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A Special Issue on 5th Annual Meeting of GSZ.
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Vol.VIIIssue: 2:

Proceedings of the Annual Symposium & Plenary Session on Regenerative Medicine (PASRM)

doi:10.46582/jsrm.0702015

(JSRM code: 007020700015)

(International Year of Chemistry - Commemorative Lecture)

Nanotechnology, Cell Culture and Tissue Engineering
Haraguchi K1
(PASRM** 2011-001)

1Kawamura Institute of Chemical Research, Japan

Abstract

We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels) and soft, polymer nanocomposites (M-NCs: solid), with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide)(PNIPA)/clay network and M-NCs consisting of poly(2-methoxyethyacrylate)(PMEA)/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2), normal human dermal fibroblast (NHDF), and human umbilical vein endothelial cells (HUVEC), could be cultured to be confluent on the surfaces of N-NC gel or dried N-NC gels and M-NC film, regardless of their thickness. Also, it was found that cells cultured on the surfaces of N-NC gels and M-NCs could be detached in the forms of sheets of cells or single cells without trypsin treatment, but by just decreasing the temperature to 200C. Thus, the serious disadvantages (intractability, mechanical fragility, optical turbidity, poor processing ability, low stimulus sensitivity, etc.) associated with the conventional, chemically-crosslinked polymeric materials were overcome in NC gels and M-NCs. Also, NC gels and M-NCs can be used as new types of substrata with ability of cell culture and subsequent thermoresponsive cell detachment.

Corresponding Author: Dr. Kazutoshi Haraguchi,
General Manager and Director,
Kawamura Institute of Chemical Research,
Sakura 285-0078,
Chiba,
JAPAN

(JSRM code: 007020700016)

Biomarkers for Early Detection of Post-Transplantation Rejection
Levy G1
(PASRM** 2011-002)

1University of Toronto Transplantation Institute, Toronto, Canada

Abstract

Although transplantation is one of the major medical achievements of the last half century, the shortage of organs and need for long term immunosuppressive therapy limits its usefulness. Advances in stem cell therapy has the potential both to overcome the shortage of organs but also to provide novel ways of reintroducing a tolerogenic state in patients who require life saving transplantation therapy.

Understanding mechanisms of rejection which involve both innate and adaptive immunity would allow for novel therapeutic approaches to eliminate or avoid the use of toxic immunosuppressive agents. The evolving era of functional genomics in organ transplantation has been supported by advances in gene profiling, sequencing, proteomics, antibody profiling and bioinformatics. Thus, heralding a new era of intelligent and personalized monitor and therapy. Molecular and cell based biomarkers and now emerging which may be useful to monitor the immune status of the patient and it is anticipated that over the next several years these will detect rejection for immune events before a transplanted organ or cell is damaged. Patterns of genomic biomarkers are also being developed which may predict patients, who achieve tolerance which may not only be useful in the setting of transplantation, but also in patients with autoimmune disease. The ultimate goal of future studies will be to identify markers with sufficient predictive value to improve graft survival, limit graft injury from under immunosuppression and reduce patient morbidity from over immunosuppression.

These approaches will also be critical for successful stem cell therapy where it is known that immunologic barriers limit its adoption.

** Proceedings of Annual Symposium on Regenerative Medicine

Corresponding Author: Gary Levy, MD, FRCP(C), AGAF
Director, University of Toronto Transplantation Institute;
Director, Training Program in Regenerative Medicine,
University of Toronto,
CANADA

(JSRM Code: 007020700017)

Olfactory Mucosa Transplantation for Spinal Cord Injury
Iwatsuki K1
(PASRM** 2011-003)

1Osaka University Hospital, Osaka, Japan

Abstract

Carlos Lima et al. who are pioneers in this field reported their clinical pilot study of olfactory mucosa transplantation for chronic spinal cord injury. They showed the safety and feasibility of it. Olfactory mucosa contains the olfactory ensheathing cells and neural stem cells. Recent studies have demonstrated the potential therapeutic role of both cells in spinal cord injury. We have already reported the effectiveness of olfactory mucosa transplantation for rat spinal cord injury. Furthermore we indicated the reconstruction of cortico-spinal tract by BDA (biotinylated dextran amine) tracer study with the olfactory mucosa transplantation. We elucidated how grafts of nasal olfactory mucosa repair the injured rat spinal cord as compared with the nasal respiratory mucosa containing no olfactory ensheathing cell and neural stem cell. The spinal cord of recipient rats (adult female Sprague-Dawlley rats; 10 rats; 160-180g) was exposed at The 8-9 level, and a contusion injury was produced using the weight drop device developed at New York University. The exposed cord was moderately contused by a 10g weight that dropped from a height of 75 mm. A couple of weeks after injury, the injury site were exposed and posterior sulcus of the spinal cord was opened. Minced olfactory mucosa or respiratory mucosa derived from GFP rats were transplanted into the sulcuses. The BBB score in each animal was observed at 1, 2, 4 and 8 weeks after the transplantation. The recovery of the hind limb movement in the olfactory mucosa transplanted rats improved significantly compared to the respiratory mucosa transplanted rats. In histological assessment, the expression of p75NGFR and GFAP was strong in the olfactory mucosa grafts at 1 and 2 weeks after the transplantation and it was decreased at 8 weeks after the transplantation. The expression of p75NGFR and GFAP was not observed in the respiratory mucosa graft. The expression of Neurofilament was observed strongly at the site in the olfactory mucosa transplanted rats. The numerous fibres strongly stained with Neurofilament were surrounding the GFP positive cells and penetrating the transplanted olfactory mucosa. There were no apparent Neurofilament stained fibres at the marginal spinal cord. As we have already reported, olfactory mucosa transplantation for spinal cord injury has a certain effectiveness for the hind limb motor recovery. In this study, we recognized the numerous axons which surround the transplanted cells and penetrate the mucosa at the transplanted site without marginal spinal white matter. Olfactory mucosa might be more suitable niche than white matter which contains inhibiting factor for axonal regeneration in spinal cord. To succeed the neuronal regenerative therapy, cells, factors and scaffold have been required. Olfactory mucosa might have all of them. We are now performing the clinical trial of olfactory mucosa transplantation for chronic complete spinal cord injuries in Japan. We could have four patients so far and recognize the voluntary EEG of their thigh.

Corresponding Author: Dr. Koichi Iwatsuki,
Dept. Of Neurosurgery,
Osaka University Hospital,
Osaka, JAPAN

(JSRM Code: 007020700018)

Repair of Cartilage injuries using in vitro engineered 3D cartilage tissue- Preliminary Results of Our Animal Studies
Arumugam S1 , Manjunath S2, Senthilkumar R2, Rajendiran S3, Yoshioka H4, Mori Y4,
Abraham S2,5

(PASRM** 2011-004)

1. Dept. of Arthroscopy & Sports Medicine, Sri Ramachandra University, Chennai, India
2. Nichi-In Centre for Regenerative Medicine, Chennai, India
3. Central Laboratory, Sri Ramachandra University, Chennai, India
4. Waseda University, Tokyo, Japan 5. Yamanashi University-School of Medicine, Chuo, Japan

Abstract

Introduction: The cartilage injuries demand novel therapeutic approaches as the success rates of the current conventional strategies for the repair of injured articular cartilages are not that encouraging. Earlier we have reported that the Thermoreversible Gelation Polymer (TGP) is an ideal scaffold for human chondrocyte expansion in vitro. In this study, we report the preliminary results of the in vitro expansion, characterization and experimental in vivo transplantation of chondrocytes in a rabbit model of cartilage injury

Materials & Methods: Nine rabbits were included in this study scheduled for two years, after approval by the ethics committee. In the first animal, Chondrocytes were isolated from the weight bearing area of patellar groove in the left hindlimb and cultured in TGP Scaffold and maintained at 37°C in 5% carbon dioxide incubator for 64 days without growth factors. Then the TGP-Chondrocyte construct was transplanted into an experimental defect created in the knee of the right forelimb of the same rabbit. After a period of 10 weeks, a biopsy was taken from the transplanted region and subjected to morphological analysis, characterization by histopathology (H&E stain) and Immunohistochemistry (S-100 staining).

Results: The chondrocytes in the 3D TGP culture had round to oval shaped morphology without any de-differentiation which is otherwise observed in Conventional 2D cultures. A macroscopic structure which resembled cartilage was appreciated in the TGP construct in vitro after 64 days which was then transplanted to the rabbit. The H&E and Immunohistochemistry studies confirmed the presence of chondrocytes in the biopsy tissue.

Conclusion: Based on the results, we conclude that the TGP significantly supports the in vitro expansion of chondrocytes for a longer period and the 3D culture using TGP preserves the phenotype of the articular chondrocytes. The tissue thus grown when implanted with the TGP has engrafted well without any adverse reactions and upon confirmation of safety following completion of the entire study with adequate follow-up, human applications could be considered.

(JSRM Code: 007020700019)

Successful transplantation of in vitro expanded human corneal endothelial precursors to corneal endothelial surface using a nanocomposite sheets
Parikumar P1 , John S2, Senthilkumar R2, Manjunath S2, Baskar S2, Haraguchi K3, Abraham S2,4
(PASRM** 2011-005)

1. The Light Eye Hospital, Dharmapuri, India
2. Nichi-In Centre for Regenerative Medicine, Chennai, India
3. Kawamura Institute of Chemical Research, Chiba, Japan
4. Yamanashi University - Faculty of Medicine, Chuo, Japan

Abstract

Background: Though the transplantation of in vitro expanded human corneal endothelial precursors in animal models of endothelial damage by injecting into the anterior chamber has been reported, the practical difficulties of accomplishing such procedure in human patients have been a hurdle to clinical translation. Here we report the successful transplantation of in vitro expanded human corneal precursor cells to an animal eye using a transparent Nano-composite sheet and their engraftment.

Materials and Methods: Human Corneal endothelial cells (HCEC) were isolated from human cadaver eyes with informed consent and expanded in the lab using a sphere forming assay in a novel Thermoreversible Gelation Polymer (TGP) for 26 days. HCEC obtained by sphere forming assay were seeded in a novel Nano-composite sheet, which was made of PNIPA-NC gels by in-situ, free-radical polymerization of NIPA monomer in the presence of exfoliated clay (synthetic hectorite “Laponite XLG”) uniformly dispersed in aqueous media. After a further seven days in vitro culture of HCEC in the Nano-composite sheet, cells were harvested and transplanted on cadaver-bovine eyes (n=3). The cells were injected between the corneal endothelial layer and the Nano-composite sheet that had been placed prior to the injection in close proximity to the endothelial layer. After three hours, the transplanted Nano-composite sheets were removed from the bovine eyes and subjected to microscopic examination. The corneas were subjected to Histo-pathological studies along with controls.

Results: HCEC formed sphere like colonies in TGP which expressed relevant markers as confirmed by RT-PCR. Microscopic studies of the Nanosheets and histopathological studies of the cornea of the Bull’s eye revealed that the HCEC got engrafted to the corneal endothelial layer of the bovine eyes with no remnant cells in the Nanosheet.

Conclusion: Transplantation of in vitro expanded donor human corneal endothelial cells using a transparent Nano-composite sheet was feasible in bovine eyes and the HCEC an engrafted within three hours of transplantation. Pilot human studies could be planned for utilization of this material and strategy.

(JSRM Code: 007020700020)

Autologous Immune Enhancement Therapy for cancer using NK cells and CTLs without feeder layers; our six year experience in India
Dedeepiya V1 , Terunuma H2, Manjunath S1, Senthilkumar R1, Thamaraikannan P1, Srinivasan T1, HelenReena C 1,Preethy S 1, Abraham S1,4
(PASRM** 2011-006)

1. Nichi-In Centre for Regenerative Medicine, Chennai, India
2. Biotherapy Institute of Japan, Tokyo, Japan
3. Hope Foundation, Chennai, India
4. Yamanashi University - Faculty of Medicine, Chuo, Japan

Abstract

Background: Autologous Natural Killer (NK) cells and Cytotoxic T Lymphocytes (CTLs) based immune-cell therapy, otherwise called as Autologous Immune enhancement therapy (AIET), though has been in clinical practice in several developed nations since early 90s, in India it is in infancy due to lack of technological knowhow. Our institute has been providing the AIET cell expansion services since 2005 and we here in report our experience in 30 such patients of both solid tumours and hematological malignancies.

Materials and Methods: The number of AIET transfusions in each patient ranged from one to six. All the patients included had Stage III to IV malignancy. AIET was either given along with the chemotherapy or after the completion of a minimum of six cycles of chemotherapy in all the patients. 70 ml of Peripheral Blood was collected each time. The protocol followed was as per Terunuma et al (Breast Cancer 2010) which uses only the patients’ autologous plasma for expansion of the Natural Killer Cells and Cytotoxic T lymphocytes from the peripheral blood. The cells were cultured for a period of 10 to 16 days and then transfused to the patients intravenously. The cells were subjected to Flow cytometry before and after the in vitro expansion. Feeder layers were not used in the procedure of in vitro expansion at any stage.

Results: The percentage of NK cells and CTLs after expansion by flow cytometry ranged from 60 to 82 %. There were no adverse reactions in any of the patients following transfusion. The mean prolonged survival time was 15 months and 27% of the patients had Static non-progressive disease after the therapy. Two patients reported significant decrease in Cancer marker levels after AIET and among the terminally ill, two had more than two years survival. All the patients reported improvement in quality of life and resumption of appetite following AIET.

Conclusion: Optimal in vitro expansion of NK cells and CTLs of patients with stage III-IV cancer either concurrently or after chemotherapy could be accomplished using autologous serum without use of feeder layers. The In vitro expanded NK cells and CTLs when given intravenously decrease the tumor size and prolong the survival without any adverse effect in our experience.

(JSRM Code: 007020700021)

Stem Cell Therapy for Cardiovascular Disorders - Our Clinical Experience
Jayakrishnan AG1,2 , Madhusankar N3, Rao YY 4, Manjunath S5, Dedeepiya V5, Abraham S5,6
(PASRM** 2011-007)

1. Omega Hospital, Mangalore, India
2. Fr. Mullers Hospital, Mangalore, India
3. Frontier Lifeline, Chennai, India *
4. KG Hospital, Coimbatore, India
5. Nichi-In Centre for Regenerative Medicine, Chennai, India
6. Yamanashi University-Faculty of Medicine, Chuo, Japan

Abstract

Background: Autologous Bone Marrow stem Cell transplantation is a viable therapeutic option for patients with end stage heart failure due to cardiomyopathy of varied etiology as there are only limited treatment options other than cardiac transplantation.
The rationale behind the application of stem cells in these patients include

Materials and Methods: In this presentation we describe our study on a series of 13 patients who received isolated and expanded CD 34 cells from the bone marrow. Seven had ischemic dysfunction, three had dilated cardiomyopathy and three had primary pulmonary hypertension. Five patients received the stem cells via intracoronary injection, three directly into the myocardium and three intrapulmonary.

Results: All patients showed functional improvement of the myocardium recorded by non-invasive investigations and improvement in the quality of life. Follow up period ranged from 6 months to 2 years.

Conclusion: Our experience with bone marrow derived stem cells in patients with cardiomyopathy has been encouraging. More studies are planned in the future.

*Former affiliation, when the study was done

(JSRM Code: 007020700022)

Our experience of application of Autologous Bone Marrow Stem Cells in critical limb ischemia in six diabetic patients – A five-year follow-up
Subrammaniyan R1 , Amalorpavanathan J 1, Shankar R 1, Rajkumar M 1, Baskar S 2,Manjunath S 2, Senthilkumar R 2, Abraham S2,3
(PASRM** 2011-007)

1. Vascular Surgery Department, Vijaya Hospital, Chennai, India
2. Nichi-In Centre For Regenerative Medicine, Chennai, India
3. Yamanashi University-Faculty of Medicine, Chuo, Japan

Abstract

Background: Numerous Clinical studies have reported the safety and efficacy of injection of one Marrow and Peripheral Blood Mononuclear cells in patients with lower limb ischemia. Earlier we have reported the six months follow-up of successful application of autologous bone marrow mononuclear cells in patients with Fontaine Stage IV critical limb ischemia due to diabetes. As a continuation of the previous study, herein we report the long term results of the six patients after a follow-up for five years.

Materials and Methods: Six Diabetic patients with Fontaine Stage IV critical limb ischemia with ulcers were given intra-lesional injections of their autologous bone marrow mononuclear cells (BMMNC), isolated following the cGMP protocols. The patients have been followed up at regular intervals for five years after the treatment with all relevant clinical investigations.

Results: Six months follow-up results revealed that all the patients showed improvements with appearance of healthy granulation tissue and uniform revascularization. Complete healing was reported at a mean duration of nine months in five patients and one patient died due to a complication of renal failure, peritoneal dialysis and cardiac failure, which were unrelated to the BMMNC injection. Five year continuous follow-up revealed that the healed tissue with or without skin grafting remained healthy in all the five patients and two of the patients are able to walk without support with a pain free walking distance of greater than 100m.There were no adverse effects in any of the patients.

Conclusion: Autologous bone marrow stem cell therapy has been found to be salvaging the affected limb in patients with Fontaine Stage IV Critical Limb ischemia patients where revascularization was not feasible. Hence with our experience of six patients we recommend that the same should be considered in patients of similar clinical parameters before considering an amputation.


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