bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Therapy for Parkinson's Disease: Only Young Onset Patients Allowed? Reflections about the Results of Recent Clinical Trials with Cell Therapy and the Progression of Parkinson's Disease
Authors: Linazasoro Gurutz
Published: April 2008

The selection of the best candidates for surgery among Parkinson's disease (PD) patients is a debated topic. This could be particularly important for transplantation studies in which patients with advanced PD and motor complications refractory to conventional pharmacological treatments are usually included. The development of lesions in nondopaminergic structures, which apparently are unaffected by the intervention, could eventually lead to the appearance of disabling, treatment-resistant symptoms. This has been considered as the crucial factor responsible for the outcome of any therapeutic procedure. However, other factors might be involved. It is suggested in this article that the rate of progression of PD and the effects of ageing are more important than the extradopaminergic involvement in the final outcome. Rate of progression of PD is critically related to the power of compensatory mechanisms, which are age related and under the control of still unknown genes. Thus, patients with young onset parkinsonism (YOP), either caused by gene mutations or not, could be the best candidates for surgery because they have a slower disease progression and more competent compensatory mechanisms. On the other hand, this can also explain the appearance of unexpected side effects such as the “runaway” dyskinesias reported following transplantation.
Suppression of Human T-Cell Responses to β-Cells by Activation of B7-H4 Pathway
Authors: Ou Dawei , Wang Xiaojie , Metzger Daniel L. , Ao Ziliang , Pozzilli Paolo , James Roger F. L. , Chen Lieping , Warnock Garth L.
Published: April 2008

B7-H4, a recently described member of the B7 family of cosignal molecules, is thought to be involved in the regulation of cellular and humoral immune responses through receptors on activated T and B cells. Human islet cells express positive B7-H4 mRNA in RT-PCR assays, but not B7-H4 protein on cell surface in flow cytometric analyses. To investigate the regulatory effects of activation of the B7-H4 pathway on the function of activated T cells of patients with type 1 diabetes (T1D), we have used our in vitro human experimental system, including human β-cell antigen-specific T-cell clones and human β-cell lines CM and HP62, as well as primary islet cells. B7-H4.Ig protein was purified from the culture supernatant of 293T cells transfected by a B7-H4.Ig plasmid (pMIgV, containing a human B7-H4 cDNA and a mouse IgG2a Fc cDNA). Our preliminary studies showed that immobilized fusion protein human B7-H4.Ig (coated with 5 μg/ml for 2 h at 37°C), but not control Ig, clearly inhibited the proliferation of activated CD4+ and CD8+ T cells of patients induced by anti-CD3 antibody in CFSE assays. B7-H4.Ig also arrested cell cycle progression of T cells in G0/G1 phase and induced T-cell apoptosis as measured by BrdU-7-AAD flow cytometric analysis. To determine the cytoprotective effects of B7-H4, we developed transfectants of human β-cell lines CM and HP62 and islet cells transfected with the B7-H4.Ig plasmid, using empty vector transfectants as controls. The results demonstrate that cell-associated B7-H4.Ig expressed on human β-cells clearly inhibits the cytotoxicity of the T-cell clones to targeted human β-cells in 51Cr release cytotoxicity assays. Activation of the B7-H4 pathway may represent a novel immunotherapeutic approach to inhibit T-cell responses for the prevention of β-cell destruction in T1D.
Ectopic Ossification in the Scar Tissue of Rats with Myocardial Infarction
Authors: Ribeiro Karla Consort , Mattos Elisabete César , Werneck-de-Castro João Pedro Saar , Ribeiro Vanessa Pinho , Costa-e-Sousa Ricardo Henrique , Miranda Amarildo , Olivares Emerson Lopes , Farina Marcos , Mill José Geraldo , dos Santos Goldenberg Regina Coeli , Masuda Masako Oya , de Carvalho Antônio Carlos Campos
Published: April 2008

We describe the occurrence of bone-like formations in the left ventricular wall of infarcted rats treated or not with bone marrow cells injected systemically or locally into the myocardium. The incidence of ectopic calcification in hearts has been reported in rare cases in children with infarcts without previous coronary artery disease. Recently, ventricular calcification has been correlated with unselected bone marrow cell transplantation into infarcted rat hearts. Echocardiographic analysis of large infarction in rats frequently reveals the presence of echogenic structures in the left ventricular wall, sometimes projecting to the lumen of the chamber. The histological examination of these echogenic structures exhibited bone, cartilage, and marrowlike formations extending from the collagen-rich matrix of the ventricle wall. Microanalytical techniques verified the presence of hydroxyapatite in the mineral phase. Ossification was found in 25 out of 30 hearts evaluated 90 days postinfarct, being observed in 14 out of 17 animals submitted to cell therapy and in 11 out of 13 infarcted rats not submitted to cell therapy. Our study indicates that chondro-osteogenic differentiation can take place in the pathological rat heart independent of animal treatment with marrow cells.
The Development and Characterization of an HEK293-Derived Cell Line for Use in an Intratumoral Cytokine Delivery System
Authors: Hamilton M. J. , Huang Q. X. , Li C. L. , Ellem K. A. O.
Published: April 2008

As part of ongoing work to develop a method of cytokine delivery for use as an intratumoral depot, we noted that HEK293 cells, encapsulated in alginate, died within 24–48 h after in vivo, intratumoral implantation. We hypothesized that the highly hypoxic and acidic conditions found inside the tumor was the cause of the cells' premature demise. Therefore, we set out to develop a cell line, derived from HEK293, that would survive these hostile conditions. The HEK293 line was selected in 0.3–0.5% oxygen conditions over several weeks, followed by a further 6-week period of culture in alternating hypoxic and normoxic conditions. The most rapidly growing clones were selected and grown in normoxic conditions for several weeks to ensure their stability. The clones were then compared to the original line in terms of cell proliferation in normoxia and hypoxia, colony-forming efficiency, and morphological characteristics. The resulting line was able to proliferate in the harshest of conditions and continues to release its biological payload after alginate microencapsulation.
Survival and Function of Transplanted Islet Cells on an in Vivo, Vascularized Tissue Engineering Platform in the Rat: A Pilot Study
Authors: Brown David L. , Meagher Peter J. , Knight Kenneth R. , Keramidaris Effie , Romeo-Meeuw Rosalind , Penington Anthony J. , Morrison Wayne A.
Published: April 2008

As in vivo tissue engineering of complex tissues and organs progresses, there is a need for an independently vascularized, alterable, and recoverable model. Current models of islet cell transplantation (release into the portal venous system, placement under the renal capsule, and microencapsulation) lack these qualities. We have developed a model of angiogenesis and spontaneous tissue generation in the rat that lends itself as a potential platform for tissue engineering. In this experiment, we examined the effectiveness of such a model in addressing some of the shortcomings of endocrine pancreatic transplantation. An arteriovenous loop was created in the groins of five adult inbred Sprague-Dawley rats, and placed within polycarbonate chambers. Isolated pancreatic islet cell clusters were placed within the chambers, suspended in a matrix of Matrigel®. The chambers were recovered at 3 weeks, and the newly generated tissue was processed for histologic and immunohistochemical analysis. By 3 weeks, spontaneous generation of angiogenesis and collagen matrix and deposition of a collagen matrix was observed. Surviving islet cells were identified by histology and their viability was confirmed via immunohistochemistry for insulin and glucagon. This study demonstrates the ability to maintain viability and functionality of transplanted islet cells on a tissue-engineered platform with an independent vascular supply. The model provides the ability to alter the graft environment via matrix substitution, cellular coculture, and administration of growth factors. The transplanted tissues are recoverable without animal sacrifice and are microsurgically transferable. This model may provide an in vivo culture platform for the study of islet transplantation.
Porcine Islet Graft Function is Affected by Pretreatment with a Caspase-3 Inhibitor
Authors: Brandhorst Daniel , Kumarasamy Vidya , Maatoui Adel , Alt Alexandra , Bretzel Reinhard G. , Brandhorst Heide
Published: April 2008

During the isolation procedure and after transplantation islets are subjected to numerous variables associated with the induction of apoptosis. The present study investigated the effect of transient pretreatment with caspase inhibitors on function and survival of transplanted pig islets. Isolated porcine islets (3000 IEQ) were incubated overnight in 200 μM of the caspase-3 inhibitor DEVD-CMK prior to transplantation into diabetic nude mice. Glucose-stimulated insulin release of pretreated islets was assessed during static incubation. DEVD-CMK successfully prevented the expression of capase-3 and DFF as demonstrated in heat-shocked pig islets. Nevertheless, transient pretreatment of freshly isolated pig islets with DEVD-CMK resulted in a significantly decreased final graft function of 50.0% (n = 16) compared to 85.7% (n = 14) in control islets (p < 0.05). Glucose-stimulated insulin release of porcine islets (n = 6) was not significantly effected by overnight culture with DEVD-CMK. Morphological assessment revealed that this caspase-3 inhibitor significantly increased the percentage of necrosis to a small, but nevertheless significant, extent in comparison to control islets (p < 0.05). The study demonstrates that short-time pretreatment with the caspase-3 inhibitor DEVD-CMK reduces the capacity of transplanted porcine islets to restore normoglycemia in diabetic nude mice.
Differentiation of Human Embryonic Stem Cells to Hepatocytes Using Deleted Variant of HGF and Poly-amino-urethane-Coated Nonwoven Polytetrafluoroethylene Fabric
Authors: Soto-Gutierrez Alejandro , Navarro-Alvarez Nalú , Rivas-Carrillo Jorge David , Chen Yong , Yamatsuji Tomoki , Tanaka Noriaki , Kobayashi Naoya
Published: April 2008

Human embryonic stem (hES) cells have recently been studied as an attractive source for the development of a bioartificial liver (BAL). Here we evaluate the differentiation capacity of hES cells into hepatocytes. hES cells were subjected to suspension culture for 5 days, and then cultured onto poly-amino-urethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric in the presence of fibroblast growth factor-2 (bFGF) (100 ng/ml) for 3 days, then with deleted variant of hepatocyte growth factor (dHGF) (100 ng/ml) and 1% dimethyl sulfoxide (DMSO) for 8 days, and finally with dexamethasone (10–7 M) for 3 days. The hES cells showed gene expression of albumin in a time-dependent manner of the hepatic differentiation process. The resultant hES-derived hepatocytes metabolized the loaded ammonia and lidocaine at 7.8% and 23.6%, respectively. A million of such hepatocytes produced albumin and urea at 351.2 ng and urea at 7.0 μg. Scanning electron microscopy showed good attachment of the cells on the surface of the PTFE fabric and well-developed glycogen rosettes and Gap junction. In the present work we have demonstrated the efficient differentiation of hES cells to functional hepatocytes. The findings are useful to develop a BAL.
Survival of Microencapsulated Islets at 400 Days Posttransplantation in the Omental Pouch of NOD Mice
Authors: Kobayashi Tsunehiro , Aomatsu Yukio , Iwata Hiroo , Kin Tatsuya , Kanehiro Hiromichi , Hisanga Michiyoshi , Ko Saiho , Nagao Mitsuo , Harb George , Nakajima Yoshiyuki
Published: April 2008

The long-term durability of agarose microencapsulated islets against autoimmunity was evaluated in NOD mice. Islets were isolated from 6-8-week-old prediabetic male NOD mice and microencapsulated in 5% agarose hydrogel. Microencapsulated or nonencapsulated islets were transplanted into the omental pouch of spontaneously diabetic NOD mice. Although the diabetic NOD mice that received nonencapsulated islets experienced a temporary reversal of their hyperglycemic condition, all 10 of these mice returned to hyperglycemia within 3 weeks. In contrast, 9 of 10 mice transplanted with microencapsulated islets maintained normoglycemia for more than 100 days. Islet grafts were removed at 100, 150, 200, 300, and 400 days posttransplantation. A prompt return to hyperglycemia was observed in the mice after graft removal, indicating that the encapsulated islet grafts were responsible for maintaining euglycemia. Histological examination revealed viable islets in the capsules at all time points of graft removal. In addition, β-cells within the capsules remained well granulated as revealed by the immunohistochemical detection of insulin. No immune cells were detected inside the microcapsules and no morphological irregularities of the microcapsules were observed at any time point, suggesting that the microcapsules successfully protected the islets from cellular immunity. Sufficient vascularization was evident close to the microcapsules. Considerable numbers of islets showed central necrosis at 400 days posttransplantation, although the necrotic islets made up only a small percentage of the islet grafts. Islets with central necrosis also showed abundant insulin production throughout the entire islets, except for the necrotic part. These results demonstrate the long-term durability of agarose microcapsules against autoimmunity in a syngeneic islet transplantation model in NOD mice.
Progressive Dopamine Neuron Loss in Parkinson's Disease: The Multiple Hit Hypothesis
Authors: Carvey Paul M. , Punati Ashok , Newman Mary B.
Published: April 2008

Animal models have been an essential tool for researchers and clinicians in their efforts to study and treat Parkinson's disease (PD). Thus, the various ways 6-hydroxydopamine is employed, the use of MPTP in rodents and nonhuman primates, the prenatal exposure to bacterial endotoxin, the postnatal exposure to environmental toxins such as paraquat and rotenone, the assessment of dopamine (DA) neurons in genetic knockout mouse, and even the behavioral analysis of fruit flies and worms have added significantly to our knowledge base of PD—or have they? Are these animal models manifesting a true model of PD? Have the 7786 published studies (to date) on PD with animal models led to a clearer understanding of its etiology, treatment, or progression? In this review we critically assess this question. We begin with a succinct history of the major contributions, which have led to the current animal models of PD. We then evaluate the primary issue of the progressive loss of DA neurons, which, except for a few studies, has not been addressed in animal models of PD, even though this is the major pathological characteristic of the disease. Lastly, we discuss the possibility that more than one risk factor for PD may be necessary to develop an animal model that shows synergy—the progressive loss of DA neurons. Thus, the multiple hit hypothesis of PD—that is, the effect of more then one risk factor—may be the start of new era in animal models of PD that is one step closer to mimicking the pathology of PD in humans.
Timing of Cord Blood Treatment after Experimental Stroke Determines Therapeutic Efficacy
Authors: Newcomb Jennifer D. , Ajmo Craig T. , Sanberg Cyndy D. , Sanberg Paul R. , Pennypacker Keith R. , Willing Alison E.
Published: April 2008

Embolic stroke is thought to cause irreparable damage in the brain immediately adjacent to the region of reduced blood perfusion. Therefore, much of the current research focuses on treatments such as anti-inflammatory, neuroprotective, and cell replacement strategies to minimize behavioral and physiological consequences. In the present study, intravenous delivery of human umbilical cord blood cells (HUCBC) 48 h after a middle cerebral artery occlusion (MCAo) in a rat resulted in both behavioral and physiological recovery. Nissl and TUNEL staining demonstrated that many of the neurons in the core were rescued, indicating that while both necrotic and apoptotic cell death occur in ischemia, it is clear that apoptosis plays a larger role than first anticipated. Further, immunohistochemical and histochemical analysis showed a diminished and/or lack of granulocyte and monocyte infiltration and astrocytic and microglial activation in the parenchyma in animals treated with HUCBC 48 h poststroke. Successful treatment at this time point should offer encouragement to clinicians that a therapy with a broader window of efficacy may soon be available to treat stroke.
Pig-to-Nonhuman Primate Islet Xenotransplantation: A Review of Current Problems
Authors: Rood P. P. M. , Buhler L. H. , Bottino R. , Trucco M. , Cooper D. K. C.
Published: April 2008

Islet allotransplantation has been shown to have potential as a treatment for type 1 diabetic patients. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited number of islets. This comprehensive review focuses on experience obtained in pig-to-nonhuman primate models, particularly with regard to the different types of islets (fetal, neonatal, adult) and isolation procedures used, and the methods to determine islet viability. The advantages and disadvantages of the methods to induce diabetes (pancreatectomy, streptozotocin) are discussed. Experience in pig-to-nonhuman primate islet transplantation studies is reviewed, including discussion of the possible mechanisms of rejection and the immunosuppressive regimens used. The research carried out to date has led to workable animal models to study islet xenotransplantation, but several questions regarding methodology remain unanswered, and details of these practicalities require to be adequately addressed. The encouraging porcine islet survival reported recently provides an indicator for future immunosuppressive regimens.
One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B12 and Heparin
Authors: Yamazoe Hironori , Kobori Masato , Murakami Yoshinobu , Yano Keiichi , Satoh Mitsuo , Mizuseki Kenji , Sasai Yoshiki , Iwata Hiroo
Published: April 2008

We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B12 and heparin, but without any expensive cytokines. After 14 days in culture, β-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B12/heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B12/heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B12/heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.
Estimation of Pancreas Weight from Donor Variables
Authors: Kin Tatsuya , Murdoch Travis B. , Shapiro A. M. James , Lakey Jonathan R. T.
Published: April 2008

Previous studies have identified several donor factors affecting the outcome of islet isolation. Pancreas weight has not been considered as a donor selection criterion, because a value cannot be obtained prior to organ procurement. However, a larger pancreas will likely contain a higher number of islets. Therefore, the prediction of pancreas weight would be helpful in donor selection, benefiting cost and efficiency of the islet isolation laboratory. The purpose of this study was to investigate normal pancreas weight in cadaveric donors and identify pancreas weight predictors from demographic data of cadaveric organ donors. We retrospectively analyzed data on pancreas weight from 354 cadaveric donors with respect to gender, age, body weight, body height, body mass index (BMI), and body surface area (BSA). In men, pancreas weight correlated more closely with body weight than with age, height, or BMI. BSA was as strong a correlate of pancreas weight as body weight. In women, pancreas weight had a similar pattern of relationships, with generally lower correlation coefficients. On the basis of the observation of gender-specific pancreas weight difference in elderly donors, stepwise multiple linear regression analyses were conducted separately for younger (≥40 years) and elderly (≤41 years) donors. In younger donors, body weight and age were the major predictors of pancreas weight [pancreas weight (g) = 4.355 + 0.742 × body weight (kg) + 0.837 × age (years) (R2 = 0.564, p < 0.001)]. In contrast, pancreas weight of elderly donors was best predicted by BSA and gender [pancreas weight (g) =-17.624 + 60.036 × BSA (m2) − 7.152 × gender (R2 = 0.372, p< 0.001; “gender”: 1 = female, 0 = male)]. Pancreas weight was found to be positively associated with pre- and postpurification islet yields. These formulae should contribute to the estimation of pancreas weight, and thus improve donor selection for islet isolation and transplantation.
Coinhibitory T-Cell Signaling in Islet Allograft Rejection and Tolerance
Authors: Truong Wayne , Hancock Wayne W. , Anderson Colin C. , Merani Shaheed , Shapiro A. M. James
Published: April 2008

Autoaggressive T cells directed against insulin secreting pancreatic β-cells mediate the development of type 1 diabetes. Islet transplantation offers superior glycemic control over exogenous insulin, but chronic immunosuppression limits its broad application. Pathogenic T cells are also important in allograft rejection. Inducing and maintaining antigen-specific peripheral T-cell tolerance toward β-cells is an attractive strategy to prevent autoimmune disease, and to facilitate treatment of diabetes with islet allografts without long-term immunosuppression. Recent efforts have focused on blocking costimulatory T-cell signals for tolerance induction. Although costimulatory blockade can prolong graft survival, true immunological tolerance remains elusive. Costimulatory signals may even be required for the maintenance of peripheral tolerance. The discovery of novel coinhibitory T-cell pathways, including CTLA-4, PD-1, and BTLA, offers an alternative approach. Stimulating negative T cell cosignals alone or in combination may help induce tolerance. The focus of this review is to summarize the strategies directed at turning off the immune response by exploiting these negative cosignaling pathways in tolerance induction in islet transplantation. Activating several coinhibitory pathways together may be synergistic in preventing pathogenic T-cell responses. Tolerance induction will likely rely on understanding the balance of positive and negative signals affecting the state of T-cell activation.
Genetic Modification of Hepatocytes towards Hepatocyte Transplantation and Liver Tissue Engineering
Authors: Kuge Hiroyuki , Ohashi Kazuo , Yokoyama Takashi , Kanehiro Hiromichi , Hisanaga Michiyoshi , Koyama Fumikazu , Bumgardner Ginny L. , Kosai Ken-Ichiro , Nakajima Yoshiyuki
Published: April 2008

Cell-based therapies, including liver tissue engineering following hepatocyte transplantation, have therapeutic potential for several types of liver diseases. Modifications in the methodology to manipulate the donor hepatocytes in a more simple and timely manner prior to transplantation would enhance the therapeutic efficacy of this procedure. Conventional approach for vector-mediated gene transduction to the isolated hepatocytes has been performed under primary culture conditions that routinely require several days to complete. In our study, we have established a clinically feasible approach that requires only 1 h of infection time with an adenoviral vector system that results in an extremely efficient transduction efficiency (>80%). To optimize transduction efficiency and sustain normal cellular function, we determined that the isolated hepatocytes should be maintained in UW solution as a suspension medium and infected with adenoviral vectors (Ad) for no more than 1 h at a MOI of 1. To establish if the isolated hepatocytes could be used as a source for cell-based therapies, we transplanted the Ad-transduced hepatocytes into the liver or under the kidney capsule. When the cells were transplanted into the liver, Ad-transduced hepatocytes cultured in suspension conditions were found to have a significantly higher survival rate (p < 0.01) than Ad-transduced hepatocytes cultured under standard conditions. We also confirmed that these Ad-transduced hepatocytes have ability to survive long term and were able to engineer a biologically active hepatic tissue under the kidney capsule. Finally, we obtained high level of transduction into canine, porcine, and human isolated hepatocytes in a suspension solution mixed with Ad. In conclusion, the present studies demonstrate that isolated hepatocytes could be genetically modified using Ad when kept in a suspension solution. For this reason, this cell-modified technique could be used for the treatment of liver-targeted diseases and/or disorders.
Effects of Simulated Microgravity on the Morphology and Function of Neonatal Porcine Cell Clusters Cultured with and without Sertoli Cells
Authors: Luca G. , Calvitti M. , Nastruzzi C. , Macchiarulo G. , Becchetti E. , Neri L. M. , Capitani S. , Basta G. , Brunetti P. , Calafiore R. , Cameron D. F.
Published: April 2008

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of β-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation β-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.
Molecular Imaging Reveals Skeletal Engraftment Sites of Transplanted Bone Marrow Cells
Authors: Mayer-Kuckuk Philipp , Doubrovin Mikhail , Bidaut Luc , Budak-Alpdogan Tulin , Cai Shangde , Hubbard Vanessa , Alpdogan Onder , Van Den Brink Marcel , Bertino Joseph R. , Blasberg Ronald G. , Banerjee Debabrata , Gelovani Juri
Published: April 2008

Molecular imaging holds great promise for the in vivo study of cell therapy. Our hypothesis was that multimodality molecular imaging can identify the initial skeletal engraftment sites post-bone marrow cell transplantation. Utilizing a standard mouse model of bone marrow (BM) transplantation, we introduced a combined bioluminescence (BLI) and positron emission tomography (PET) imaging reporter gene into mouse bone marrow cells. Bioluminescence imaging was used for monitoring serially the early in vivo BM cell engraftment/expansion every 24 h. Significant cell engraftment/expansion was noted by greatly increased bioluminescence about 1 week posttransplant. Then PET was applied to acquire three-dimensional images of the whole-body in vivo biodistribution of the transplanted cells. To localize cells in the skeleton, PET was followed by computed tomography (CT). Co-registration of PET and CT mapped the sites of BM engraftment. Multiple, discrete BM cell engraftment sites were observed. Taken together, this multimodality approach may be useful for further in vivo characterization of various therapeutic cell types.
Bioreactors for Extracorporeal Liver Support
Authors: Gerlach Jörg C.
Published: April 2008

Hybrid extracorporeal liver support is an option to assist liver transplantation therapy. An overview on liver cell bioreactors is given and our own development is described. Furthermore, the prospects of the utilization of human liver cells from discarded transplantation organs due to steatosis, cirrhosis, or traumatic injury, and liver progenitor cells are discussed. Our Modular Extracorporeal Liver Support (MELS) concept proposes an integrative approach for the treatment of hepatic failure with appropriate extracorporeal therapy units, tailored to suit the actual clinical needs of each patient. The CellModule is a specific bioreactor (charged actually with primary human liver cells, harvested from human donor livers found to be unsuitable for transplantation). The DetoxModule enables albumin dialysis for the removal of albumin-bound toxins, reducing the biochemical burden of the liver cells and replacing the bile excretion of hepatocytes in the bioreactor. A Dialysis Module for continuous veno-venous hemofiltration can be added to the system if required in hepato-renal syndrome.
Towards the Development of a Pediatric Ventricular Assist Device
Authors: Borovetz Harvey S. , Badylak Stephen , Boston J. Robert , Johnson Carl , Kormos Robert , Kameneva Marina V. , Simaan Marwan , Snyder Trevor A. , Tsukui Hiro , Wagner William R. , Woolley Joshua , Antaki James , Diao Chenguang , Vandenberghe Stijn , Keller Bradley , Morell Victor , Wearden Peter , Webber Steven , Gardiner Jeff , Li Chung M. , Paden Dave , Paden Bradley , Snyder Shaun , Wu Jingchun , Bearnson Gill , Hawkins John A. , Jacobs Gordon , Kirk John , Khanwilkar Pratap , Kouretas Peter C. , Long James , Shaddy R. E.
Published: April 2008

The very limited options available to treat ventricular failure in children with congenital and acquired heart diseases have motivated the development of a pediatric ventricular assist device at the University of Pittsburgh (UoP) and University of Pittsburgh Medical Center (UPMC). Our effort involves a consortium consisting of UoP, Children's Hospital of Pittsburgh (CHP), Carnegie Mellon University, World Heart Corporation, and LaunchPoint Technologies, Inc. The overall aim of our program is to develop a highly reliable, biocompatible ventricular assist device (VAD) for chronic support (6 months) of the unique and high-risk population of children between 3 and 15 kg (patients from birth to 2 years of age). The innovative pediatric ventricular assist device we are developing is based on a miniature mixed flow turbodynamic pump featuring magnetic levitation, to assure minimal blood trauma and risk of thrombosis. This review article discusses the limitations of current pediatric cardiac assist treatment options and the work to date by our consortium toward the development of a pediatric VAD.
The Use of Extracellular Matrix as an Inductive Scaffold for the Partial Replacement of Functional Myocardium
Authors: Badylak Stephen F. , Kochupura Paul V. , Cohen Ira S. , Doronin Sergey V. , Saltman Adam E. , Gilbert Thomas W. , Kelly Damon J. , Ignotz Ronald A. , Gaudette Glenn R.
Published: April 2008

Regenerative medicine approaches for the treatment of damaged or missing myocardial tissue include cell-based therapies, scaffold-based therapies, and/or the use of specific growth factors and cytokines. The present study evaluated the ability of extracellular matrix (ECM) derived from porcine urinary bladder to serve as an inductive scaffold for myocardial repair. ECM scaffolds have been shown to support constructive remodeling of other tissue types including the lower urinary tract, the dermis, the esophagus, and dura mater by mechanisms that include the recruitment of bone marrow-derived progenitor cells, angiogenesis, and the generation of bioactive molecules that result from degradation of the ECM. ECM derived from the urinary bladder matrix, identified as UBM, was configured as a single layer sheet and used as a biologic scaffold for a surgically created 2 cm2 full-thickness defect in the right ventricular free wall. Sixteen dogs were divided into two equal groups of eight each. The defect in one group was repaired with a UBM scaffold and the defect in the second group was repaired with a Dacron patch. Each group was divided into two equal subgroups (n = 4), one of which was sacrificed 15 min after surgical repair and the other of which was sacrificed after 8 weeks. Global right ventricular contractility was similar in all four subgroups groups at the time of sacrifice. However, 8 weeks after implantation the UBM-treated defect area showed significantly greater (p < 0.05) regional systolic contraction compared to the myocardial defects repaired with by Dacron (3.3 ± 1.3% vs. −1.8 ± 1.1%; respectively). Unlike the Dacron-repaired region, the UBM-repaired region showed an increase in systolic contraction over the 8-week implantation period (–4.2 ± 1.7% at the time of implantation vs. 3.3 ± 1.3% at 8 weeks). Histological analysis showed the expected fibrotic reaction surrounding the embedded Dacron material with no evidence for myocardial regeneration. Histologic examination of the UBM scaffold site showed cardiomyocytes accounting for approximately 30% of the remodeled tissue. The cardiomyocytes were arranged in an apparently randomly dispersed pattern throughout the entire tissue specimen and stained positive for α-sarcomeric actinin and Connexin 43. The thickness of the UBM graft site increased greatly from the time of implantation to the 8-week sacrifice time point when it was approximately the thickness of the normal right ventricular wall. Histologic examination suggested complete degradation of the originally implanted ECM scaffold and replacement by host tissues. We conclude that UBM facilitates a constructive remodeling of myocardial tissue when used as replacement scaffold for excisional defects.
Vascular Tissue Engineering
Authors: Arrigoni Chiara , Camozzi Davide , Remuzzi Andrea
Published: April 2008

Reconstructive surgery using autologous vessels is the conventional approach for substitution of diseased vessels or for generation of bypass to improve blood supply downstream of stenosed vessels. In some circumstances the use of autologous material is not possible due to concomitant diseases or previous use, and artificial grafts must be used. Unfortunately, these grafts cannot substitute small-caliber arterial vessels because of thrombotic complications. The objective of tissue engineering at the vascular level is then to generate biological substitutes of arterial conduits with functional characteristics of native vessels, combining cellular components with biodegradable scaffolds. These research projects started in several laboratories, in the late 1990s, and have expanded in different directions using a number of experimental approaches. The objective of this review is to give an overview of the results so far obtained in this area of research, and to discuss the problems related to these investigations, at the experimental and clinical level. The article provides an overview of different biodegradable scaffolds used, experimental techniques for vessels maturation in vitro under mechanical stimulation, and of differentiated as well as precursors of vascular cells, which opens new opportunities for further development of this form of cell transplantation. Finally, the current available results in clinical research will be discussed.
Comparison of Successful and Unsuccessful Islet/Sertoli Cell Cotransplant Grafts in Streptozotocin-Induced Diabetic Mice
Authors: Dufour Jannette M. , Lord Sarah J. , Kin Tatsuya , Rayat Gina R. , Dixon Doreen E. , Bleackley R. Chris , Korbutt Gregory S. , Rajotte Ray V.
Published: April 2008

Sertoli cells (SC) protect islet allografts from immune destruction in diabetic rodents. In this study, we examined the difference between successful and rejected islet/SC cografts in order to further improve this procedure for optimal extension of islet allograft survival. We cotransplanted 500 BALB/c islets with 1–8 million BALB/c SC under the kidney capsule of diabetic BALB/c, C3H-HeJ, and C57BL/6 mice. Cotransplantation of islets with up to 8 million SC was not detrimental to long-term islet graft function in syngeneic mice. However, large numbers of SC were detrimental to islet graft survival in allogeneic mice with the optimal dose for cotransplantation of 4 or 1 million SC in C3H-HeJ or C57BL/6 mice, respectively. Examination of successful grafts, from euglycemic recipients, revealed the presence of SC arranged in tubule structures with islets surrounding these tubules. Cellular infiltrate in successful grafts revealed CD4 T cells and macrophages along the periphery and within the grafts, and very few CD8 T cells. Conversely, examination of unsuccessful grafts, harvested from hyperglycemic recipients at the time of rejection, revealed the presence of SC arranged randomly with islets adjacent to the Sertoli cells, when present, and massive CD4 and CD8 T cell as well as macrophage cell infiltration. Prolongation of islet allograft survival appeared to be a function of SC transplant mass and recipient genetic background. A consequence of long-term graft acceptance is the formation of SC tubule structures, which may be an additional requirement for optimal protection of islet allografts.
Long-Term Culture of Japanese Human Embryonic Stem Cells in Feeder-Free Conditions
Authors: Navarro-Alvarez Nalú , Soto-Gutierrez Alejandro , Yuasa Takesui , Yamatsuji Tomoki , Shirakawa Yasuhiro , Nagasaka Takeshi , Sheng Sun D , Javed Muhammad Shahid , Tanaka Noriaki , Kobayashi Naoya
Published: April 2008

Human pluripotent embryonic stem cells (hESCs) have great promise for research into human developmental biology, development of cell therapies for the treatment of diseases, toxicology, and drug discovery. Traditionally, undifferentiated hESCs are maintained on mouse embryonic fibroblasts (MEFs), which impede the clinical applications of hESCs. Here we have examined the long-term stability of the Japanese hESC line (KhES-1) in feeder-free culture. KhES-1 cells were cultured with MEF conditioned medium (CM) and different doses of basic fibroblast growth factor (bFGF) in six-well-plates of which the surface was coated with Matrigel. KhES-1 cells were maintained for at least 40 passages. In this culture system, the cells maintained stable proliferation rates and steadily expressed Oct-4, Nanog, and alkaline phosphatase. In addition, KhES-1 cells maintained without direct feeder contact formed embryonic bodies with expression of markers from the three germ layers. Here we demonstrated that Japanese human embryonic stem cells KhES-1 were cultured long term in a feeder-free method, while retaining pluripotency in vitro.
The Local Injection of Peritoneal Macrophages Induces Neovascularization in Rat Ischemic Hind Limb Muscles
Authors: Hirose Nobuyuki , Maeda Hironori , Yamamoto Morio , Hayashi Yoshihiro , Lee Gang-Hong , Chen Liyan , Radhakrishnan Geethalakshmi , Rao Parijatha , Sasaguri Shiro
Published: April 2008

Macrophages play a pivotal role in the development of newly formed vascular networks, in addition to their normal immunological functions. This research focuses on peritoneal macrophages as a novel source in cell implantation therapy for ischemic diseases. In this study, production of angiogenic growth factors by peritoneal macrophages and its in vivo effect of neovascularization were evaluated. Mononuclear cells from the peritoneal cavity (P-MNCs) enriched with macrophages were isolated and stimulated with hypoxia and interleukin-1β (IL-1β) to mimic an ischemic tissue environment in vitro. Expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) of mRNA in P-MNCs was apparently enhanced by hypoxic stimulation, and the production of VEGF protein was also augmented by hypoxia and IL-1β. A rat ischemic hind limb model was created and P-MNCs (8 × 106/limb) were injected into the ischemic muscles. The blood flow, which was assessed using the colored microsphere method, showed that the percentage blood flow was significantly increased by P-MNCs injection 4 weeks after surgery (48.3 ± 16.8% in noninjected ischemic limb vs. 84.3 ± 13.0% in the P-MNCs-injected limb). A histological analysis revealed that the number of capillaries detected by alkaline phosphatase staining was increased in the P-MNCs group 4 weeks after injection. Furthermore, the number of α-smooth muscle actin-positive vessels also showed a significant increase following P-MNC injection. The injected P-MNCs labeled with fluorescence were detected in the interstitial space of ischemic muscles, and VEGF protein expression of the implanted cells was confirmed by immunohistochemistry. These results indicate that peritoneal macrophages stimulate capillary formation and arteriogenesis in the ischemic limbs, possibly through the production of angiogenic growth factors. These findings suggest that the physiological angiogenic property of peritoneal macrophages could therefore be utilized for neovascularization in cell implantation therapy.
Green Tea Polyphenols Affect Skin Preservation in Rats and Improve the Rate of Skin Grafts
Authors: Kawazoe Takeshi , Kim Hakhee , Tsuji Yasumi , Morimoto Naoki , Hyon Suong-Hyu , Suzuki Shigehiko
Published: April 2008

Green tea polyphenols have been recently reported to promote the preservation of tissues, such as blood vessels, corneas, nerves, islet cells, articular cartilage, and myocardium, at room temperature. These findings indicate the possibility of a new method of tissue banking without freezing. A main active ingredient of green tea, epigallocatechin-3-gallate (EGCG), is a polyphenol that possesses antioxidant, antimicrobial, antiproliferative, and free radical scavenging effects. This study examined the effects of EGCG regarding skin preservation. Skin sample biopsy specimens measuring 1 × 1 cm from GFP rats were held in sterile containers with 50 ml preserving solution at 4°C and 37°C for up to about 8 weeks. Periodically, some of the preserved skin specimens were directly examined histologically and others were transplanted into nude mice. Histological examinations of skin preserved at 4°C revealed a degeneration of the epidermal and dermal layers from 5 weeks in all groups. In the groups preserved at 37°C, degeneration and flakiness of the epidermal layer were demonstrated starting at 2 weeks preservation regardless of addition of EGCG. After 2–7 weeks of preservation the rat skin grafted to nude mice in the EGCG groups stored at 4°C showed successful engraftment. However, grafts preserved at 4°C without EGCG and at 37°C did not demonstrate GFP-positive keratinocyte or fibroblasts. In conclusion, the present findings suggest the future clinical usefulness of EGCG for skin preservation without freezing; however, the mechanism by which EGCG promotes skin preservation still remains unclear.
Amelioration of Cisplatin-Induced Acute Renal Injury by Renal Progenitor-Like Cells Derived from the Adult Rat Kidney
Authors: Kinomura Masaru , Kitamura Shinji , Tanabe Katsuyuki , Ichinose Kunihiro , Hirokoshi Kumiko , Takazawa Yuki , Kitayama Hiroyuki , Nasu Tatsuyo , Sugiyama Hitoshi , Yamasaki Yasushi , Sugaya Takeshi , Maeshima Yohei , Makino Hirofumi
Published: April 2008

The replacement of a necrotic tubular epithelium with functional tubular epithelial cells is required for recovery from acute renal failure (ARF). A rat renal progenitor-like (rKS56) cell line was recently established derived from the S3 segment of renal proximal tubules. The therapeutic efficacy of rKS56 cells was examined in a rat model of cisplatin-induced ARF. rKS56-lacZ cells expressing β-galactosidase were injected into SD rats either at the subcapsule of the left kidney (rKS-SC) or via the left renal artery (rKS-IA) 2 days after the injection of cisplatin. Bluo-gal(+) rKS56-lacZ cells were observed in the subcapsule in the rKS-SC group on day 5, and were further increased in number on day 9, accompanied by partial distribution in the corticomedullary junction, but not in the rKS-IA group. A portion of Bluo-gal(+) cells coexpressed Ki-67, aquaporin-1, hepatocyte growth factor (HGF), and c-Met. rKS-SC treatment significantly improved the tubular injury scores, ameliorated tubular cell apoptosis, and induced cell proliferation. The renal function also significantly improved in the rKS-SC group on day 5. These results demonstrate that locally implanted rKS56 cells could differentiate into tubular epithelial cells, thereby accelerating the recovery from tubular injury, most likely by producing tubular trophic factors. These results suggest the therapeutic potential of this novel approach for patients with end-stage renal failure.