ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 18, NUMBER 2, 2009

Cell Transplantation, Vol. 18, pp. 111-118, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Amnion: A Potent Graft Source for Cell Therapy in Stroke

Seong Jin Yu,1 Maddalena Soncini,2 Yuji Kaneko,1 David C. Hess,1 Ornella Parolini,2 and Cesar V. Borlongan1,3

1Department of Neurology, Medical College of Georgia, Augusta, GA, USA
2Centro di Ricerca E. Menni, Fondazione Poliambulanza Instituto Ospedaliero, Brescia, Italy
3Institute of Molecular Medicine Genetics, Medical College of Georgia, Augusta, GA, USA

Regenerative medicine is a new field primarily based on the concept of transplanting exogenous or stimulating endogenous stem cells to generate biological substitutes and improve tissue functions. Recently, amnion-derived cells have been reported to have multipotent differentiation ability, and these cells have attracted attention as a novel cell source for cell transplantation therapy. Cells isolated from amniotic membrane can differentiate into all three germ layers, have low immunogenicity and anti-inflammatory function, and do not require the destruction of human embryos for their isolation, thus circumventing the ethical debate commonly associated with the use of human embryonic stem cells. Accumulating evidence now suggests that the amnion, which had been discarded after parturition, is a highly potent transplant material in the field of regenerative medicine. In this report, we review the current progress on the characterization of MSCs derived from the amnion as a remarkable transplantable cell population with therapeutic potential for multiple CNS disorders, especially stroke.

Key words: Adult stem cells; Progenitors; Placenta; Mesenchymal stromal cells; Transplantation; Cerebral ischemia

Address correspondence to Cesar V. Borlongan, Ph.D., at his current address: Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida, 12901 Bruce B. Downs Blvd., MDC78, Tampa, FL 33612, USA. E-mail: cborlongan@health.usf.edu

Cell Transplantation, Vol. 18, pp. 119-133, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Microtransplantation of Dopaminergic Cell Suspensions: Further Characterization and Optimization of Grafting Parameters

Guido Nikkhah,1 Christoph Rosenthal,1 Gero Falkenstein,1 Alexandra Roedter,1 Anna Papazoglou,1 and Almuth Brandis2

1Department of Stereotactic and Functional Neurosurgery, Laboratory of Molecular Neurosurgery, Neurocenter, Albert-Ludwigs-University, Freiburg, Germany
2Institute of Neuropathology, Medical School Hanover, Hanover, Germany

Intracerebral transplantation of dopaminergic (DA) cells is currently further explored as a potential restorative therapy for Parkinson's disease (PD). However, before they can be considered for a more widespread clinical use a number of critical issues have to be resolved, including an optimized transplantation protocol. This study has been performed in a rat 6-hydroxydopamine model of PD and is based on the microtransplantation approach. The results demonstrate a reduced survival (threefold) for a single cell suspension of E14 rat ventral mesencephalon compared to a fragment suspension when a metal cannula is used for implantation. However, fragment suspensions result in a more variable graft survival and ectopically placed cells along the implantation tract. When a glass capillary is used for implantation, the survival of the single cell suspension (so-called "micrograft") improved by fourfold (vs. single cells/metal cannula) and is superior to the combination of the metal cannula and fragment suspension (+40%). The micrografts show a reduced variability in DA neuron survival as well as fewer ectopically placed cells. Moreover, the implantation time can significantly be reduced from 19 to 7 min in micrografted animals without a compromise in DA graft survival and functional behavioral outcome. Using the microtransplantation approach graft size can be tailored effectively by varying the density of the final cell suspension at least between 11,000 and 320,000 cells/ml, resulting in comparable survival of tyrosine hydroxylase (TH)-positive neurons in the range of 2-4%. With this approach no more than 100 surviving TH-positive neurons are necessary to produce functional effects in the amphetamine-induced rotation test. Interestingly, we found that DA micrografts into lesion striatum present 20% higher survival rates of TH neurons in comparison to the intact striatum. In summary, these results provide further evidence for the usefulness of the microtransplantation approach and allow for a more precise and tailored adaptation of the implantation parameters for further studies on DA, and possibly also other neural-, glial-, and stem cell-derived grafts.

Key words: 6-Hydroxydopamine lesion; Nigrostriatal system; Transplantation protocol; DA neuron survival

Address correspondence to Guido Nikkhah, M.D., Ph.D., Department of Stereotactic and Functional Neurosurgery, Neurocenter, Albert-Ludwigs-University, Breisacher Str. 64, D-79106 Freiburg, Germany. Tel: 49-761-270-5063; Fax: 49-761-270-5010; E-mail: guido.nikkhah@uniklinik-freiburg.de

Cell Transplantation, Vol. 18, pp. 135-144, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effective Cryopreservation of Neural Stem or Progenitor Cells Without Serum or Proteins by Vitrification

L. L. Kuleshova,1* F. C. K. Tan,2,3* R. Magalhães,1 S. S. Gouk,1 K. H. Lee,1 and G. S. Dawe2,3

1Low Temperature Preservation Unit, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
2Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
3Neurobiology and Ageing Programme, Centre for Life Sciences, National University of Singapore, Singapore

Development of effective cryopreservation protocols will be essential to realizing the potential for clinical application of neural stem and progenitor cells. Current cryopreservation protocols have been largely employed in research, which does not require as stringent consideration of viability and sterility. Therefore, these protocols involve the use of serum and protein additives, which can potentially introduce contaminants, and slow cooling with DMSO/glycerol-based cryopreservation solutions, which impairs cell survival. We investigated whether serum- and protein-free vitrification is effective for functional cryopreservation of neurosphere cultures of neural stem or progenitor cells. To protect the samples from introduction of other contaminants during handling and cryostorage, an original "straw-in-straw" method (250 ml sterile straw placed in 500 ml straw) for direct immersion into liquid nitrogen and storing the samples was also introduced. The protocol employed brief step-wise exposure to vitrification solution composed of ethylene glycol (EG) and sucrose (40% v/v EG, 0.6 M sucrose) and removal of vitrification solution at room temperature. Evaluation of the effects of vitrification revealed that there were no differences between control and vitrified neural stem or progenitor cells in expression of the neural stem or progenitor cell markers, proliferation, or multipotent differentiation. This sterile method for the xeno-free cryopreservation of murine neurospheres without animal or human proteins may have the potential to serve as a starting point for the development of cryopreservation protocols for human neural stem and progenitor cells for clinical use.

Key words: Vitrification; Cryopreservation; Neural progenitor cells; Neural stem cells; Neurospheres

Address correspondence to L. L. Kuleshova, Ph.D., Low Temperature Preservation Unit, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Block MD 11 #03-01C, 10 Medical Drive, Singapore 117597. Tel: +65-6516-3359; Fax: +65-6773-5461; E-mail: nmikl@nus.edu.sg or G. S. Dawe, Ph.D., Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 10 Medical Drive, Singapore 117597. Tel: +65-6516-8864; Fax: +65-6873-7690; E-mail: gavindawe@nus.edu.sg

*These authors contributed equally to this work.

Cell Transplantation, Vol. 18, pp. 145-158, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Delivery of Adipose-Derived Precursor Cells for Peripheral Nerve Repair

Lizzie Y. Santiago,1,2 Julio Clavijo-Alvarez,1 Candace Brayfield,1,4 J. Peter Rubin,1,3 and Kacey G. Marra1,3,4

1Division of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA
2Developmental Neurobiology Section, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
3McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA
4Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA

To test the hypothesis that the transplantation of adipose precursor cells (APCs) improves nerve regeneration and functional recovery, human APCs were transplanted into the lumen of a nerve guide in a 6-mm unilateral sciatic nerve defect in athymic rats. The three control groups for the study were biodegradable, polycaprolactone-based nerve conduit without APCs, autograft, and empty defect. Behavioral tests were performed every 3 weeks, and the sciatic functional index (SFI) was calculated based on measurements from the hindlimb prints. After 12 weeks, the nerve as well as right and left gastrocnemius muscles were removed and preserved for histological evaluation. Full regeneration of the sciatic nerve occurred on the rats that received the autograft, the guide, and the guide with APCs; no regeneration was observed on any of the rats in which the defect was left untreated (empty defect). APCs survived transplantation for up to 12 weeks in the injured peripheral nerve. No significant colocalization was observed between the immunostaining for glial fibrillary protein and anti-human lamin A/C, implying that the APCs did not differentiate into Schwann cells at the site of injury. In comparison with the rats with untreated defects, a decrease in muscle atrophy was observed on those rats that received the autograft and the guide with cells as indicated by the gastrocnemius muscle weight ratio and the muscle fiber ratio. Significant differences in SFI were observed 3 weeks postinjury between the rats in which the guide was left empty and those that received the guide with APCs; however, these differences were not observed at 12 weeks. The transplantation of APCs promoted the formation of a more robust nerve as evidenced by the results from the cross-sectional area of regenerated nerve, and the transplantation of APCs produced a decrease in muscle atrophy.

Key words: Adipose; Progenitor cells; Peripheral nerve repair; Nerve guide; Polycaprolactone

Address correspondence to Kacey G. Marra, Ph.D., Departments of Surgery and Bioengineering, University of Pittsburgh, 1655E BST, 200 Lothrop St., Pittsburgh, PA 15261, USA. Tel: (412) 383-8924; Fax: (412) 648-2821; E-mail: marrak@upmc.edu

Cell Transplantation, Vol. 18, pp. 159-170, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cartilage Regeneration of Adipose-Derived Stem Cells in a Hybrid Scaffold From Fibrin-Modified PLGA

Yiyong Wei,1 Hunyu Hu,2 Haiqiang Wang,2 Yinsong Wu,2 Lianfu Deng,1 and Jin Qi1

1Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
2Institute of Orthopaedics and Traumatology, Xijing Hospital, The Fourth Military Medical University, Xi'an, People's Republic of China

Adipose-derived stem cells (ASCs) appear to be a useful stem cell population, which has been shown to possess multipotentiality. The aim of this study was to evaluate the utility of ASCs in tissue-engineered cartilage using a hybrid scaffold from fibrin-modified PLGA scaffold. ASCs were isolated from rabbit adipose tissue. The PLGA scaffold was prepared by low-temperature deposition technology and the hybrid scaffold was fabricated by a freeze-drying method. When ASCs were seeded onto fibrin-modified PLGA scaffold in vitro, enhanced cellular viability was observed compared to unmodified PLGA scaffold. The analysis of proteoglycan and collagen II revealed that fibrin-modified scaffold succeeded in inducing ASCs to differentiate into chondrocytes in vitro. A preliminary study on cartilage regeneration was also performed in vivo. Observation of histology and immunoblotting demonstrated that ASCs containing the hybrid scaffold promoted cartilage regeneration in the defects of articular cartilage much better than other groups. These results indicated that ASCs containing the hybrid scaffold are a more effective way to potentially enhance articular cartilage regeneration.

Key words: Cartilage regeneration; Adipose; Stem cells; PLGA; Fibrin; Scaffold

Address correspondence to Yiyong Wei, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025. Tel: 86-21-54370065; E-mail: yiyong_wei@hotmail.com

Cell Transplantation, Vol. 18, pp. 171-181, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Assessments of Proliferation Capacity and Viability of New Zealand Rabbit Peripheral Blood Endothelial Progenitor Cells Labeled With Superparamagnetic Particles

Xiao-Li Mai,1 Zhan-Long Ma,1 Jun-Hui Sun,1 Sheng-Hong Ju,1 Ming Ma,2 and Gao-Jun Teng1

1Jiangsu Key Laboratory of Molecular Imaging and Functional Imaging, Department of Radiology, Zhongda Hospital, Southeast University, Nanjing, China
2Laboratory of Molecular and Biomolecular Electronics, Southeast University, Nanjing, China

Magnetic resonance imaging (MRI) has proven to be effective in tracking the distribution of transplanted stem cells to target organs by way of labeling cells with superparamagnetic iron oxide particles (SPIO). However, the effect of SPIO upon labeled cells is still unclear on a cellular level. With this study, the proliferation and viability of New Zealand rabbit peripheral blood endothelial progenitor cells (EPCs) labeled with SPIO were evaluated and in vitro images were obtained using a 1.5 T MR scanner. Mononuclear cells (MNCs) were isolated from peripheral blood of the adult New Zealand rabbit and cultured in fibronectin-coated culture flasks, in which EPCs were identified from cell morphology, outgrowth characteristics, and internalization of DiI-Ac-LDL and binding to FITC-UEA I. EPCs were incubated with the self-synthesized poly-L-lysine-conjugated SPIO (PLL-SPIO) particles in a range of concentrations. The prevalence of iron-containing vesicles or endosomes in the cytoplasm of labeled cells was confirmed with Prussian blue staining and transmission electron microscopy. Tetrazolium salt (MTT) assay, cell apoptosis, and cycle detection were assessed to evaluate proliferation and function of various concentrations, magnetically labeled EPCs. The quantity of iron per cell was determined by atomic absorption spectrometry. The cells underwent MRI with different sequences. The result showed that rabbit EPCs were efficiently labeled with the home synthesized PLL-SPIO. There was found to be no statistically significant difference in the MTT values of light absorption measured on the third and fifth days. Between labeled and unlabeled cells, there were also no aberrations found in the cell cycles, apoptosis, or growth curves. The atomic absorption spectrophotometer showed that the intracellular content of Fe decreased as more time elapsed after labeling. The labeled EPCs demonstrated a loss of MRI signal intensity (SI) when compared with the SI of unlabeled cells. These signal changes (DSI) were visible when cells were labeled with more than 5 × 104/ml of SPIO. The change in SI corresponded to the amount of iron in the EPCs, which reached a maximum at T2*WI. These data demonstrate that EPCs from the peripheral blood of the New Zealand rabbit can be effectively labeled with self-synthesized PLL-SPIO with minimal effects on cell proliferation and activity. Magnetically labeled EPCs can be imaged at 1.5 T MR and can therefore be used as an MR tracker of implanted EPCs.

Key words: Endothelial progenitor cells; Cell labeling; Superparamagnetic particles; Magnetic resonance imaging; Toxicity

Address correspondence to Gao-Jun Teng, M.D., Department of Radiology, Zhong-Da Hospital, Southeast University, 87 Ding Jia Qiao Road, Nanjing 210009, China. Tel: 086 25 83272121; Fax: 086 25 83311083; E-mail: gjteng@vip.sina.com

Cell Transplantation, Vol. 18, pp. 183-193, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Generation of Hepatocyte-Like Cells From In Vitro Transdifferentiated Human Fetal Pancreas

Suchitra Sumitran-Holgersson,1 Greg Nowak,1 Shifaan Thowfeequ,2 Setara Begum,1 Meghnad Joshi,1 Marie Jaksch,3 Anders Kjaeldgaard,4 Carl Jorns,1 Bo-Göran Ericzon,1 and David Tosh2

1Division of Transplantation Surgery, Karolinska University Hospital-Huddinge, Karolinska Institutet, Stockholm, Sweden
2Centre for Regenerative Medicine, Department of Biology & Biochemistry, University of Bath, Bath, UK
3The Burnham Institute for Medical Research, La Jolla, CA, USA
4Division of Obstetrics and Gynecology, Karolinska University Hospital-Huddinge, Karolinska Institutet, Stockholm, Sweden

Although the appearance of hepatic foci in the pancreas has been described in animal experiments and in human pathology, evidence for the conversion of human pancreatic cells to liver cells is still lacking. We therefore investigated the developmental plasticity between human embryonic pancreatic cells and liver cells. Cells were isolated and expanded from 7-8-week-old human fetal pancreata (HFP) and were characterized for the absence and presence of pancreatic and hepatic markers. In vitro expanded HFP were treated with fibroblast growth factor 2 (FGF2) and dexamethasone (DX) to induce a liver phenotye in the cells. These treated cells in various passages were further studied for their capacity to be functional in hepatic parenchyma following retrorsine-induced injury in nude C57 black mice. Amylase- and EPCAM-positive-enriched cells isolated from HFP and treated with FGF2 and DX lost expression of pancreatic markers and gained a liver phenotype. Hepatic differentiation was based on the expression (both at the mRNA and protein level) of liver markers albumin and cytokeratin 19. When transplanted in vivo into nude mice treated with retrorsine, both cell types successfully engrafted and functionally differentiated into hepatic cells expressing human albumin, glycogen, dipeptidyl peptidase, and g-glutamyltranspeptidase. These data indicate that human fetal pancreatic cells have a capacity to alter their gene expression profile in response to exogenous treatment with FGF2 and DX. It may be possible to generate an unlimited supply of hepatocytes in vitro for cell therapy.

Key words: Hepatocytes; Cholangiocytes; Retrorsine; Dexamethasone; Fibroblast growth factor

Address correspondence to Suchitra Sumitran-Holgersson Dr. Med. Sci. at his current address: Professor of Transplantation Biology, Department of Transplantation Surgery, Sahlgrenska University Hospital, 431 45 Gothenburg, Sweden. E-mail: suchitra.holgersson@surgery.gu.se

Cell Transplantation, Vol. 18, pp. 195-201, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effect of Micro- and Macroencapsulation on Oxygen Consumption by Pancreatic Islets

Roberta Cornolti,1 Marina Figliuzzi,1 and Andrea Remuzzi1,2

1Department of Biomedical Engineering, Mario Negri Institute for Pharmacological Research, Bergamo, Italy
2Department of Industrial Engineering, Faculty of Engineering, University of Bergamo, Dalmine (BG), Italy

Immunoisolation of pancreatic islets is extensively investigated for glycemic control in diabetic experimental animals. We previously reported that subcutaneous xenotransplantation of bovine islets protected by a selective polysulfone membrane successfully controlled glycemia in diabetic rats for up to 20 days. We then wondered whether immunoisolated islets have adequate oxygen supply in this device, where only diffusive transport allows cell function and survival. Here we set up an experimental technique to measure oxygen consumption rate (OCR) using a Clark's electrode inserted in a glass thermostated chamber connected to a data recorder and acquisition system. Bovine islets were isolated from 6-month-old calves, encapsulated in sodium alginate microcapsules or inserted in polysulfone hollow fibers. After 1 and 2 days in culture a series of measurements was performed using free islets (at normal or high-glucose concentration), islets encapsulated in microcapsules, or in hollow fibers. In free islets OCR averaged from 2.0 ± 0.8 pmol/IEQ/min at low-glucose concentration and from 2.5 ± 1.0 pmol/IEQ/min at high-glucose concentration (p < 0.01). OCR in islets encapsulated in microcapsules and in hollow fibers was comparable, and not significantly different from that measured in free islets. Two days after isolation OCR averaged 2.3 ± 0.6 in free islets, 2.3 ± 0.9 in alginate microcapsules, and 2.2 ± 0.7 pmol/IEQ/min in hollow fibers. These results show that OCR by bovine islets is comparable to that previously reported for other species. OCR increases in islets stimulated with high glucose and may be considered as a functional index. Moreover, islet encapsulation in alginate microcapsule, as well as in hollow fiber membranes, did not significantly affect in vitro OCR, suggesting adequate islet oxygenation in these conditions.

Key words: Oxygen consumption rate; Immunoisolation; Bovine pancreatic islet; Microcapsule; Hollow fiber

Address correspondence to Andrea Remuzzi, Department of Biomedical Engineering, Mario Negri Institute for Pharmacological Research, Via Gavazzeni, 11, 24125 Bergamo, Italy. Tel: +39 (035) 319888; Fax: +39 (035) 319331; E-mail: aremuzzi@marionegri.it

Cell Transplantation, Vol. 18, pp. 203-206, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Collagenase Isoforms for Pancreas Digestion

Federico Bertuzzi,6 Silvia Cainarca,2 Simona Marzorati,1 Angela Bachi,3 Barbara Antonioli,1 Rita Nano,1 Roberto Verzaro,4 and Camillo Ricordi5

1Unit of Cell Therapy for Type 1 Diabetes Mellitus, San Raffaele Scientific Institute, Milan, Italy
2Axxam SpA, San Raffaele Biochemical Science Park, Milan, Italy
3Biological Mass Spectrometry, DIBIT, San Raffaele Scientific Institute, Milan, Italy
4Surgery Department, ISMETT, Palermo, Italy
5Diabetes Research Institute, University of Miami, Miami, FL, USA
6Diabetes Department, Niguarda Hospital, Milan, Italy

The available information concerning the characteristics and composition of collagenase batches, which are effective in the digestion of human pancreas for islet transplants, is scarce and incomplete. A large inter- and intrabatched variability in activity and efficiency of blend enzymes available for isolation has been observed. The aim of this study was to characterize enzyme blend components. Liberase batches were characterized by SDS-PAGE analyses, microelectrophoresis, and then by MALDI-TOF MS analysis. Three main bands were detected by SDS-PAGE analysis and submitted to MALDI-TOF MS analysis. Two bands were found to correspond to class I (isoform b and another of 106 kDa) and one to class II (isoform d) collagenase. These results represent an important step towards a complete characterization of enzymes, with the final aim of identifying key components for a standardized product.

Key words: Islet isolation; Purification; Collagenase; Proteases

Address correspondence to Camillo Ricordi, Diabetes Research Institute, University of Miami, 1450 NW 10th Avenue, Miami, FL 33136, USA. E-mail: cricordi@med.miami.edu