|ognizant Communication Corporation|
VOLUME 13, NUMBER 3, 2004
Cell Transplantation, Vol. 13, pp. 197-211, 2004
0963-6897/04 $20.00 + 00
Copyright © 2004 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Differentiation of Human and Mouse Embryonic Stem Cells Along a Hepatocyte Lineage
Hitoshi Shirahashi,1 Jian Wu,1 Naoki Yamamoto,1 Andreea Catana,1 Henning Wege,1 Brook Wager,1 Kiwamu Okita,2 and Mark A. Zern1
1Transplant Research Institute, University of California,
Davis Medical Center, Sacramento, CA, 95817
2Department of Gastroenterology and Hepatology, Yamaguchi University, School of Medicine, Ube, 755-8505, Japan
Embryonic stem (ES) cells may differentiate along a hepatocyte lineage; however, currently there are no reports of culture conditions yielding high levels of hepatocyte-specific gene expression in these cells. We investigated culture conditions for differentiating ES cells into hepatocyte-like cells in vitro. Various combinations of culture media, growth and differentiation factors, and substratum precoatings were evaluated, and it was determined that a combination of Iscove's modified Dulbecco's medium with 20% fetal bovine serum, human insulin, dexamethasone, and collagen type I precoating was optimal for directing mouse ES cells along a hepatocyte lineage. Treatment of mouse ES cell with the optimal condition led to prealbumin gene expression 20% as high, and albumin synthesis 7% as high, as in mouse liver. The optimal culture condition also induced albumin gene expression in differentiated human ES cells 1% as high as in normal human hepatocytes as shown by Western blot analysis, and cells were positive for human albumin by immunocytochemistry. In addition, our optimal condition led to high levels of albumin gene expression in primary mouse hepatocytes after 35 days of culture, levels 10-fold higher than with other hepatocyte differentiation media. In conclusion, our optimal condition directed both mouse and human ES cells along a hepatocyte lineage. This represents the initial step in establishing cell lines that can be employed in cell-based therapeutics in humans and for toxicology and pharmacology studies.
Key words: Albumin; Embryonic stem cells; Hepatocyte; Insulin; Dexamethasone
Address correspondence to Mark A. Zern, M.D., Transplant Research Institute, UC Davis Medical Center, 4635 2nd Ave. Suite 1001, Sacramento, CA 95817. Tel: (916) 734-8063; Fax: (916) 734-8097; E-mail: email@example.com
Comparison of Bioenergetic Activity of Primary Porcine Hepatocytes Cultured in Four Different Media
Konstantinos J. Dabos,1 Leonard J. Nelson,1 Chandralal H. Hewage,2 John A. Parkinson,2 A. Forbes Howie,3 Ian H. Sadler,2 Peter C. Hayes,1 and John N. Plevris1
1Liver Cell Biology Laboratory, Department of Hepatology, 2Department of Chemistry, and 3Department of Clinical Biochemistry, University of Edinburgh, Edinburgh EH16 4SU, Scotland, UK
Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were William's E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 x 1010; viability 87 ± 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 x 106 viable cells per plate. Using H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis, ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. William's E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.
Key words: Glucolysis; Gluconeogenesis; Ketogenesis; Ureagenesis; Albumin synthesis
Address correspondence to Dr. Konstantinos Dabos, Endoscopy Unit, Royal Infirmary of Edinburgh, 51 Little France Crescent, EH16 4SU Scotland, UK. Tel: +44 131 2421603; Fax: +44 131 2421618; E-mail: firstname.lastname@example.org
Engraftment Measurement in Human Liver Tissue After Liver Cell Transplantation by Short Tandem Repeats Analysis
Valeria R. Mas,1,2 Daniel G. Maluf,1 Melissa Thompson,1 Andrea Ferreira-Gonzalez,2 and Robert A. Fisher1
1Division of Transplantation, Department of Surgery, and 2Division of Molecular Diagnostics, Department of Pathology, Virginia Commonwealth University, Richmond, VA
Hepatocyte transplantation has been proposed as a technique for bridging patients to whole-organ transplantation, for providing metabolic support during liver failure, and for replacing whole-organ transplantation in certain metabolic liver diseases. Assessment of hepatocyte engraftment has been difficult to measure, and the degree of engraftment needed to correct various liver disorders is still unknown. A sensitive, simple, and specific method of monitoring engraftment of transplanted hepatocytes for the purpose of bridging human liver failure to native regeneration using short tandem repeats (STRs) was evaluated. The analytical sensitivity of the test was evaluated using DNA mixing curves and established as 0.5% (percentage of donor DNA/recipient DNA). Sex-matched and mismatched cases were included during the validation. The clinical evaluation of the assay was performed using liver samples from two patients who underwent hepatocyte transplantation. We concluded from this study that the AmpFLSTR® Profiler PlusTM PCR Amplification Kit, a well-established technique in forensic medicine, is specific, sensitive, and a reproducible assay for measurement of engraftment after hepatocyte transplantation in both sex-matched and sex-mismatched cases.
Key words: Hepatocyte transplantation; Engraftment; Short tandem repeats; Liver transplantation
Address correspondence to Robert A. Fisher, M.D., FACS, Professor of Surgery, Division of Transplant, Department of Surgery, Virginia Commonwealth University, Box 980254, Sanger Hall 8012A, Richmond, VA 23298-0248. Tel: (804) 828-2461; E-mail: email@example.com
Polyvinyl Pyrrolidone: A Novel Cryoprotectant in Islet Cell Cryopreservation*
Hesham M. El-Shewy, William F. Kendall, Jr., Marcus Darrabie, Bradley H. Collins, and Emmanuel C. Opara
Department of Surgery, Duke University Medical Center and Durham VA Medical Center, Durham, NC 27710
The present study was performed on the basis of the hypothesis that the low molecular weight (MW) compounds, DMSO and glycerol, permeate the cell and interact hydrophobically with intracellular proteins, thereby perturbing the cytoskeletal architecture of frozen cells and diminishing islet cell integrity and function. Isolated rat islets were cultured overnight (18-24 h) at 37°C in RPMI medium supplemented with 10% fetal calf serum and 1% mixture of penicillin/streptomycin. Using a programmable temperature controller, samples of precounted islets were then frozen under liquid nitrogen, in the presence of either 2 M DMSO (MW = 0.078 kDa), 3 M glycerol (MW = 0.092 kDa), 5% polyethylene glycol (PEG, MW = 20 kDa), or 10% polyvinylpyrrolidone (PVP, MW = 40 kDa), and stored at -80°C for 1 week. Following thawing and overnight (18-24 h) culture, intact islet recovery was determined by islet counting after dithizone staining. Islet function was assessed by determination of glucose-stimulated insulin secretion in perifusion experiments with Krebs-Ringer bicarbonate buffer, pH 7.4, containing either basal (3.3 mM) or high (16.7 mM) glucose concentrations. The assessment of islet recovery and function of all cryopreserved samples was performed only after thawing and overnight culture (18-24 h) of islets. The mean ± SEM percent intact islet recovery was higher with PVP compared with DMSO (82 ± 4.6 vs. 62.7 ± 3.1%, respectively, p < 0.005, n = 9). Furthermore, the glucose stimulation index of insulin secretion by islets taken from samples frozen with PEG and PVP, after thawing and overnight culture, was comparable to that of freshly isolated islets, in contrast to DMSO and glycerol. There was no significant difference in intact islet recovery and function between samples frozen with PVP and those frozen with PEG. Samples frozen with DMSO and glycerol had similar results in islet recovery and function. These data show that PVP is a new and potent cryoprotectant for islet cell freezing.
Key words: Islet cells; Polyvinylpyrrolidone; Cryopreservation; Transplantation
Address correspondence to Dr. Emmanuel C. Opara at his present address: Pritzker Institute of Biomedical Science & Engineering, Illinois Institute of Technology, 10 West 32nd Street, E1-116, Chicago, IL 60616. E-mail: firstname.lastname@example.org
*This study was partly funded by the Robert Leet & Clara Guthrie Patterson Trust, and was presented at the Cell Transplant Society meeting held in Atlanta, GA, in March 2003.
The Effects of Transforming Growth Factor-b2 on Dopaminergic Graft Survival
Shannon L. Macauley,1 Alexander D. Horsch,2 Marinus Oterdoom,3 Ming H. Zheng,1 and Gregory R. Stewart1
1Genzyme Corporation, Framingham, MA 01701
2University Hospital Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands
3VU Medisch Centrum, Amsterdam, The Netherlands
Dopaminergic cell transplantation is a promising therapeutic approach for the treatment of Parkinson's disease, the potential of which is limited due to poor survival and low dopamine content within engrafted tissue. In this study, the ability of transforming growth factor-b2 (TGF-b2) to influence transplant survival was evaluated. Cell suspensions containing fetal rat ventral mesencephalon (VM) cells were incubated prior to surgery with vehicle (DPBS), varying concentrations of TGF-b2 (5-1000 ng/ml), or a pan-specific antibody against TGF-b (1D11, 100 ng/ml). VM cell suspensions (200,000 cells) were unilaterally implanted into the striatum of adult Sprague-Dawley rats (n = 5-11 animals/group). Following a 3-week survival period, small but viable VM grafts containing tyrosine hydroxylase-positive (TH+) neurons and fibers were present in all animals. Addition of TGF-b2 resulted in a steep, bell-shaped dose-response curve with a significant effect on TH+/dopamine cell survival. At 50 ng/ml TGF-b2, the number of surviving dopamine neurons was increased twofold compared with controls. Addition of TGF-b2 or 1D11 did not significantly influence graft volume. Further studies, possibly in combination with other neurotrophic factors, need to be performed to obtain a greater understanding of the effects of TGF-b on dopamine neurons and fetal VM cell engraftment.
Key words: Transplantation; Parkinson's disease; Neurotrophic; Neuroprotection; TGF-b
Address correspondence to Gregory R. Stewart, Ph.D., Genzyme Corporation, One Mountain Road, Framingham, MA 01701. Tel: (508) 271-2646; Fax: (508) 872-9080; E-mail: email@example.com
Neuroprotective Effects of Encapsulated CNTF-Producing Cells in a Rodent Model of Huntington's Disease Are Dependent on the Proximity of the Implant to the Lesioned Striatum*
Dwaine F. Emerich1 and Shelley R. Winn2
1LCT BioPharma, Cranston, RI 02921
2Department of Surgery, Oregon Health and Science University, Portland, OR 97239
Huntington's disease (HD) is a devastating genetic disorder with no effective treatments for preventing or lessening the underlying neuronal degeneration. Intracerebral delivery of CNTF in animal models of HD has shown considerable promise as a means of protecting striatal neurons that would otherwise be destined to die. The present study examines whether the neuroprotective effects of CNTF require that the delivery be immediately proximal to the lesion site or whether protective effects can be exerted when the delivery site is more distal to the site of injury. Encapsulated CNTF-producing cells were implanted into the lateral ventricle either ipsilateral or contralateral to an intrastriatal quinolinic acid (QA) injection. A robust neuroprotective effect was observed only in those animals receiving CNTF implants ipsilateral to the QA injection. In these animals, the loss of striatal ChAT and GAD activity as well as the behavioral impairments that resulted from QA were completely prevented. In contrast, no neurochemical or behavioral benefits were produced by implants of CNTF-producing cells in the contralateral ventricle. These data continue to support the use of cellular delivery of CNTF for HD but caution that delivery directly to the striatum may be needed if any clinical benefits are to be seen.
Key words: Polymer encapsulation; Huntington's disease; CNTF; Quinolinic acid; Fibroblasts; Neurotrophic factors; Gene therapy
Address correspondence to Dwaine F. Emerich, 245 Armington Street, Cranston, RI 02905. Tel: (401) 785-2895; E-mail: firstname.lastname@example.org
*These data were collected while the authors were employees at CytoTherapeutics, Inc., Providence RI.
Liposomal Formulations of Tacrolimus and Rapamycin Increase Graft Survival and Fiber Outgrowth of Dopaminergic Grafts
Aylin Y. Alemdar,1 Damaso Sadi,1 Vivian C. McAlister,2 and Ivar Mendez1,3
1Department of Anatomy and Neurobiology, Dalhousie University,
Halifax, Nova Scotia, Canada, B3H 4H7
2Department of Surgery, Division of General Surgery, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 2Y9
3Department of Surgery, Division of Neurosurgery, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4H7
The immunosuppressive drugs tacrolimus (TAC) and rapamycin (RAPA) have both been found to have neuroprotective effects on dopaminergic neurons. The purpose of the present study was to investigate whether liposomal formulations of these drugs administered directly into the brain improve cell survival and fiber outgrowth. Rats with unilateral 6-hydroxydopamine lesions were transplanted with 800,000 fetal rat ventral mesencephalic cells and randomly divided to one of four groups. Group 1 received a transplant containing cells only; group 2 received a cell suspension containing 0.68 mM liposomal RAPA (LRAPA); group 3 received a cell suspension containing 2.0 mM liposomal TAC (LTAC); and group 4 received a cell suspension containing a liposomal formulation of both 0.68 mM RAPA and 2.0 mM TAC (LRAPATAC). Rats were sacrificed after 6 weeks, and cell survival and fiber outgrowth were assessed using tyrosine hydroxylase (TH) immunohistochemistry. The animals receiving a cell suspension containing either LTAC or LRAPATAC were found to have significantly more surviving TH-immunoreactive (TH-ir) cells than the control group receiving cells only. The group receiving LTAC had significantly longer fibers, the group receiving LRAPA had significantly more fibers close to the graft, and the group receiving LRAPATAC had significantly more fibers at all distances. This study shows the feasibility of using liposomal formulations of neuroimmunophilins directly in the brain at the time of implantation to improve graft survival and fiber outgrowth. Furthermore, we have shown that the combination of LTAC and LRAPA has a synergistic effect. These compounds may play an important role in optimizing graft survival and host reinnervation in cell-mediated brain repair strategies for the treatment of neurological conditions.
Key words: Transplantation; Parkinson's disease; Tacrolimus; Rapamycin; Liposomes
Address correspondence to Dr. Ivar Mendez, Neural Transplantation Laboratory, Department of Anatomy and Neurobiology, Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5. Tel: (902) 494-8896; Fax: (902) 494-1212; E-mail: email@example.com
Reassessment of Caspase Inhibition to Augment Grafted Dopamine Neuron Survival
Deanna M. Marchionini, Timothy J. Collier, Mark R. Pitzer, and Caryl E. Sortwell
Department of Neurological Sciences, Research Center for Brain Repair, Rush University Medical Center, Chicago, IL 60612
One experimental therapy for Parkinson's disease (PD) is the transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted neurons die, many via apoptosis. Activated caspases play a key role in execution of the apoptotic pathway; therefore, exposure to caspase inhibitors may provide an effective intervention strategy for protection against apoptotic cell death. In the present study we examined the efficacy of two different caspase inhibitors, caspase-1 inhibitor Ac-YVAD-CMK and caspase-3 inhibitor Ac-DEVD-CMK, to augment mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival in culture and following implantation into the denervated striatum of rats. We report that treatment with Ac-YVAD-CMK provided partial but nonsignificant protection for TH-ir neurons against serum withdrawal in mesencephalic cultures plated at low density, while neither caspase inhibitor promoted TH-ir neuron survival in higher density cultures, simulating graft density. We demonstrate that plating procedures (full well vs. microislands) and cell density directly affect the degree of insult experienced by TH-ir neurons following serum withdrawal. This varying degree of insult directly impacts whether caspase inhibition will augment TH-ir neuron survival. Our grafting experiments demonstrate that Ac-YVAD-CMK does not augment grafted TH-ir neuron survival when added to mesencephalic cell suspensions prior to grafting or to mesencephalic reaggregates for 3 days in vitro prior to transplantation. These experiments provide further evidence of the failure of these caspase inhibitors to augment TH-ir neuron survival. Furthermore, we suggest that cell culture paradigms used to model grafting paradigms must more closely approximate the cell densities of mesencephalic grafts to effectively screen potential augmentative treatments.
Key words: Apoptosis; Parkinson's disease; Transplant; Cell density
Address correspondence to Deanna Marchionini, Department of Neurological Sciences, Rush University Medical Center, 1735 West Harrison St., Suite 300, Chicago, IL 60612. Tel: (312) 563-3578; Fax: (312) 563-3571; E-mail: Deanna_Marchionini@rush.edu
Intracerebral Xenotransplantation of GFP Mouse Bone Marrow Stromal Cells in Intact and Stroke Rat Brain: Graft Survival and Immunologic Response
H. Irons,1* J. G. Lind,1 C. G. Wakade,2 G. Yu,1 M. Hadman,1 J. Carroll,1,4 D. C. Hess,1,3,4 and C. V. Borlongan1,2,3,4
1Department of Neurology and 2Institute of Molecular
Medicine and Genetics, 3School of Graduate Studies, Medical
College of Georgia, Augusta, GA 30912
4Research and Affiliations Service Line, Augusta Veterans Administration Medical Center, Augusta, GA 30912
The present study characterized survival and immunologic response of bone marrow stromal cells (BMSCs) following transplantation into intact and stroke brains. In the first study, intrastriatal transplantation of BMSC (60,000 in 3 ml) or vehicle was performed in normal adult Sprague-Dawley male rats that subsequently received daily cyclosporin A (CsA, 10 mg/kg, IP in 3 ml) or vehicle (olive oil, similar volume) starting on day of surgery up to 3 days posttransplantation. Animals were euthanized at 3 or 30 days posttransplantation and brains were processed either for green fluorescent protein (GFP) microscopy or flow cytometry (FACS). Both GFP epifluorescence and FACS scanning revealed GFP+ BMSCs in both groups of transplanted rats with or without CsA, although significantly increased (1.6- to 3-fold more) survival of GFP+ BMSCs was observed in the immunosuppressed animals. Further histologic examination revealed widespread dispersal of BMSCs away from the graft core accompanied by many long outgrowth processes in non-CsA-transplanted animals, whereas a very dense graft core, with cells expressing only sporadic short outgrowth processes, was observed in CsA-transplanted animals. There were no detectable GFP+ BMSCs in nontransplanted rats that received CsA or vehicle. Immunologic response via FACS analysis revealed a decreased presence of cytotoxic cells, characterized by near complete absence of CD8+ cells, and lack of activation depicted by low CD69 expression in CsA-treated transplanted animals. In contrast, elevated levels of CD8+ cells and increased activation of CD69 expression were observed in transplanted animals that received vehicle alone. CD4+ helper cells were almost nondetectable in transplanted rats that received CsA, but also only minimally elevated in transplanted rats that received vehicle. Nontransplanted rats that received either CsA or vehicle displayed very minimal detectable levels of all three lymphocyte markers. In the second study, a new set of male Sprague-Dawley rats initially received bilateral stereotaxic intrastriatal transplantation of BMSCs and 3 days after were subjected to unilateral transient occlusion of middle cerebral artery. The animals were allowed to survive for 3 days after stroke without CsA immunosuppression. Epifluorescence microscopy revealed significantly higher (5-fold more) survival of transplanted GFP+ BMSCs in the stroke striatum compared with the intact striatum. The majority of the grafts remained within the original dorsal striatal transplant site, characterized by no obvious migration in intact striatum, but with long-distance migration along the ischemic penumbra in the stroke striatum. Moreover, FACS scanning analyses revealed low levels of immunologic response of grafted BMSCs in both stroke and intact striata. These results, taken together, suggest that xenotransplantation of mouse BMSCs into adult rats is feasible. Immunosuppression therapy can enhance xenograft survival and reduce graft-induced immunologic response; however, in the acute phase posttransplantation, BMSCs can survive in intact and stroke brain, and may even exhibit long-distance migration and increased outgrowth processes without immunosuppression.
Key words: Neural transplantation; Cerebral ischemia; Immunosuppression; Striatum; Lymphocytes; Cyclosporin A; Graft; Cell migration; Neuronal phenotype
Address correspondence to Dr. Cesar V. Borlongan, BI-3080, Department of Neurology, Medical College of Georgia, Augusta, GA 30912-3200. Tel: (706) 733-0188, ext. 2485; Fax: (706) 823-3949; E-mail: firstname.lastname@example.org
*H. Irons is an M.D./Ph.D. student and 2003 ASNTR travel awardee.
Comparison of Embryonic Stem Cell-Derived Dopamine Neuron Grafts and Fetal Ventral Mesencephalic Tissue Grafts: Morphology and Function
David M. Yurek and Anita Fletcher-Turner
Department of Surgery/Neurosurgery, University of Kentucky College of Medicine, Lexington, KY
In this study we compared the function and morphology of two types of neural grafts: allografts of fetal ventral mesencephalic (VM) tissue and xenografts of embryonic stem cell (ESC)-derived dopamine neurons. Mouse embryonic stem cells were cultured and exposed to differentiation factors that induced approximately 10% of the cells to express a dopaminergic phenotype. These cells were then harvested and implanted into the denervated striatum of rats with unilateral lesions of the nigrostriatal pathway. Another group of lesioned rats received allografts of fetal ventral mesencephalic tissue. While both types of grafts yield a similar number of tyrosine hydroxylase (TH)-positive cells, amphetamine-induced rotational behavior was differentially affected by these grafts: rotational behavior was significantly reduced in lesioned rats receiving allografts of fetal VM tissue while ESC grafts had slight but insignificant effects on rotational scores. Densitometry measures of TH+ fiber outgrowth revealed a similar area of reinnervation and a comparable number of TH+ cells for ESC graft when compared with VM grafts. These data suggest there are similarities and also distinct differences in the manner in which ESC and VM grafts interact with the denervated striatum.
Key words: Parkinson's disease; Neural transplantation; Stem cells; Dopamine; Rodent
Address correspondence to David Yurek, Ph.D., Department of Surgery/Neurosurgery, University of Kentucky College of Medicine, Health Sciences Research Building, Lexington, KY 40536-0305. Tel: (859) 257-8219; Fax: (859) 323-6343; E-mail: David.Yurek@uky.edu
Graft-Induced Plasticity in the Mammalian Host CNS
Jitka Ourednik and Václav Ourednik
Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011
In this review we trace back the history of an idea that takes a new approach in restorative neurotransplantation by focusing on the "multifaceted dialogue" between graft and host and assigns a central role to graft-evoked host plasticity. In several experimental examples ranging from the transfer of solid fetal tissue grafts into mechanical cortical injuries to deposits of neural stem cells into hemisectioned spinal cord, MPTP-damaged substantia nigra or mutant cerebella supportive evidence is provided for the hypothesis, that in many CNS disorders regeneration of the host CNS can be achieved by taking advantage of the inherent capacity of neural grafts to induce protective and restorative mechanisms within the host. This principle might once allow us to spare even complex circuitry from neurodegeneration.
Key words: CNS; Regeneration; Neuroprotection; Rescue; Graft; Neurotransplantation; Neural stem cell
Address correspondence to Dr. Jitka Ourednik, Department of Biomedical
Sciences, College of Veterinary Medicine, Iowa State University, Ames,
IA 50011. Tel/Fax: +1-515-294-6449; E-mail: email@example.com