ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 17, NUMBER 8, 2008

Cell Transplantation, Vol. 17, pp. 877-886, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Mature Adipocyte-Derived Cells, Dedifferentiated Fat Cells (DFAT), Promoted Functional Recovery From Spinal Cord Injury-Induced Motor Dysfunction in Rats

Yuki Ohta,1 Mitsuko Takenaga,1 Yukie Tokura,1 Akemi Hamaguchi,1 Taro Matsumoto,2 Koichiro Kano,3 Hideo Mugishima,2 Hideyuki Okano,4 and Rie Igarashi1

1Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki 216-8512, Japan
2Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, Nihon University School of Medicine, Tokyo 173-8610, Japan
3Department of Animal Sciences, College of Bioresource Sciences, Nihon University, Fujisawa 252-8510, Japan
4Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan

Transplantation of mature adipocyte-derived cells (dedifferentiated fat cells) led to marked functional recovery from spinal cord injury (SCI)-induced motor dysfunction in rats. When mature adipocytes were isolated from rat adipose tissue and grown in ceiling culture, transformation into fibroblast-like cells without lipid droplets occurred. These fibroblast-like cells, termed dedifferentiated fat cells (DFAT), could proliferate and could also differentiate back into adipocytes. DFAT expressed neural markers such as nestin, bIII tubulin, and GFAP. Allografting of DFAT into SCI-induced rats led to significant recovery from hindlimb dysfunction. Grafted cells were detected at the injection site, and some of these cells expressed bIII tubulin. DFAT expressed neurotrophic factors such as BDNF and GDNF prior to transplantation, and grafted cells were also positive for these factors. Therefore, these neurotrophic factors derived from grafted DFAT might have contributed to the promotion of functional recovery. These findings also suggest that mature adipocytes could become a new source for cell replacement therapy to treat central nervous system disorders.

Key words: Mature adipocytes; Dedifferentiated fat cells (DFAT); Spinal cord injury; Transplantation; Neurotrophic factor

Address correspondence to Yuki Ohta, Institute of Medical Science, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8512, Japan. Tel: +81 44 977 8111; Fax: +81 44 976 3747; E-mail: yuki-o@marianna-u.ac.jp

Cell Transplantation, Vol. 17, pp. 887-897, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Evaluation of Drug-Metabolizing and Functional Competence of Human Hepatocytes Incubated Under Hypothermia in Different Media for Clinical Infusion

María José Gómez-Lechón,1,2 Agustín Lahoz,3 Nuria Jiménez,1,2 Ana Bonora,1,2 José V. Castell,1,2,4 and María Teresa Donato1,2,4

1Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Valencia, Spain
3Unidad Mixta Hospital La Fe-Advancell, Valencia, Spain
4Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. Key factors for clinical cell transplantation to progress is to prevent hepatocyte damage, loss of viability and cell functionality, factors that depend on the nature of the tissue used for isolation to a large extent. The main sources of tissue for hepatocyte isolation are marginal livers that are unsuitable for transplantation, and segments from reduced cadaveric grafts. Hepatocellular transplantation requires infusing human hepatocytes in suspension over a period of minutes to hours. The beneficial effect of hypothermic preservation of hepatocytes in infusion medium has been reported, but how critical issues towards the success of cell transplantation, such as the composition of infusion medium and duration of hepatocyte storage will affect hepatocyte quality for clinical cell infusion has not been systematically investigated. Infusion media composition is phosphate-buffered saline containing anticoagulants and human serum albumin. The supplementation of infusion media with glucose or N-acetyl-cystein, or with both components at the same time, has been investigated. After isolation, hepatocytes were suspended in each infusion medium and a sample at the 0 time point was harvested for cell viability and functional assessment. Thereafter, cells were incubated in different infusion media agitated on a rocker platform to simulate the clinical infusion technique. The time course of hepatocyte viability, funtionality (drug-metabolizing enzymes, ureogenic capability, ATP, glycogen, and GSH levels), apoptosis (caspase-3 activation), and attachment and monolayer formation were analyzed. The optimal preservation of cell viability, attaching capacity, and functionality, particularly GSH and glycogen levels, as well as drug-metabolizing cytochrome P450 enzymes, was found in infusion media supplemented with 2 mM N-acetyl-cystein and 15 mM glucose.

Key words: Apoptosis; Hepatocytes; Glycogen; Hypothermia; Infusion medium; N-Acetyl-cystein; P450 enzymes; Ureogenesis

Address correspondence to María J. Gómez-Lechón, Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Avda de Campanar 21, 46009-Valencia, Spain. Tel: +34 96 1973048; Fax: +34 96 1973018; E-mail: gomez_mjo@gva.es

Cell Transplantation, Vol. 17, pp. 899-909, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Use of Bioluminescent Imaging to Assay the Transplantation of Immortalized Human Fetal Hepatocytes Into Mice

Moon Seok Choi,1,2* Andreea M. Catana,1* Jian Wu,1 Young Seok Kim,1 Sang Jeong Yoon,1 Alexander D. Borowsky,3 Sanjiv S. Gambhir,4 Sanjeev Gupta,5 and Mark A. Zern1

1Transplant Research Institute, UC Davis Medical Center, Sacramento, CA, USA
2Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 135-710, Korea
3Department of Pathology and Laboratory Medicine, UC Davis, Davis, CA, USA
4Molecular Imaging Program at Stanford, Departments of Radiology and Bioengineering, Bio-X Program, Stanford University, Stanford, CA, USA
5Marion Bessin Liver Research Center, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

Noninvasive serial monitoring of the fate of transplanted cells would be invaluable to evaluate the potential therapeutic use of human hepatocyte transplantation. Therefore, we assessed the feasibility of bioluminescent imaging using double or triple fusion lentiviral vectors in a NOD-SCID mouse model transplanted with immortalized human fetal hepatocytes. Lentiviral vectors driven by the CMV promoter were constructed carrying reporter genes: firefly luciferase and green fluorescence protein with or without herpes simplex virus type 1 thymidine kinase. Human fetal hepatocytes immortalized by telomerase reconstitution (FHhTERT) were successfully transduced with either of these fusion vectors. Two million stably transduced cells selected by fluorescence-activated cell sorting were injected into the spleens of NOD-SCID mice pretreated with methylcholanthrene and monocrotaline. The transplanted mice were serially imaged with a bioluminescence charged-coupled device camera after D-luciferin injection. Bioluminescence signal intensity was highest on day 3 (6.10 ± 2.02 × 105 p/s/cm2/sr, mean ± SEM), but decreased to 2.26 ± 1.54 × 105 and 7.47 ± 3.09 × 104 p/s/cm2/sr on day 7 and 10, respectively (p = 0.001). ELISA for human albumin in mice sera showed that levels were similar to those of control mice on day 2 (3.25 ± 0.92 vs. 2.84 ± 0.59 ng/ml, mean ± SEM), peaked at 18.04 ± 3.11 ng/ml on day 7, and decreased to 8.93 ± 1.40 and 3.54 ± 0.87 ng/ml on day 14 and 21, respectively (p = 0.02). Real-time quantitative RT-PCR showed gene expression levels of human albumin, a1-antitrypsin, and transferrin in mouse liver were 60.7 ± 6.5%, 26.0 ± 1.4%, and 156.8 ± 62.4% of those of primary human adult hepatocytes, respectively, and immunohistochemistry revealed cells with human albumin and a1-antitrypsin expression in the mouse liver. In conclusion, our study demonstrated that bioluminescent imaging appears to be a sensitive, noninvasive modality for serial monitoring of transplanted hepatic stem cells.

Key words: Hepatocytes; Stem cells; Transplantation; Luciferase; Imaging techniques

Address correspondence to Mark A. Zern, M.D., Transplant Research Institute, UC Davis Medical Center, 4635 Second Avenue, Suite 1001, Sacramento, CA 95817, USA. Tel: (916) 734-8063; Fax: (916) 734-8097; E-mail: mazern@ucdavis.edu

*These authors contributed equally to the work.

Cell Transplantation, Vol. 17, pp. 911-922, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

Merced Leiker, Gen Suzuki, Vijay S. Iyer, John M. Canty, Jr., and Techung Lee

Center for Research in Cardiovascular Medicine, University at Buffalo, Buffalo, NY, USA

Therapeutic implantation of mesenchymal stem cells (MSCs) is entering the realm of clinical trials for several human diseases, and yet much remains uncertain regarding their dynamic distribution and cell fate after in vivo application. Discrepancies in the literature can be attributed in part to the use of different cell labeling/tracking methods and cell administration protocols. To identify a stem cell detection method suitable for myocardial implantation in a large animal model, we experimented on three different MSC labeling methods: adenovirus-mediated expression of enhanced green fluorescence protein (EGFP) and b-galactosidase (LacZ), and nuclear staining with DAPI. Intramuscular and intracoronary administrations of labeled porcine MSCs identified the nuclear affinity dye to be a reliable stem cell tracking marker. Stem cell identification is facilitated by an optimized live cell labeling condition generating bright blue fluorescence sharply confined to the nucleus. DAPI-labeled MSCs retained full viability, ceased proliferation, and exhibited an increased differentiation potential. The labeled MSCs remained fully active in expressing key growth factor and cytokine genes, and notably exhibited enhanced expression of the chemokine receptor CXCR4 and its ligand SDF1, indicating their competency in response to tissue injury. Histological analysis revealed that approximately half a million MSCs or ~2% of the administered MSCs remained localized in the normal pig heart 2 weeks after coronary infusion. That the vast majority of these identified MSCs were interstitial indicated the ability of MSCs to migrate across the coronary endothelium. No evidence was obtained indicating MSC differentiation to cardiomyocyte.

Key words: Mesenchymal stem cells; Heart; Tracking; Implantation

Address correspondence to Techung Lee, Department of Biochemistry, SUNY at Buffalo, 3435 Main Street, Buffalo, NY 14214, USA. Tel/fax: (716) 829-3106; E-mail: chunglee@buffalo.edu

Cell Transplantation, Vol. 17, pp. 923-941, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Use of Clinically Approved Small Particles of Iron Oxide (SPIO) for Labeling of Mesenchymal Stem Cells Aggravates Clinical Symptoms in Experimental Autoimmune Encephalomyelitis and Influences Their In Vivo Distribution

Richard Schäfer,1 Miriam Ayturan,2 Rüdiger Bantleon,3 Rainer Kehlbach,3 Georg Siegel,1 Joerg Pintaske,4 Sabine Conrad,5 Hartwig Wolburg,6 Hinnak Northoff,1 Jakub Wiskirchen,3 and Robert Weissert2

1Institute of Clinical and Experimental Transfusion Medicine, University Hospital Tübingen, Tübingen, Germany
2Experimental Neuroimmunology, Department of General Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany
3Department of Diagnostic Radiology, University Hospital Tübingen, Tübingen, Germany
4Department of Diagnostic Radiology, Section on Experimental Radiology, University Hospital Tübingen, Tübingen, Germany
5Department of Anatomy, University of Tübingen, Tübingen, Germany
6Department of Pathology, University of Tübingen, Tübingen, Germany

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Mesenchymal stem cells (MSC) have been shown to ameliorate symptoms in experimental autoimmune encephalomyelitis (EAE), a model of MS. Using cloned MSC labeled with clinically approved small particles of iron oxide (SPIO) for treatment of EAE we analyzed the tissue localization of transferred cells. Treatment with unlabeled MSC led to disease amelioration compared to controls. In contrast, treatment with SPIO-labeled MSC lead to increase in disease severity. Treatment with SPIO alone did not alter disease course. After transplantation labeled and nonlabeled MSC were detected in the CNS and the liver with significantly more SPIO-labeled cells present in the CNS. Iron deposition was present in the group treated with SPIO-labeled MSC, indicating that in vivo the initially cell surface-bound iron detached from the MSC. These results could be of great importance for imaging of patients in the clinical setting, indicating that in vivo application of SPIO-labeled MSC needs to be performed with caution because the cell-derived exposure of iron can lead to disease aggravation.

Key words: Experimental autoimmune encephalomyelitis; Multiple sclerosis; Mesenchymal stem cells; Small particles of iron oxide; Imaging; Homing

Address correspondence to Richard Schäfer, M.D., Institute of Clinical and Experimental Transfusion Medicine, University Hospital Tübingen, D-72076 Tübingen, Germany. Tel: +49-7071-2981665; Fax: +49-7071-295240; E-mail: richard.schaefer@med.uni-tuebingen.de

Cell Transplantation, Vol. 17, pp. 943-953, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Bone Marrow Cell Transplant Does Not Prevent or Reverse Murine Liver Cirrhosis

L. F. Quintanilha,1 E. G. Mannheimer,1 A. B. Carvalho,1 B. D. Paredes,1 J. V. Dias,1 A. S. Almeida,2 B. Gutfilen,3 L. M. Barbosa da Fonseca,3 C. M. C. Resende,3 G. F. M. Rezende,3 A. C. Campos de Carvalho,1 and R. C. S. Goldenberg1

1Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, 21941-902, Brazil
2Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, 21941-902, Brazil
3Faculdade de Medicina, Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, 21941-902, Brazil

We tested the effect of bone marrow cell (BMC) transplantation in either preventing or reversing cirrhosis on an experimental model of chronic liver disease. Female Wistar rats were fed a liquid alcohol diet and received intraperitoneal injections of carbon tetrachloride (CCl4) over 15 weeks. Ten animals (cell-treated group) received five injections of BMCs during the cirrhosis induction protocol (on the 4th, 6th, 8th, 10th, and 12th weeks) and four animals received the cells after liver injury was established through tail vein. Nine animals (nontreated group) were submitted to the previously described protocols; however, they received vehicle injections. Analyses were performed to verify whether the infusion of cells was effective in preventing the development of cirrhosis in our model of induction, and if the cells could reverse cirrhosis once it was established. Hepatic architecture and fibrotic septa were analyzed in liver slices stained with hematoxilin & eosin and Sirius red, respectively. Fibrosis quantification was measured by Sirius red histomorphometry. Indirect immunofluorescence was performed to detect the amount of tissue transglutaminase 2. Blood analyses were performed to assess liver injury and function by the assessment of alanine aminotransferase and albumin. Ultrasound was performed to analyze the portal vein caliber and presence of ascitis. Cirrhosis features (regenerative nodules and fibrous septa) were observed in histopathology after 15 weeks of continuous hepatic injury in nontreated and cell-treated groups. Collagen content, immunofluorescence analysis, and biochemical and ultrasound parameters were similar in nontreated and cell-treated groups; however, both groups showed significant differences compared to a normal control group. Cell infusions with bone marrowderived cells seem to be ineffective in improving morphofunctional parameters of the liver when applied to chronic cases either during or after establishment of the hepatic lesion.

Key words: Cirrhosis; Bone marrow cell; Biochemistry; Histology; Ultrasound

Address correspondence to Regina Coeli dos Santos Goldenberg, Avenida Carlos Chagas Filho, 373, Edifício do Centro de Ciências da Saúde, Bloco G (Instituto de Biofísica Carlos Chagas Filho), 2° andar/sala 53, Cidade Universitária, 21941-902, Rio de Janeiro, Brazil. Tel: +55 2125626559; E-mail: rcoeli@biof.ufrj.br

Cell Transplantation, Vol. 17, pp. 955-968, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Comparative Characterization of Cultured Human Term Amnion Epithelial and Mesenchymal Stromal Cells for Application in Cell Therapy

Grozdana Bilic,1* Steffen M. Zeisberger,1* Ajit S. Mallik,1,2 Roland Zimmermann,1 and Andreas H. Zisch1,2

1Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland
2Zurich Center for Integrative Human Physiology, Zurich, Switzerland

Emerging evidence suggests human amnion tissue as a valuable source of two distinct types of pluripotent cells, amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs), for applications in cell replacement therapy. For some approaches, it may be necessary to culture and differentiate these cells before they can be transplanted. No systematic attempt has been yet made to determine the quantity and quality of amnion cells after isolation and culture. We looked at amnion cell isolates from 27 term placentas. Following our optimized protocol, primary yields were 6.3 × 106 hAECs and 1.7 × 106 hAMSCs per gram amnion. All 27 cases gave vital cultures of hAMSCs, while one third of cases (9 of 27) failed to give adherent cultures of hAECs. Primary cultures contained significantly more proliferating than apoptotic cells (hAECs: 16.4% vs. 4.0%; hAMSCs: 9.5% vs. 2.4%). Neither hAECs nor hAMSCs were clonogenic. They showed slow proliferation that almost stopped beyond passage 5. Microscopic follow-up revealed that hAEC morphology gradually changed towards mesenchymal phenotype over several passages. Flow cytometric characterization of primary cultures showed expression of mesenchymal progenitor markers CD73, CD90, CD105, and CD166, as well as the embryonic stem cell markers SSEA-3 and -4 on both amnion cell types. These profiles were grossly maintained in secondary cultures. Reverse transcriptase-PCR analysis exhibited transcripts of Oct-3/4 and stem cell factor in primary and secondary cultures of all cases, but no telomerase reverse transcriptase. Immunocytochemistry confirmed translation into Oct-3/4 protein in part of hAEC cultures, but not in hAMSCs. Further, both amnion cell types stained for CD90 and SSEA-4. Osteogenic induction studies with amnion cells from four cases showed significantly stronger differentiation of hAECs than hAMSCs; this capacity to differentiate greatly varied between cases. In conclusion, hAECs and hAMSCs in culture exhibit and maintain a similar marker profile of mesenchymal progenitors. hAECs were found as a less reliable source than hAMSCs and altered morphology during subculture.

Key words: Human amnion epithelial cells; Human amnion mesenchymal cells; Culture; Stemness markers; Cell transplantation

Address correspondence to Grozdana Bilic, Ph.D. or Steffen M. Zeisberger, Ph.D., Department of Obstetrics, University Hospital Zurich, Frauenklinikstrasse 10, 8091 Zurich, Switzerland. Tel: +41 44 2558585; Fax: +41 44 2554430; E-mail: grozdana.bilic@usz.ch or steffen.zeisberger@usz.ch

*Both authors contributed equally to this work.

Cell Transplantation, Vol. 17, pp. 969-975, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Local Effects of Retrovirally Transduced Endostatin-Expressing Human Umbilical Cord Blood CD34+ Cells on Transplanted Malignancy in a Mouse Model of Hepatic Cancer

Zao Jiang,1 Gao-Jun Teng,2 Bao-An Chen,3 Zheng-Feng Xu,4 Jie Hu,1 and Ze-Ye Shao3

1Department of Oncology, Zhongda Hospital, Southeast University, Nanjing, China
2Jiangsu Provincial Key Laboratory of Molecular Imaging, Zhongda Hospital, Southeast University, Nanjing, China
3Laboratory of Hematology , Clinical Medical Collage, Southeast University, Nanjing, China
4The Center of Prenatal Diagnosis, Nanjing Maternity and Child Health Hospital, Nanjing Medical University, Nanjing, China

Antiangiogenesis has been exploited as an effective approach to inhibit the growth of solid tumors. This technique has been evaluated using various vectors in several xenograft animal models to demonstrate the efficacy of endostatin gene therapy against cancer growth. However, previous studies have not examined the use of cord blood CD34+ cells as endostatin-producing cells for gene therapy against hepatoma. This exploratory study was done to investigate the local effects of CD34+ cells transduced with the endostatin gene on a mouse xenograft tumor model. The human endostatin gene was transferred into CD34+ cells using the recombinant retrovirus plasmid, pLncx/endo. Expression was verified by RT-PCR and Western blot analyses, confirming the stable expression and secretion of endostatin from the transferred CD34+ cells. The proliferation of vascular endothelial cells was evaluated by MTT assay and found to decrease by about 59.9% when treated with the supernatant of cultured transfected CD34+ cells in vitro. These genetically modified cord blood CD34+ cells were implanted intratumorally and tumor regression was evaluated after 2 weeks. The average size of a xenograft tumor in the CD34+/endo group was reduced 31.39% compared to that in the untreated mice or those transplanted with CD34+ cells transduced with a control vector. The microvascular density of the tumor decreased 62.45% in the treated group. The expression of proliferation cell nuclear antigen (PCNA) also decreased significantly in the treated group. Moreover, the apoptotic index (AI) of tumors, as evaluated by TUNEL staining, was significantly enhanced in the treatment group. Our findings indicate that angiogenesis of the xenograft tumor in mice may be inhibited by local administration of genetically modified CD34+ cells expressing the endostatin gene. This novel approach may lead to a new direction of cell-based gene therapy for malignancy.

Key words: Endostatin; Gene therapy; Angiogenesis; CD34+ cells; Tumor

Address correspondence to Dr. Zao Jiang, Department of Oncology, Zhongda Hospital, Southeast University, 87# Ding-Jia-Qiao Road, Nanjing, 210009, China. Tel: 86-25-83275407; Fax: 86-25-83272011; E-mail: jiangzao2003@yahoo.com.cn

Cell Transplantation, Vol. 17, pp. 977-986, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Long-Term Survival and Bipotent Terminal Differentiation of Human Mesenchymal Stem Cells (hMSC) in Combination With a Commercially Available Three-Dimensional Collagen Scaffold

S. Neuss,1,2 R. Stainforth,1 J. Salber,3 P. Schenck,4 M. Bovi,5 R. Knüchel,1 and A. Perez-Bouza1

1Institute of Pathology, RWTH Aachen University, 52074 Aachen, Germany
2Interdisciplinary Centre for Clinical Research, IZKF "Biomat.," RWTH Aachen University, 52074 Aachen, Germany
3Department of Textile and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen, Germany
4Dr. Suwelack Skin & Health Care AG, 48727 Billerbeck, Germany
5Electron Microscopic Facility, RWTH Aachen University, 52074 Aachen, Germany

Researchers working in the field of tissue engineering ideally combine autologous cells and biocompatible scaffolds to replace defect tissues/organs. Due to their differentiation capacity, mesenchym-derived stem cells, such as human mesenchymal stem cells (hMSC), are a promising autologous cell source for the treatment of human diseases. As natural precursors for mesenchymal tissues, hMSC are particularly suitable for bone, cartilage, and adipose tissue replacement. In this study a detailed histological and ultrastructural analysis of long-term cultured and terminally differentiated hMSC on 3D collagen scaffolds was performed. Standardized 2D differentiation protocols for hMSC into adipocytes and osteoblasts were adapted for long-term 3D in vitro cultures in porous collagen matrices. After a 50-day culture period, large numbers of mature adipocytes and osteoblasts were clearly identifiable within the scaffolds. The adipocytes exhibited membrane free lipid vacuoles. The osteoblasts were arranged in close association with hydroxyapatite crystals, which were deposited on the surrounding fibers. The collagen matrix was remodeled and adopted a contracted and curved form. Human MSC survive long-term culture within these scaffolds and could be terminally differentiated into adipocytes and osteoblasts. Thus, the combination of hMSC and this particular collagen scaffold is a possible candidate for bone and adipose tissue replacement strategies.

Key words: Mesenchymal stem cells; Autologous cells; Adipose tissue engineering; Bone tissue engineering; Collagen; Electron microscopy

Address correspondence to Sabine Neuss, Ph.D., Institute of Pathology, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany. Tel: +49 241 8080622; Fax: +49 241 8082439; E-mail: sneuss-stein@ukaachen.de

Cell Transplantation, Vol. 17, pp. 987-996, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Redifferentiation of In Vitro Expanded Adult Articular Chondrocytes by Combining the Hanging-Drop Cultivation Method With Hypoxic Environment

Inigo Martinez,1 Jan Elvenes,1 Randi Olsen,2 Kjell Bertheussen,3 and Oddmund Johansen1

1Department of Orthopaedic Surgery, Institute of Clinical Medicine, University of Tromsø, 9037 Tromsø, Norway
2Department of Electron Microscopy, Medicine Faculty, University of Tromsø, 9037 Tromsø, Norway
3Department of Obstetrics and Gynaecology, University Hospital of Northern Norway, N-9038 Tromsø, Norway

The main purpose of this work has been to establish a new culturing technique to improve the chondrogenic commitment of isolated adult human chondrocytes, with the aim of being used during cell-based therapies or tissue engineering strategies. By using a rather novel technique to generate scaffold-free three-dimensional (3D) structures from in vitro expanded chondrocytes, we have explored the effects of different culture environments on cartilage formation. Three-dimensional chondrospheroids were developed by applying the hanging-drop technique. Cartilage tissue formation was attempted after combining critical factors such as serum-containing or serum-free media and atmospheric (20%) or low (2.5%) oxygen tensions. The quality of the formed microtissues was analyzed by histology, immunohistochemistry, electron microscopy, and real-time PCR, and directly compared with native adult cartilage. Our results revealed highly organized, 3D tissue-like structures developed by the hanging-drop method. All culture conditions allowed formation of 3D spheroids; however, cartilage generated under low oxygen tension had a bigger size, enhanced matrix deposition, and higher quality of cartilage formation. Real-time PCR demonstrated enhanced expression of cartilage-specific genes such us collagen type II and aggrecan in 3D cultures when compared to monolayers. Cartilage-specific matrix proteins and genes expressed in hanging-drop-developed spheroids were comparable to the expression obtained by applying the pellet culture system. In summary, our results indicate that a combination of 3D cultures of chondrocytes in hanging drops and a low oxygen environment represent an easy and convenient way to generate cartilage-like microstructures. We also show that a new specially tailored serum-free medium is suitable for in vitro cartilage tissue formation. This new methodology opens up the possibility of using autogenously produced solid 3D structures with redifferentiated chondrocytes as an attractive alternative to the currently used autologous chondrocyte transplantation for cartilage repair.

Key words: Cartilage; Chondrocytes; Hypoxia; Serum-free medium; Spheroids; Transplantation

Address correspondence to Inigo Martinez, Department of Orthopaedic Surgery, Institute of Clinical Medicine, University of Tromsø, 9037 Tromsø, Norway. Tel: +47 77 64 46 86; Fax: +47 77 62 71 64; E-mail: inigo.martinez@fagmed.uit.no