bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolated Hepatocyte Transplantation for Crigler-Najjar Syndrome Type 1
Authors: Ambrosino Giovanni , Varotto Sergio , Strom Stephen C. , Guariso Graziella , Franchin Elisa , Miotto Diego , Caenazzo Luciana , Basso Stefano , Carraro Paolo , Valente Maria Luisa , D'amico Davide , Zancan Lucia , D'antiga Lorenzo
Published: April 2008

Crigler-Najjar syndrome type 1 (CN1) is an inherited disorder characterized by the absence of hepatic uridine diphosphoglucuronate glucuronosyltransferase (UDPGT), the enzyme responsible for the conjugation and excretion of bilirubin. We performed allogenic hepatocyte transplantation (AHT) in a child with CN1, aiming to improve bilirubin glucuronidation in this condition. A 9-year-old boy with CN1 was prepared with plasmapheresis and immunosuppression with prednisolone and tacrolimus. When a graft was made available, 7.5 × 109 hepatocytes were isolated and infused into the portal vein percutaneously. After 2 weeks phenobarbitone was added to promote the enzymatic activity of UDPGT of the transplanted hepatocytes. Nocturnal phototherapy was continued throughout the studied period. Total bilirubin was considered a reliable marker of allogenic cell function. There was no significant variation of vital signs nor complications during the infusion. Mean ± SD bilirubin level was 530 ± 38 μmol/L before and 359 ± 46 μmol/L after AHT (t-test, p < 0.001). However, the introduction of phenobarbitone was followed by a drop of tacrolimus level with increase of alanine aminotransferase (ALT) and increase of bilirubin. After standard treatment of cellular rejection bilirubin fell again but from then on it was maintained at a greater level. After discharge the patient experienced a further increase of bilirubin that returned to predischarge levels after readmission to the hospital. This was interpreted as poor compliance with phototherapy. Only partial correction of clinical jaundice and the poor tolerability to nocturnal phototherapy led the parents to refuse further hepatocyte infusions and request an orthotopic liver transplant. After 24 months the child is well, with good liver function on tacrolimus and prednisolone-based immunosuppression. Isolated AHT, though effective and safe, is not sufficient to correct CN1. Maintenance of adequate immunosuppression and family compliance are the main factors hampering the success of this procedure.
Monocrotaline, an Alternative to Retrorsine-Based Hepatocyte Transplantation in Rodents
Authors: Witek Rafal P. , Fisher Samantha H. , Petersen Bryon E.
Published: April 2008

Retrorsine has been used extensively to inhibit proliferation of resident hepatocytes in various transplantation models. Here we report a successful alternative to currently unavailable retrorsine that can be used in cellular transplantation models. Based on structural and molecular similarities, we investigate the use of monocrotaline (MCT) in cell transplantation studies in rodents. In this study, MCT was given to rats intraperitoneally in two injections 2 weeks apart. Two weeks after the final injection, a partial hepatectomy followed by splenic hepatocyte transplantation was performed. The results indicate that MCT, at two doses of 30 mg/kg, highly enhances liver repopulation by donor hepatocytes following partial hepatectomy and produces 15.3 ± 4.9% liver repopulation within the first 6 weeks following transplantation. Additionally, we tested the effectiveness of MCT in a murine model. Using two injections of 50 mg/kg each, given 2 weeks apart, hepatocyte proliferation in the native liver was inhibited and subsequent oval cell transplants engrafted at 18 ± 21.3% after 16 weeks posttransplantation. In conclusion, MCT can be used as an effective selective pressure for donor hepatocytes in cell transplantation to the liver in rodents.
hVEGF165Increases Survival of Transplanted Hepatocytes Within Portal Radicles: Suggested Mechanism for Early Cell Engraftment
Authors: Shani-Peretz Hagit , Tsiperson Vladislav , Shoshani Gideon , Veitzman Ella , Neufeld Gera , Baruch Yaacov
Published: April 2008

VEGF is a potent angiogenic factor that promotes hepatocyte growth, increases permeability of blood vessels, and induces vasodilatation, and may accelerate engraftment and function of transplanted hepatocytes. The aim was to study the effect of VEGF on early hepatocyte engraftment. Thirty-two Lewis syngeneic female rats underwent 70% partial hepatectomy. Eighteen received 240 ng VEGF165and 14 received saline for control. Thereafter, intrasplenic transplantation of 107 male hepatocytes was done. Semiquantitative analysis of PCR product of the SRY region of the Y-chromosome was performed. Paraffin-embedded sections were stained for H&E and for PCNA immunostaining. By PCR, male hepatocytes were identified in 8 livers out of 14 VEGF-treated rats at 24 – 48 h, compared with only 1 liver out of 8 controls. Transplanted cells were seen within portal vessels radicles in 7 out of 14 VEGF-treated rats for as long as 48 h posttransplantation, compared with only one control liver at 24 h. There was no histological sign of cell injury to transplanted or adjacent cells. Two weeks after transplantation male transplanted cells were identified in two out of four rats treated with hVEGF165and in one out of six rats treated with saline. No transplanted cells were detected within portal tracts 14 days after transplantation. hVEGF165enhances the presence of transplanted hepatocytes within portal vessels after transplantation. We suggest an additional mechanism for cell engraftment, whereby transplanted hepatocytes first stick to each other in the portal radicles. Later they become included in the liver parenchyma as groups of organized cells in a process stimulated by VEGF.
Instant Hepatic Differentiation of Human Embryonic Stem Cells Using Activin a and a Deleted Variant of HGF
Authors: Chen Yong , Soto-Gutierrez Alejandro , Navarro-Alvarez Nalu , Rivas-Carrillo Jorge David , Yamatsuji Tomoki , Shirakawa Yasuhiro , Tanaka Noriaki , Kobayashi Naoya
Published: April 2008

Human embryonic stem (hES) cells have the ability to differentiate into a variety of different cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. Here we investigated an efficient method of hepatic differentiation from hES cells. A human ES cell line, KhES-1, was used and maintained by a nonfeeder method. KhES-1 cells were cultured for 5 days in the presence of human activin A (50 ng/ml) and then treated with a deleted variant of hepatocyte growth factor (dHGF) at 0, 100, or 500 ng/ml for 7 days. The resultant cells were biologically analyzed. The expression of the endodermal genes SOX17 and FOXA2 increased in KhES-1 cells after activin A treatment. In contrast, Oct4, a self-renewal undifferentiated marker, decreased in a time-dependent manner in KhES-1 cells. Following a 7-day treatment of the resultant cells with dHGF, especially at 500 ng/ml, KhES-1 cells showed an expression of the hepatic makers albumin, AFP, and CK18. Transitional electron microscopy showed well-developed glycogen rosettes and a gap junction in KhES-1 cells treated with 500 ng/ml of dHGF. We developed an efficient method to differentiate KhES-1 cells into hepatocyte-like cells in vitro using 50 ng/ml of activin A and 500 ng/ml of dHGF.
The Significant Improvement of Survival Times and Pathological Parameters by Bioartificial Liver with Recombinant HepG2 in Porcine Liver Failure Model
Authors: Enosawa Shin , Miyashita Tomoyuki , Saito Tomohiro , Omasa Takeshi , Matsumura Toshiharu
Published: April 2008

We developed a bioartificial liver (BAL) containing human hepatoblastoma cell line, HepG2, with the addition of ammonia removal activity by transfecting a glutamine synthetase (GS) gene and estimated the efficacy using pigs with ischemic liver failure. GS-HepG2 cells showed 15% ammonia removal activity of porcine hepatocytes, while unmodified HepG2 had no such activity. The established GS-HepG2 cells were grown in a circulatory flow bioreactor to 3.5–4.1 × 109 cells. Survival time of the animals treated with GS-HepG2 BAL was significantly prolonged compared to the cell-free control (14.52 ± 5.24 h vs. 8.53 ± 2.52 h) and the group treated with the BAL consisting of unmodified wild-type HepG2 (9.58 ± 4.52 h). Comparison showed the cell-containing BAL groups to have significantly fewer incidences of increased brain pressure. Thus, the GS-HepG2 BAL treatment resulted in a significant improvement of survival time and pathological parameters in pigs with ischemic liver failure.
Cell Therapy for Diabetes Mellitus
Authors: Kobayashi Naoya
Published: April 2008

The number of diabetic patients in the world is increasing in recent years and the prevention of diabetes mellitus is therefore one of the urgent medical issues. Exogenous insulin is used for the control of blood glucose in diabetic patients; however, hypoglycemic episodes are unavoidable. Over the last several decades, islet transplantation has been developed as a promising method to achieve strict control of blood glucose and a potential cure for type 1 diabetes. However, due to the shortage of donor pancreata, alternative sources of islets have been sought through the generation of beta cells from stem cells, use of porcine islets, and beta cell expansion with growth factors. However, differentiation and expansion of embryonic and pancreatic stem cells and expansion of differentiated beta cells in vitro is limited. Expansion of primary beta cells by growth factors is also hampered by the senescence of the cells. Thus, we focused on establishing a human pancreatic beta cell line that is functionally equivalent to primary beta cells and can yield large amounts of cells for transplantation. Using Cre/loxP-based reversible immortalization, we constructed a reversibly immortalized pancreatic beta cell clone (NAKT-15). The cells may overcome the limitation of primary pancreatic beta cells for transplantation to control type 1 diabetes. In order to avoid the use of immunosuppressive agents, we are currently engaged in developing an implantable bag-type bioartificial pancreas. In this article, I discuss the hurdles of the current therapy for diabetes and introduce the possible future treatment of diabetes.
Induction of Pancreatic Stem/Progenitor Cells into Insulin-Producing Cells by Adenoviral-Mediated Gene Transfer Technology
Authors: Noguchi Hirofumi , Xu Gang , Matsumoto Shinichi , Kaneto Hideaki , Kobayashi Naoya , Bonner-Weir Susan , Hayashi Shuji
Published: April 2008

β-Cell replacement therapy via islet transplantation is a promising possibility for the optimal treatment of type 1 diabetes; however, such an approach is severely limited by the shortage of donor organs. This problem could be overcome if it were possible to generate transplantable islets from stem cells. We showed previously that adult β-cells might originate from duct or duct-associated cells. Ductal progenitor cells in the pancreas would become particularly useful for therapies that target β-cell replacement in diabetic patients, because duct cell types are abundantly available in the pancreas of these patients and in donor organs. In this study, we examined which embryonic transcription factors in adult mouse and human duct cells could efficiently induce their differentiation into insulin-expressing cells. Infection with the adenovirus expressing PDX-1, Ngn3, NeuroD, or Pax4 induced the insulin gene expression. NeuroD was the most effective inducer of insulin expression in primary duct cells. Surprisingly, adenovirus Pax4 strongly induced Ngn3 expression, while Pax4 is considered the downstream target of Ngn3. These data suggest that the overexpression of transcription factors, especially NeuroD, facilitates pancreatic stem/progenitor cell differentiation into insulin-producing cells.
Development, Characterization, and Use of a Fetal Skin Cell Bank for Tissue Engineering in Wound Healing
Authors: De Buys Roessingh Anthony S. , Hohlfeld Judith , Scaletta Corinne , Hirt-Burri Nathalie , Gerber Stefan , Hohlfeld Patrick , Gebbers Jan-Olaf , Applegate Lee Ann
Published: April 2008

Wound healing in fetal skin is characterized by the absence of scar tissue formation, which is not dependent on the intrauterine environment and amniotic fluid. Fetal cells have the capacity of extraordinary expansion and we describe herein the development of a fetal skin cell bank where from one organ donation (2–4 cm2) it is possible to produce several hundred million fetal skin constructs of 9 × 12 cm2. Fetal cells grow three to four times more rapidly than older skin cells cultured in the same manner and these banked fetal cells are very resistant against physical and oxidative stress when compared to adult skin cells under the same culture conditions. They are up to three times more resistant to UVA radiation and two times more resistant towards hydrogen peroxide treatment. This mechanism may be of major importance for fetal cells when they are delivered to hostile wound environments. For fetal cell delivery to patients, cells were associated with a collagen matrix to form a three-dimensional construct in order to analyze the capacity of these cells for treating various wounds. We have seen that fetal cells can modify the repair response of skin wounds by accelerating the repair process and reducing scarring in severe burns and wounds of various nature in children. Hundreds of thousands of patients could potentially be treated for acute and chronic wounds from one standardized and controlled cell bank.
Survival of an Islet Allograft Deficient in iNOS after Implantation into Diabetic NOD Mice
Authors: Börjesson Andreas , Andersson Annika K. , Sandler Stellan
Published: April 2008

Proinflammatory cytokines play a major role in rejection of pancreatic islet allografts and in type 1 diabetes (T1D). In rodent islets, exposure to IL-1β alone or combined with IFN-γ induces expression of inducible nitric oxide synthase (iNOS). Inhibition of iNOS or a deletion of the iNOS gene has been shown to be protective in animal models of T1D. In the present study we tested the hypothesis that transplantation of pancreatic islets deficient in iNOS (iNOS–/–) would permit increased graft survival. Pancreatic islets isolated from wild-type (wt) mice and iNOS–/– mice were allogeneically transplanted beneath the kidney capsule of spontaneously diabetic NOD mice. When blood glucose increased above 12.0 mM after preceding normalization of hyperglycemia, animals were sacrificed. Histological examinations of grafts were performed and graft gene expression was analyzed by real-time PCR. Transplantations of the two types of islets could reverse hyperglycemia and the grafts functioned for on average 1 week posttransplantation. Morphological examination of both types of islet grafts showed immune cell infiltration around and within the grafts. Remaining endocrine cells could be observed in wt and iNOS–/– islet grafts. In the removed grafts iNOS-/islet tissue contained higher mRNA levels of insulin, proinsulin convertases (PC-1 and PC-2), and IL-1β compared to transplanted wt islets. The assessments of insulin, PC-1 and PC-2 mRNAs of the grafts suggest that the iNOS–/– islets may be more resistant to destruction in the transplantation model used; however, this was not sufficient to prolong the period of normoglycemia posttransplantation.
Retinal Progenitor Cell Xenografts to the Pig Retina: Immunological Reactions
Authors: Warfvinge Karin , Kiilgaard Jens F. , Klassen Henry , Zamiri Parisa , Scherfig Erik , Streilein Wayne , Prause Jan U. , Young Michael J.
Published: April 2008

We evaluated the host response to murine retinal progenitor cells (RPCs) following transplantation to the subretinal space (SRS) of the pig. RPCs from GFP mice were transplanted subretinally in 18 nonimmunosuppressed normal or laser-treated pigs. Evaluation of the SRS was performed on hematoxylin-eosin (H&E)-stained sections. Serum samples were taken from naive and RPC-grafted pigs and mouse-reactive antibody responses were assessed. At 1 week, histology showed a few perivascular lymphocytes consistent with a mild retinal vasculitis, and depigmentation of the RPE with large numbers of mononuclear inflammatory cells in the choroid near the transplantation site. Large choroidal infiltrates were evident at 2–5 weeks. Serum from naive and RPC-xenografted pigs contained significant levels of preformed IgG and IgM antibodies against murine antigens. Xenogeneic RPCs transplanted to the porcine SRS induced mononuclear infiltration in the choroid with graft rejection occurring over 2–5 weeks. Serum analysis confirmed that mice and pigs are discordant species; however, a cell-mediated acute mechanism appears to be responsible, rather than an antibody-mediated rejection.
Use of Repeating Dispensers to Increase the Efficiency of the Intramuscular Myogenic Cell Injection Procedure
Authors: Skuk Daniel , Goulet Marlyne , Tremblay Jacques P.
Published: April 2008

Intramuscular myoblast transplantation in humans and nonhuman primates requires precise repetitive cell injections very close to each other. Performed with syringes operated manually throughout large regions, this procedure takes a lot of time, becoming tiring and thus imprecise. We tested two repetitive dispensers with Hamilton syringes as cell injection devices to facilitate this procedure. Monkeys received intramuscular allotransplantations of β-galactosidase-labeled myoblasts, using either a monosyringe or a multisyringe repeating dispenser. The monosyringe repeating dispenser allowed performing cell injections faster and easier than with a manually operated syringe. The multisyringe dispenser accelerated the procedure still more, but it was not ergonomic. Biopsies of the myoblast-injected sites 1 month later showed abundant β-galactosidase-positive myofibers, with the same density and morphological pattern observed following myoblast transplantation with a syringe operated manually. We recommend the monosyringe repeating dispenser for myoblast transplantation in skeletal muscles and maybe in the heart.
Regeneration of Osteonecrosis of Canine Scapho-lunate Using Bone Marrow Stromal Cells: Possible Therapeutic Approach for Kienböck Disease
Authors: Ikeguchi Ryosuke , Kakinoki Ryosuke , Aoyama Tomoki , Shibata Kotaro Roberts , Otsuka Seiji , Fukiage Kennichi , Nishijo Koichi , Ishibe Tatsuya , Shima Yasuko , Otsuki Bungo , Azuma Takashi , Tsutsumi Sadami , Nakayama Tomitaka , Otsuka Takanobu , Nakamura Takashi , Toguchida Junya
Published: April 2008

We evaluated the ability of canine bone marrow stromal cells (cBMSCs) to regenerate bone in a cavity of the scapholunate created by curretage and freeze–thawing with liquid nitrogen (LN). Autologous BMSCs were harvested from the iliac crest and expanded in vitro. Their potential to differentiate into osteo-, chondro-, and adipogenic lineages was confirmed using a standard differentiation induction assay. LN-treated scapholunates showed no regeneration of bone tissue when the cavity was left alone, demonstrating severe collapse and deformity as observed in human Kienböck disease. A combination of β-tri-calcium phosphate and a vascularized bone graft with autologous fibroblasts failed to regenerate bone in the LN-treated cavity. When the same procedure was performed using BMSCs, however, LN-treated scapholunates showed no collapse and deformity, and the cavity was completely filled with normal cancerous bone within 4 weeks. These results suggested the potential of using BMSCs to treat Kienböck disease.
High Yields of Autologous Living Dermal Equivalents Using Porcine Gelatin Microbeads as Microcarriers for Autologous Fibroblasts
Authors: Liu Jin Yu , Hafner Jürg , Dragieva Galya , Burg Günter
Published: April 2008

Permanent skin replacement requires a dermal component to ensure adequate long-term graft stability and to prevent wound contraction. This study was to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous living dermal equivalents on a large scale. Autologous fibroblasts were isolated from split-thickness skin biopsy from a leg ulcer patient, inoculated onto macroporous porcine gelatin microbeads, and incubated in a bioreactor (Cellspin) in serum-free fibroblast growth medium or in DMEM medium containing 10% fetal calf serum (FCS). Fibroblasts rapidly adhered to and actively proliferated on the microbeads in the bioreactor in both serum-free and serum-containing medium. MTT assay showed the number of fibroblasts on the microbeads reached up to 5.3- or 4.0-fold the cells seeded in DMEM medium containing 10% FCS or serum-free medium, respectively. When removed from Bio-MCCS and cultured under static conditions, fibroblasts were able to leave the microbeads and proliferate to confluence on the bottom of tissue culture flasks. When stored at room temperature in DMEM containing 10% FBS, fibroblast cultured on the microbeads retained highest viabilities for at least 3 weeks, up to 82% of originals. This Bio-MCCS using porcine gelatin microbeads as carriers for fibroblasts offers a new option of mass production of autologous living dermal equivalents.
Assessment of in Vitro Applicability of Reversibly Immortalized NKNT-3 Cells and Clonal Derivatives
Authors: Hoekstra Ruurdtje , Deurholt Tanja , ten Bloemendaal Lysbeth , Desille Mireille , van Wijk Albert C. W. A. , Clement Bruno , Oude Elferink Ronald P. J. , van Gulik Thomas M. , Chamuleau Robert A. F. M.
Published: April 2008

In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4–20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.
Maintenance of Mouse, Rat, and Pig Pancreatic Islet Functions by Coculture with Human Islet-Derived Fibroblasts
Authors: Miki Atsushi , Narushima Michiki , Okitsu Teru , Takeno Yuichi , Soto-Gutierrez Alejandro , Rivas-Carrillo Jorge David , Navarro-Alvarez Nalú , Chen Yong , Tanaka Kimiaki , Noguchi Hirofumi , Matsumoto Shinichi , Kohara Michinori , Lakey Jonathan R. T. , Kobayashi Eiji , Tanaka Noriaki , Kobayashi Naoya
Published: April 2008

Development of an efficient preculture system of islets is ideal. Toward that goal, we constructed a human pancreatic islet-derived fibroblast cell line MNNK-1 for a source as a coculture system for freshly isolated islets to maintain islet functions. Human pancreatic islet cells were nucleofected with a plasmid vector pYK-1 expressing simian virus 40 large T antigen gene (SV40T) and hygromycin resistance gene (HygroR). One of the transduced cell lines, MNNK-1, was established and served as a feeder cell in the coculture for freshly isolated mouse, rat, and pig islets. Morphology, viability, and glucose-responding insulin secretion were analyzed in the coculture system. MNNK-1 cells were morphologically spindle shaped and were negative for pancreatic endocrine markers. MNNK-1 cells were positive for α-smooth muscle actin and collagen type I and produced fibroblast growth factor. Coculture of the mouse, rat, and pig islets with MNNK-1 cells maintained their viability and insulin secretion with glucose responsiveness. A human pancreatic islet-derived fibroblast cell line MNNK-1 was established. MNNK-1 cells were a useful means for maintaining morphology and insulin secretion of islets in the coculture system.
Short-Term Heart Retention and Distribution of Intramyocardial Delivered Mesenchymal Cells within Necrotic or Intact Myocardium
Authors: Tran Nguyen , Li Yan , Maskali Fatiha , Antunes Laurent , Maureira Pablo , Laurens Marie-Helene , Marie Pierre-Yves , Karcher Gilles , Groubatch Frederique , Stoltz Jean-François , Villemot Jean-Pierre
Published: April 2008

Cell therapy with bone marrow mesenchymal stem cells (BMSCs) is a new strategy for treating ischemic heart failure, but data concerning the distribution and retention of transplanted cells remain poor. We investigated the short-term myocardial retention of BMSCs when these cells are directly injected within necrotic or intact myocardium. 111Indium-oxine-labeled autologous BMSCs were injected within either 1-month-old infarction (n = 6) or normal myocardium (n = 6) from rats. Serial in vivo pinhole scintigraphy was scheduled during 1 week in order to track the implanted cells. The myocardial retention of BMSCs was definitely higher in myocardial infarction than in normal myocardial area (estimated percent retention at 2 h: 63 ± 3% vs. 25 ± 4%, p < 0.001) and the estimated cardiac retention values were unchanged in both groups along the 7 days of follow-up. On heart sections at day 7, labeled BMSCs were still around the injection site and appeared confined to the scarred tissue corresponding either to the infarct area or to the myocardium damaged by needle insertion. BMSCs have a higher retention when they are injected in necrotic than in normal myocardial areas and these cells appear to stay around the injection site for at least a 7-day period.
Genetically Engineered Human Mesenchymal Stem Cells Produce Met-Enkephalin at Augmented Higher Levels in Vitro
Authors: Sugaya Ikuko , Qu Tingyu , Sugaya Kiminobu , Pappas George D.
Published: April 2008

We have reported that transplantation of adrenal medullary chromaffin cells that release endogenous opioid peptides into pain modulatory regions in the CNS produce significant antinociceptive effects in patients with terminal cancer pain. However, the usefulness of this procedure is minimal because the availability of human adrenal tissue is very limited. Alternative xenogeneic materials, such as porcine and bovine adrenal chromaffin cells present problems of immune rejection and possible pathogenic contamination. In an attempt to develop opioid peptide-producing cells of autologous origin, we have transfected human mesenchymal stem cells (hMeSCs) with a mammalian expression vector containing a fusion gene of green fluorescent protein (GFP) and human preproenkephalin (hPPE), a precursor protein for enkephalin opioid peptides. Enkephalins are major neurotransmitters that play an important role in analgesia by activating peripheral opioid receptors. Following the establishment of stable transfection of hMeSCs, the expressions of hPPE and GFP were confirmed and the production of methionine enkephalin (Met-enkephalin) was significantly increased compared to control naive hMeSCs (p < 0.05). Our in vitro data demonstrated that genetically engineered hMeSCs with transfected hPPE gene can constitutively produce opioid peptide Met-enkephalin at an augmented high level. hMeSCs are relatively easy to isolate from a patient's bone marrow aspirates and expand in culture by repeated passages. Autologous hMeSCs would not require immunosuppression when transplanted back into the same patient. Through targeted gene manipulation such as hPPE gene transfection, this may offer a virtually unlimited safe cell supply for the treatment of opioid-sensitive pain in humans.
Quantitative Assay for Quality Assurance of Human Cells for Clinical Transplantation
Authors: Boyce Steven T. , Anderson Benjamin A. , Rodriguez-Rilo Horacio L.
Published: April 2008

Transplantation of human cells after isolation and culture has become an important alternative for treatment of acute or chronic skin wounds. To increase the efficacy and reduce cost for transplantation of skin cells, more efficient and accurate techniques for evaluation of cell proliferation are needed. Hemocytometer counts provide a valid assessment of cell proliferation and viability, but they are very labor intensive and require removal of the cells from their substrate. In this study, hemocytometer counts were compared with a fluorometric assay (n = 21 per condition) that uses the commercially available reagent alamarBlue™, which is reduced to a fluorescent substrate by cellular dehydrogenases. Human epidermal keratinocytes were inoculated at 200, 600, 2000, and 6000 cells/cm2 incubated for 6 days in modified MCDB 153 medium. Alamar Blue™ was incubated with cells for 2 h at 37°C, and fluorescence was measured with a microplate reader at 590 nm. Hemocytometer counts (×10-4) from the respective cell inoculation densities were 0.30 ± 0.04, 1.07 ± 0.10, 6.37 ± 0.62, and 16.99 ± 0.96. Fluorescence values (×10–3) for the respective inoculation densities were 0.14 ± 0.01, 0.34 ± 0.02, 1.20 ± 0.09, and 1.79 ± 0.12. Regression analysis showed a statistical significant (p < 0.0001) correlation (r2 = 0.87) between cell counts and optical density from the alamarBlue™ assay. These data demonstrate that alamarBlue™ provides a valid substitute for cell counts to assess cell proliferation before clinical transplantation of engineered skin. AlamarBlue™ also allows repeated, nondamaging assessment of living cells over time. These advantages are expected to increase the validity and reliability of quality assurance standards for transplanted skin cells, and to increase the efficacy of healing of cutaneous wounds.
Size-Dependent Revascularization of Transplanted Pancreatic Islets
Authors: Kampf Caroline , Mattsson Göran , Carlsson Per-Ola
Published: April 2008

For their survival and optimal function, pancreatic islets depend posttransplantation on a rapid and adequate revascularization. Native islets display a marked size-dependent heterogeneity in both angioarchitecture and degree of blood perfusion. This study evaluated whether there also are differences in the degree of revascularization of islets of different size when transplanted. Mouse pancreatic islets were isolated by collagenase digestion, and cultured in vitro for 4–7 days before transplantation. Groups of 200 islets with a diameter either exceeding or being below 100 μm were implanted beneath the left renal capsule of syngeneic C57 BL/6 mice. One month posttransplantation, graft-bearing kidneys were removed. Histological specimens were prepared and stained for endothelium with the lectin Bandeiraea simplicifolia. Pancreata from nontrans-planted control animals were prepared similarly. The vascular density in transplanted islets was markedly lower than in native islets. However, islet transplants composed of small islets (<100 μm in diameter) had a vascular density in the endocrine tissue twice that in transplants of larger islets (>100 μm). The connective tissue stroma surrounding smaller islets was also more revascularized than in corresponding grafts with large islets. The vascular density in the connective tissue stroma surrounding the individual islets in the grafts was markedly higher than in the endocrine parts per se. These combined observations indicate that smaller islets have a higher capacity to stimulate regrowth of blood vessels following transplantation. Further studies on islet differences with regard to revascularization capacity may teach us strategies for treatment of transplanted islets to improve their revascularization.
Functional MR Microimaging of Pancreatic β-Cell Activation
Authors: Gimi Barjor , Leoni Lara , Oberholzer Jose , Braun Mark , Avila Jose , Wang Yong , Desai Tejal , Philipson Louis H. , Magin Richard L. , Roman Brian B.
Published: April 2008

The increasing incidence of diabetes and the need to further understand its cellular basis has resulted in the development of new diagnostic and therapeutic techniques. Nonetheless, the quest to noninvasively ascertain β-cell mass and function has not been achieved. Manganese (Mn)-enhanced MRI is presented here as a tool to image β-cell functionality in cell culture and isolated islets. Similar to calcium, extracellular Mn was taken up by glucose-activated β-cells resulting in 200% increase in MRI contrast enhancement, versus nonactivated cells. Similarly, glucose-activated islets showed an increase in MRI contrast up to 45%. Although glucose-stimulated Ca influx was depressed in the presence of 100 μM Mn, no significant effect was seen at lower Mn concentrations. Moreover, islets exposed to Mn showed normal glucose sensitivity and insulin secretion. These results demonstrate a link between image contrast enhancement and β-cell activation in vitro, and provide the basis for future noninvasive in vivo imaging of islet functionality and β-cell mass.
Engineered Cell Therapy for Sustained Local Myocardial Delivery of Nonsecreted Proteins
Authors: Bian Jing , Kiedrowski Matt , Mal Niladri , Forudi Farhad , Penn Marc S.
Published: April 2008

Novel strategies for the treatment of congestive heart failure have taken the form of gene and cell therapy to induce angiogenesis, optimize calcium handling by cardiac myocytes, or regenerate damaged myocardial tissue. Arguably both gene- and cell-based therapies would be benefited by having the ability to locally deliver specific transcription factors and other usually nonsecreted proteins to cells in the surrounding myocardial tissue. The herpes simplex virus type 1 (HSV-1) tegument protein VP22 has been shown to mediate protein intercellular trafficking to mammalian cells and finally localize into the nucleus, which makes it a useful cargo-carrying functional protein in cell-based gene therapy. While VP22 has been studied as a means to modulate tumor growth, little is known about the distribution and transport kinetics of VP22 in the heart and its potential application in combination with autologous cell transplantation for the delivery of proteins to myocardial tissue. The aim of this study was to evaluate the efficacy of VP22 fusion protein intercellular trafficking combined with autologous cell transplantation in the heart. In an in vitro study untransfected rat heart cells were cocultured with stably transfected rat cardiac fibroblasts (RCF) with fusion constructs of VP22. The control experiment was untransfected rat heart cells co-plated with RCF stably transfected with enhanced green fluorescence protein (eGFP). The Lewis rat model was selected for in vivo study. In the in vitro studies there was a 14-fold increase in the number of GFP-positive cells 48 h after initiating coculture with VP22-eGFP RCF compared to eGFP RCF. In the rat model, transplantation of VP22-eGFP expressing RCF led to VP22-eGFP fusion protein delivery to an area of myocardial tissue that was 20-fold greater than that observed when eGFP RCF were transplanted. This area appeared to reach a steady state between 7 and 10 days after transplantation. The VP22-eGFP area consisted of eGFP-positive endothelium, smooth muscle cells, and cardiac myocytes with delivery to an area of approximately 1 mm2 of myocardial tissue. Our data suggest a viable strategy for the delivery of proteins that are not naturally secreted or internalized, and provide the first insight into the feasibility and effectiveness of cell-penetrating proteins combined with cell transplantation in the heart.
Analysis of the Effects of Cryopreservation on Rat Hepatocytes Using SELDI-TOF Mass Spectrometry
Authors: Terry Claire , Bailey Matthew , Mitry Ragai R. , Lehec Sharon C. , Dhawan Anil , Hughes Robin D.
Published: April 2008

Successful cryopreservation of hepatocytes is essential to the future of hepatocyte transplantation as a treatment for liver disease, and also for the improved in vitro use of hepatocytes for research. However, hepatocyte function is adversely affected by even the best cryopreservation protocols. To investigate possible mechanisms for these changes, total cell lysates were prepared from fresh and cryopreserved rat hepatocytes and the proteome profiles compared using SELDI-TOF-MS ProteinChip? technology. In addition, in vitro functional assays (viability, attachment efficiency, and lactate dehydrogenase leakage) were performed on the corresponding fresh and cryopreserved hepatocytes. Sixty-one peptides were identified as being significantly changed after cryopreservation. Thirty-seven peaks were significantly increased and 24 were significantly decreased after cryopreservation. The peak intensity of a number of these peptides was found to correlate with the in vitro function of the hepatocytes. Seven peptides correlated with in vitro function after cryopreservation and 10 peptides correlated with both fresh and cryopreserved function. The peptides significantly decreased after cryopreservation could include cytosolic enzymes or cofactors, which leaked out of the cells due to cryopreservation-induced membrane damage. The peptides significantly increased after cryopreservation could be retained products of cleavage of larger intracellular polypeptides and proteins or the result of aggregation of peptides caused by physical changes in the cell due to the cryopreservation process. Proteome profiling using SELDI-TOF-MS could be a useful tool to assess the effects of isolation and cryopreservation of hepatocytes, particularly if the findings are extended to human hepatocytes.
Growth Factor Enhancement of Cardiac Regeneration
Authors: Rosenthal Nadia , Santini Maria Paola , Musarò Antonio
Published: April 2008

The potential for endogenous or supplementary stem cells to restore the form and function of damaged tissues is particularly promising for overcoming the restricted regenerative capacity of the mammalian heart. To maintain blood circulation, this essential organ needs to launch a rapid response to repair damage of the muscle wall and to prevent muscle loss. The capacity of growth factors to supplement the repair process has been successfully applied to restore the integrity of damaged skeletal muscle, reducing the fibrotic response to injury, and recruiting local populations of self-renewing precursor cells and circulating stem cells. We review the recent evidence that extension of growth factor supplementation to the heart may overcome its inherent regenerative impediments through improvement of the local tissue environment and stimulation of cell replacement, and we speculate on future research directions for treatment of myocardial damage.
Protein Transduction: A Novel Approach to Induce In Vitro Pancreatic Differentiation
Authors: Domínguez-Bendala Juan , Pastori Ricardo L. , Ricordi Camillo , Inverardi Luca
Published: April 2008

It is widely believed that human embryonic stem (huES) cells may represent a valid alternative to donor pancreata as a source of islets for transplantation. Much is known about the transcription factors whose sequential activation results in the generation of islets during pancreatic development. This knowledge has been used to articulate the theoretical possibility that such process might be recapitulated in vitro from stem cells. However, our understanding of the extracellular signals that prompt the developing pancreas to follow this sequence of molecular events is very limited. Also, the simplicity of in vitro systems makes it difficult, if not impossible, to mimic the complex signaling pattern observed in living embryos. Protein transduction (PT) technology may provide researchers with a new powerful tool to sequentially induce stem cell differentiation, entirely bypassing the need for unraveling the signaling pattern that drives the process in vivo. Here we discuss this novel application of the flourishing PT technology, which may revolutionize the way we direct stem cells along any specific lineage.
Flexible Lineage Specifications of Adult Hepatic Cells, Associated Molecular Pathways, and Their Relationship to Liver Cancer
Authors: Michalopoulos George K.
Published: April 2008

The availability of new cell culture, cell transplantation, and gene expression technology has highlighted a role of hepatocytes as facultative stem cells for the biliary epithelium. In addition, hepatocytes can revert to primitive progenitor cell types and express gene profiles that appear as characteristic “signatures” in hepatocellular carcinomas.
Wound Healing in the Biliary Tree of Liver Allografts
Authors: Demetris A. J. , Fontes Paulo , Lunz John G. , Specht Susan , Murase Noriko , Marcos Amadeo
Published: April 2008

An increasing need for liver transplantation requires evaluation and triage of organs harvested from “extended criteria” donors. Although there is currently no widely accepted definition, most would agree that “extended criteria” includes organs donated by individuals that are old (>65 years), obese, infected with HBV or HCV, non-heart beating (NHBD), or had an unstable blood pressure before harvesting or the organ experienced a long cold ischemic time. These organs carry a statistical risk of dysfunction early after transplantation, but in the majority of recipients, hepatic parenchymal function recovers. Later, however, a small but significant percentage of extended criteria donors develop biliary strictures within several months after transplantation. The strictures occur primarily because of preservation injury that leads to “ischemic cholangitis” or deep wounding of the bile duct wall. Subsequent partial wound healing and wound contraction, but failed restitution of the biliary epithelial cell (BEC) lining, result in biliary tract strictures that cause progressive biliary fibrosis, increased morbidity, and decreased organ half-life. Better understanding of the pathophysiologic mechanisms that lead to biliary strictures in extended criteria donors provides an ideal proving ground for regenerative medicine; it also can provide insights into other diseases, such as extrahepatic biliary atresia and primary sclerosing cholangitis, that likely share certain pathogenic mechanisms. Possible points of therapeutic intervention include limiting cold and warm ischemic times, donor and/or donor organ treatment, ex vivo, to minimize the ischemic/preservation injury, maximize blood flow after transplantation, promote BEC wound healing, and limit myofibroblasts activation and proliferation in the bile duct wall. The pathobiology of biliary wound healing and therapeutic potential of interleukin-6 (IL-6) are highlighted.
Hepatocyte Transplantation: Clinical Experience and Potential for Future Use
Authors: Strom Stephen C. , Bruzzone Paolo , Cai Hongbo , Ellis Ewa , Lehmann Thomas , Mitamura Keitaro , Miki Toshio
Published: April 2008

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. There are three main areas where the transplantation of isolated hepatocytes has been proposed and used for clinical therapy. Cell transplantation has been used: 1) for temporary metabolic support of patients in end-stage liver failure awaiting whole organ transplantation, 2) as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and 3) in a manner similar to gene therapy, as a “cellular therapy” for patients with genetic defects in vital liver functions. We will briefly review the basic research that leads to clinical hepatocyte transplantation, the published clinical experience with this experimental technique, and some possible future uses of hepatocyte transplantation.
Tissue Engineering and the Challenges Within
Authors: Wargo Sara L. , Kumar Thangappan Ravi , Russell Alan J.
Published: April 2008

Researchers face many challenges, both scientific and societal, in the field of tissue engineering. Herein we discuss the challenges in material design, selection of therapeutic cell source, the in vitro culturing of cells and materials, and finally the integration of the cultured construct into the body. We focus special attention on a new approach to the design of a biomaterial that would bridge synthetic and biologic materials seamlessly. The scaffolds we have developed serve as a transitional material between biotic and abiotic systems.
Development of Composite Porous Scaffolds Based on Collagen and Biodegradable Poly(ester urethane)urea
Authors: Guan Jianjun , Stankus John J. , Wagner William R.
Published: April 2008

Our objective in this work was to develop a flexible, biodegradable scaffold for cell transplantation that would incorporate a synthetic component for strength and flexibility and type I collagen for enzymatic lability and cytocompatibility. A biodegradable poly(ester urethane)urea was synthesized from poly(caprolactone), 1,4-diisocyanatobutane, and putrescine. Using a thermally induced phase separation process, porous scaffolds were created from a mixture containing this polyurethane and 0%, 10%, 20%, or 30% type I collagen. The resulting scaffolds were found to have open, interconnected pores (from 7 to >100 um) and porosities from 58% to 86% depending on the polyurethane/collagen ratio. The scaffolds were also flexible with breaking strains of 82–443% and tensile strengths of 0.97–4.11 MPa depending on preparation conditions. Scaffold degradation was significantly increased when collagenase was introduced into an incubating buffer in a manner that was dependent on the mass fraction of collagen present in the scaffold. Mass losses could be varied from 15% to 59% over 8 weeks. When culturing umbilical artery smooth muscle cells on these scaffolds higher cell numbers were observed over a 4-week culture period in scaffolds containing collagen. In summary, a strong and flexible scaffold system has been developed that can degrade by both hydrolysis and collagenase degradation pathways, as well as support cell growth. This scaffold possesses properties that would make it attractive for future use in soft tissue applications where such mechanical and biological features would be advantageous.
Multifunctional Pancreatic Islet Encapsulation Barriers Achieved via Multilayer PEG Hydrogels
Authors: Weber Laney M. , Cheung Charles Y. , Anseth Kristi S.
Published: April 2008

The diverse requirements for a successful islet encapsulation barrier suggest the benefit of a barrier system that presents differing functionalities to encapsulated cells and host cells. Initially, multifunctional hydrogels were synthesized via the sequential photopolymerization of PEG hydrogel layers, each with different isolated functionalities. The ability to achieve localized biological functionalities was confirmed by immunostaining of different entrapped antibodies within each hydrogel layer. Survival of murine islets macroencapsulated within the interior gel of two-layer hydrogel constructs was then assessed. Maintenance of encapsulated islet survival and function was observed within multilayer hydrogels over 28 days in culture. Additionally, the functionalization of the islet-containing interior PEG gel layer with cell–matrix moieties, with either 100 μg/ml laminin or 5 mM of the adhesive peptide IKVAV found in laminin, resulted in increased insulin secretion from encapsulated islets similar to that in gels without an exterior hydrogel layer. Finally, through cell seeding experiments, the ability of an unmodified, exterior PEG layer to prevent interactions, and thus attachment, between nonencapsulated fibroblasts and entrapped ECM components within the interior PEG layer was demonstrated. Together the presented results support the potential of multilayer hydrogels for use as multifunctional islet encapsulation barriers that provide a localized biologically active islet microenvironment, while presenting an inert, immunoprotective exterior surface to the host environment, to minimize graft–host interactions.