets on day 3 after transplantation (0.28 ± 0.02%) compared with freshly isolated islets (0.08 ± 0.02%, p< 0.001), and was partially reduced in transplanted islets preincubated with z-VAD.fmk 200 μM (0.14 ± 0.02%, p = 0.003) or with z-VAD.fmk 500 μM (0.17 ± 0.01%, p = 0.012), but not with a lower z-VAD.fmk (100 μM) concentration. Diabetic mice transplanted with islets preincubated with z-VAD.fmk 500 μM showed an improved metabolic evolution compared with control and z-VAD.fmk 200 μM groups. The z-VAD.fmk 500 μM group showed an overall lower blood glucose after transplantation (p = 0.02), and at the end of the study blood glucose values were reduced compared with transplantation day (15.7 ± 3.6 vs. 32.5 ± 0.5 mmol/L, p = 0.001). In contrast, blood glucose was not significantly changed in control and z-VAD.fmk 200 μM groups. Four weeks after transplantation beta cell mass was higher in z-VAD.fmk 500 μM group (0.15 ± 0.02 mg) than in the control group (0.10 ± 0.02 mg) (p = 0.043). In summary, the treatment of freshly isolated islets with the caspase inhibitor z-VAD.fmk reduced the subsequent apoptosis of the islets once they were transplanted and improved the outcome of the graft.
Central Necrosis in Isolated Hypoxic Human Pancreatic Islets: Evidence for Postisolation Ischemia
Authors: Giuliani Mauro , Moritz Wolfgang , Bodmer Elvira , Dindo Daniel , Kugelmeier Patrick , Lehmann Roger , Gassmann Max , Groscurth Peter , Weber Markus
Published: April 2008

A variety of explanations have been provided to elucidate the requirement of the large islet mass that is essential for a successful treatment of patients with type I diabetes by intrahepatic transplantation. The purpose of this study was to investigate islet cell survival under the effect of prolonged hypoxia and/or nutrient withdrawal, which mimics posttransplantation environment of transplanted islets in the liver. We studied the influence of 24 h of hypoxia (1% O2) in intact isolated human and rat islets as well as the effect of combined oxygen/nutrient deprivation in a mouse insulinoma cell line (MIN6). In intact human islets, 24 h of hypoxia led to central necrosis combined with apoptotic features such as nuclear pyknosis and DNA fragmentation. In the course of hypoxic treatment, ultrastructural analysis demonstrated a gradual transition from an apoptotic to a necrotic morphology particularly pronounced in central areas of large islets. In MIN6 cells, on the other hand, hypoxia led to a twofold (p < 0.01) increase in caspase-3 activity, an indicator of apoptosis, but not to necrosis, as determined by release of lactate dehydrogenase (LDH). Only in combination with nutrient/serum deprivation was a marked increase in LDH release observed (sixfold vs. control, p < 0.01). We therefore conclude that, similar to MIN6 cells, central necrosis in isolated hypoxic islets is the result of the combined effects of hypoxia and nutrient/serum deprivation, most likely due to limited diffusion. Provided that transplanted islets undergo a similar fate as shown in our in vitro study, future emphasis will require the development of strategies that protect the islet graft from early cell death and accelerate the revascularization process.
Transplantation of Fetal Kidney Cells: Neuroprotection and Neuroregeneration
Authors: Chiang Yung-Hsiao , Borlongan Cesario V. , Zhou Feng C. , Hoffer Barry J. , Wang Yun
Published: April 2008

Various trophic factors in the transforming growth factor-β (TGF-β) superfamily have been reported to have neuroprotective and neuroregenerative effects. Intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) or bone morphogenetic proteins (BMPs), both members of the TGF-β family, reduce ischemia- or 6-hydroxydopamine (6-OHDA)-induced injury in adult rat brain. Because BMPs and GDNF are highly expressed in fetal kidney cells, transplantation of fetal kidney tissue could serve as a cellular reservoir for such molecules and protect against neuronal injury induced by ischemia, neurotoxins, or reactive oxygen species. In this review, we discuss preclinical evidence for the efficacy of fetal kidney cell transplantation in neuroprotection and regeneration models.
Liver Repopulation after Hepatocellular Transplantation: Integration and Interaction of Transplanted Hepatocytes in the Host
Authors: Koenig Sarah , Stoesser Claudia , Krause Petra , Becker Heinz , Markus Peter M.
Published: April 2008

The mechanisms of donor hepatocyte integration into recipient liver are not fully understood. We investigated mechanisms of both the integration and interaction of transplanted hepatocytes with host liver cells as well as the repopulation of the host organ following intraportal transplantation. Mature hepatocytes were injected into the portal vein of dipeptidylpeptidase IV (DPPIV)-deficient rats pretreated with retrorsine and subjected to 30% partial hepatectomy to ensure selective donor growth. The degree of integration and proliferation was studied by colocalizing transplanted cells (DPPIV positive) with connexin 32, MMP-2, and OX-43 (multilayer immunofluorescence imaging). FACS analysis was established to assess the extent of repopulation quantitatively. Transplanted hepatocytes reached the distal portal spaces and sinusoids within 1 h after injection. A small proportion of cells succeeded in traversing the endothelial barrier through mechanical disruption in both locations. Transplanted hepatocytes lost their membrane-bound gap junctions (connexin 32) during this process. Successful integration of the donor cells required up to 5 days, heralded by gap junction reconstitution and the specific basolateral membrane expression of DPPIV. MMP-2 degraded the extracellular matrix in close proximity to donor cells, providing space for cell division. FACS analysis revealed that more than 37% of the liver was repopulated by cells derived from donors at 2 months after transplantation. Our data demonstrate a high degree of donor cell repopulation of the host organ and provide valuable insight into the specific mechanisms of donor cell integration. Connexin 32 expression in transplanted hepatocytes may serve as an indicator of their effective incorporation and communication within the recipient liver. FACS analysis reveals an accurate method to determine quantitatively the extent of liver repopulation.
In Vitro Functionality of Human Fetal Liver Cells and Clonal Derivatives under Proliferative Conditions
Authors: Deurholt Tanja , Ten Bloemendaal Lysbeth , Chhatta Aniska A. , Van Wijk Albert C. W. A. , Weijer Kees , Seppen Jurgen , Elferink Ronald P. J. Oude , Chamuleau Robert A. F. M. , Hoekstra Ruurdtje
Published: April 2008

Mature human hepatocytes are not suitable for large-scale in vitro applications that rely on hepatocyte function, due to their limited availability and insufficient proliferation capacity in vitro. In contrast, human fetal liver cells (HFLC) can be easily expanded in vitro. In this study we evaluated the hepatic function of HFLCs under proliferative conditions, to determine whether HFLCs can replace mature hepatocytes for in vitro applications. HFLCs were isolated from fetal livers of 16 weeks gestation. Hepatic functions of HFLCs were determined in primary culture and after expansion in vitro. Clonal derivatives were selected and tested for hepatic functionality. Results were compared to primary mature human hepatocytes in vitro. No differences were observed between primary HFLCs and mature human hepatocytes in albumin production and mRNA levels of various liver-specific genes. Ureagenesis was 4.4-fold lower and ammonia elimination was absent in HFLCs. Expanding HFLCs decreased hepatic functions and increased cell stretching. In contrast, clonal derivatives had stable functionality and morphology and responded to differentiation stimuli. Although their hepatic functions were higher than in passaged HFLCs, functionality was at least 20 times lower compared to mature human hepatocytes. HFLCs cannot replace mature human hepatocytes in in vitro applications requiring extensive in vitro expansion, because this is associated with decreased hepatic functionality. Selecting functional subpopulations can, at least partly, prevent this. In addition, defining conditions that support hepatic differentiation is necessary to obtain HFLC cultures suitable for in vitro hepatic applications.
Transplantation of Bioreactor-Produced Neural Stem Cells into the Rodent Brain
Authors: Mcleod M. , Hong M. , Sen A. , Sadi D. , Ulalia R. , Behie L. A. , Mendez I.
Published: April 2008

The development of new cell replacement strategies using neural stem cells (NSC) may provide an alternative and unlimited cell source for clinical neural transplantation in neurodegenerative diseases such as Parkinson's and Huntington's disease. The clinical application of neural transplantation using NSC will therefore depend upon the availability of clinical grade NSC that are generated in unlimited quantities in a standardized manner. In order to investigate the utility of NSC in clinical neural transplantation, undifferentiated murine NSC were first expanded for an extended period of time in suspension bioreactors containing a serum-free medium. Following expansion in suspension bioreactors, NSC were still able to differentiate in vitro into both astrocytes and neurons after exposure to brain-derived neurotrophic factor (BDNF), suggesting that bioreactor expansion does not alter cell lineage potentiality. Undifferentiated bioreactor-expanded NSC were then transplanted into the rodent striatum. Immunohistochemical examination revealed undifferentiated bioreactor-expanded NSC survived transplantation for up to 8 weeks and expressed the astrocytic immunohistochemical marker glial fibrillary acidic protein (GFAP), suggesting that the host striatal environment influences NSC cell fate upon transplantation. Moreover, no tumor formation was observed within the graft site, indicating that NSC expanded in suspension bioreactors for an extended period of time are a safe source of tissue for transplantation. Future studies should focus on predifferentiating NSC towards specific neuronal phenotypes prior to transplantation in order to restore behavioral function in rodent models of neurodegenerative disease.
Amelioration of Diabetes in Mice after Single-Donor Islet Transplantation Using the Controlled Release of Gelatinized FGF-2
Authors: Rivas-Carrillo Jorge David , Navarro-Alvarez Nalu , Soto-Gutierrez Alejandro , Okitsu Teru , Chen Yong , Tabata Yasuhiko , Misawa Haruo , Noguchi Hirofumi , Matsumoto Shinichi , Tanaka Noriaki , Kobayashi Naoya
Published: April 2008

Fibroblast growth factor (FGF)-2 has been recognized to be a key element involved in angiogenesis and a putative factor involved in stem cell-mediated islet regeneration. However, the usefulness of FGF-2 in an islet transplantation setting has not yet been explored. We therefore evaluated the effect of FGF-2 on both islet culture and islet transplantation. Isolated islets were cultured in the presence of 100 ng/ml FGF-2 for a week and then the glucose-responding insulin secretion and insulin contents were measured. Gelatinized FGF-2 (100 ng), which allowed the controlled release of FGF-2, was used for islet transplantation of streptozotocin-induced diabetic mice. Islets (150 IEQ), obtained from a single donor, mixed with gelatinized FGF-2, were transplanted into the subrenal capsule of the mice and the animals were observed for 30 days. Revascularization around the islet grafts was examined. The blood glucose levels were measured and the intraperitoneal glucose tolerance test (IPGTT) was performed. The supplementation of FGF-2 maintained proper insulin secretion and insulin contents in an in vitro culture. The use of gelatinized FGF-2 facilitated revascularization and favorable islet engraftment, thus resulting in an amelioration of the blood glucose levels in diabetic mice. The utilization of FGF-2 showed increased contents of insulin in the islet grafts and revealed a similar pattern as that of normal healthy mice in IPGTT. In contrast, the transplantation of islets without FGF-2 supplementation showed poor revascularization and failed to control the blood glucose levels in the diabetic mice.
Functional Hepatocyte Culture and its Application to Cell Therapies
Authors: Tanaka Kimiaki , Soto-Gutierrez Alejandro , Navarro-Alvarez Nalu , Rivas-Carrillo Jorge David , Jun Hee-Sook , Kobayashi Naoya
Published: April 2008

Since Berry and Friend developed methods to isolate hepatocytes from the liver by a collagenase digestion technique in 1969, studies in laboratory animals have demonstrated that hepatocyte transplantation could potentially be used for the treatment of liver failure and inborn errors of liver-based metabolism. Healthy human hepatocytes are an ideal source for hepatocyte transplantation; however, their relative scarcity is one of the major drawbacks, further compounded by the competing demands of liver transplantation. Notably, most of the hepatocytes are isolated from discarded livers that are not suitable for organ transplantation for a variety of reasons, including excessive fat content. Importantly, the hepatocyte isolation procedure itself exerts major stress on hepatocytes by the disruption of cell-to-cell and cell-to-matrix contacts, resulting in hepatocytic apoptosis. Prevention of apoptosis would maximize yield of healthy cells and maintain hepatocyte differentiated function in culture. In this review, we describe methods to prevent apoptosis by utilizing both antiapoptotic molecules and matrices. We also introduce a new type of liver tissue engineering, hepatocyte sheet transplantation, which utilizes unwoven cloth having a cellular adhesive property.
Double-Compartment Cell Culture Apparatus: Construction and Biochemical Evaluation for Bioartificial Liver Support
Authors: Takahashi Masahiro , Sakurai Masayuki , Enosawa Shin , Omasa Takeshi , Tsuruoka Shuichi , Matsumura Toshiharu
Published: April 2008

Functional demands on a bioartificial liver support (BAL) device are not limited to biosynthetic activities, but must also encompass metabolic removal of potentially toxic substances. For most BALs, however, the concept and design are exclusively directed to biosynthetic support. To add the ability to metabolize and remove toxic substances, we designed a double-compartment cell culture apparatus (DCCA). Two compartments are separated from each other by a compact epithelial cell sheet spread over a synthetic microporous membrane. When a renal proximal convoluted tubular cell line that had been transduced with the human multidrug-resistant (MDR) gene, PCTL-MDR, was introduced into one of the compartments (hereafter referred to as the “inner” compartment) of the DCCA, a compact cellular monolayer was formed on the membrane. Ammonium ions passed across the membrane, but glucose and its metabolite lactate could not, indicating that the DCCA allowed selective transportation of cellular metabolites. In addition to PCTL-MDR, HepG2, a cell line of hepatic-origin, transduced with CYP3A4 (designated GS-3A4-HepG2), was seeded on the opposite side of the membrane, and the metabolism and transportation of lidocaine were studied. The lidocaine metabolite, monoethylglycinexylidide, was detected in the inner compartment across the PCTL-MDR cell layered membrane, indicating that metabolism and the selective transportation of metabolites between the two compartments occurred by cooperation of renal and hepatic cells. These results suggest that this type of DCCA represents a novel BAL that possesses biotransporting activities, as well as biosynthetic and metabolic activities.