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A Special Issue on 5th Annual Meeting of GSZ.
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Vol.IVIssue: 1:

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

doi:10.46582/jsrm.0401003

(JSRM code: 004010700003)

Nanobiotechnology : What? Why? How?

Jagannadh B1
(PASRM** 2008-001)

1 Indian Institute of Chemical Technology, Hyderabad, India

Nanotechnology is the manipulation or self-assembly of individual atoms, molecules, or molecular clusters into structures to create materials and devices with new or vastly different properties. Nanotechnology can work from the top down (which means reducing the size to the nanoscale e.g. marble block to a statue or the bottom up (which involves manipulating individual atoms and molecules into nanostructures ie. we add matter till we end with the final or the desired product. Nanotechnology has been described as the new industrial revolution and both developed and developing countries are investing heavily in this technology to secure a market share.

The economy of the future will be a “knowledge-based economy” and maximizing knowledge through education, research and innovation will be important goals. Nanotechnology has a vast potential to revolutionize the critical sectors of the economy viz. health and agriculture: the subjects chosen for this talk. New tools for the treatment of diseases, disease detection, smart sensors and smart delivery systems for both mankind and agriculture are being developed and it will help to combat the viruses and other pathogens that effect both humans and the agriculture sector. It is expected that In the near future it will become possible through nanotechnology to increase the efficiency of drugs and pesticides and yet using lower doses. In the recent years applications of nanoscale biotechnology to food systems have been identified and are being pursued. Some of the studies are directed towards developing nano-sensing technology for detecting trace components in food, preserving food nutrients for an extended term and developing technology for preserving functional components without negatively impacting the palatability of food.

The talk will cover what is nanobiotechnology, why we study nanobiotechnology and how to create the nanoengineered materials and what we can expect to get out of nanobiotechnology. The work in some of these areas being carried out at the Indian Institute of Chemcical Technology will also be covered. 

Correspondign Author: Dr. B. Jagannadh, Scientist - Nanoscoience and Technology Group, Indian Institute of Chemical Technology, Hyderabad 500007. India. Tel: +9140 27193155. bj.nano@gmail.com; jagan@iict.res.in

(JSRM code: 004010700004)

Islet neogenesis potential of human adult stem cells and its applications in cell replacement therapy for diabetes

Bhonde RR1
(PASRM** 2008-002)

1 National Centre for Cell Science, Pune, India

In recent years regenerative biology has reached to greater heights due to its therapeutic potential in treating degenerative diseases; as they are not curable by modern medicine. With the advent of research in stem cells and developmental biology the regenerative potential of adult resident stem cells is becoming clearer. The long term objective of regenerative medicine or cell therapy is to treat patients with their own stem cells. These stem cells could be derived from the diseased organs such as skin, liver, pancreas etc. or from reservoirs of multipotent stem cells such as bone marrow or cord blood.

Manipulating the ability of tissue resident stem cells as well as from multipotent reservoirs such as bone marrow, umbilical cord and cord blood to give rise to endocrine cells may open new avenues in the treatment of diabetes. A better understanding of stem cell biology would almost certainly allow for the establishment of efficient and reliable cell transplantation experimental programs in the clinic. We show here that multipotent mesenchymal stem cells can be isolated from various sources such as the bone marrow, placenta, umbilical cord. Upon stimulation with specific growth factors they differentiate into islet like clusters (ILCs). When ILCs obtained from the above mentioned sources were transplanted in experimental diabetic mice, restoration of normoglycemia was observed within three weeks of transplantation with concomitant increase in the body weight. These euglycemic mice exhibited normal glucose tolerance test indicating normal utilization of glucose.

Allthough the MSCs isolated from all the sources had the same characteristics; they showed significant differences in their islet differentiation potential. ILCs isolated for the human bone marrow did not show any pancreatic hormones in vitro, but upon transplantation they matured into insulin and somatostatin producing hormones. Placental MSCs as well as ILCs showed insulin trascripts indicating their readiness towards islet lineage.

These studies point towards futuristic therapeutic approach of auto-transplantation of bone marrow for diabetes. Our studies demonstrate that human bone marrow, umbilical cord and placenta have the potential to differentiate into islets. These alternative sources of stem cells for islet neogenesis will form the basis for generating large number of islets required for transplantation for diabetes reversal.

Correspondign Author: Dr. R.R.Bhonde, Scientist ‘F’ Tissue engineering & Banking Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007, India. Tel : +9120 25690922. E-Mail: rrbhonde@nccs.res.in

(JSRM Code: 004010700005)

Clinical application of mesenchymal stem cells for aseptic bone necrosis

Aoyama T1

(PASRM** 2008-003)

1 Institute for Frontier Medical Sciences, Kyoto University, Japan

Since 2007, we had started clinical trial using mesenchymal stem cell (MSCs) for the treatment of aseptic bone necrosis as a first clinical trial permitted by Japanese Health, Labour and Welfare Ministry.

Aseptic bone necrosis of the femoral head commonly occurs in patients with two to four decades, causing severe musculoskeletal disability. Although its diagnosis is easy with X-ray and MRI, there has been no gold standard invented for treatment of this disease. MSCs represent a stem cell population in adult tissues that can be isolated and expanded in culture, and differentiate into cells with different nature. Combination with β-tri-calcium phosphate and vascularized bone graft, we succeeded to treat bone necrosis of the femoral head.

Regenerative medicine using stem cells is hopeful and shed a light on intractable disease. To become widespread, Basic, Translational, Application, and Developmental study is needed.? From an experience of cell therapy using MSCs, we started to research induced pluripotent stem cell (iPS) for clinical application.

Correspondign Author: Dr. Tomoki Aoyama, Institute for Frontier Medical Sciences, Kyoto University, Japan.

(JSRM Code: 004010700006)

Autologous Immune Enhancement therapy for Advanced Carcinoma of Pancreas-A Case Report

Sivaraman G1, Pandian A2, Baskar S3, Senthil KR3, Senthilnagarajan R3, Srinivasan V3, Dedeepiya V3, Abraham S3,4

(PASRM** 2008-004)

1. Shrusti Hospital, Chennai, India. 2. Deepam Hospital, Chennai, India. 3. Nichi-In Centre for Regenerative Medicine, Chennai, India. 4. Yamanashi University - Faculty of Medicine, Chuo, Japan

Introduction: Autologous Immune Enhancement therapy (AIET) is one of the lesser used modality of therapy in our country for advanced cancers. AIET uses patient’s own immune cells such as NK cells and T-Lymphocytes to fight against cancer.

Aim: To study the effectiveness of AIET in Advanced Carcinoma of pancreas.

Case History: A 40 years old male was diagnosed with inoperable locally advanced Carcinoma of the head of pancreas when he was investigated for obstructive jaundice. Palliative bypass surgery was done, since his tumor was inoperable. Following his surgery he received 6 cycles of adjuvant chemotherapy and 3 cycles of AIET.? After completion of chemotherapy and 2 cycles of AIET therapy his tumor marker decreased significantly with decrease in tumor size.? So re-laparotomy was attempted for removal of tumor, which was unsuccessful. After a long gap he received his 3rd cycle of AIET.? He was irregular in receiving his AIET treatment. He survived for 18 months after the diagnosis.

Conclusion: The median life span of patients following diagnosis of advanced Carcinoma of pancreas is usually not more than 3-6 months. Longer survival of our patient could be due to AIET, but we need further studies to prove it. There were no adverse effects encountered due to AIET. 

Corresponding Author: Dr.G.M.Sivaraman, MS, MRCS (UK) Consultant General & Laparoscopic Surgeon, Srushti Hospital, #1 Padmavathy Street, Thirumalai Nagar, Ramapuram, Chennai-600089. India. Tel:+91-44-24864748. gurunathan_mani@rediffmail.com;

(JSRM Code: 004010700007)

Autologous Stem Cell Therapy in Spinal Cord Injury - Our Initial Experience

Jasper JG 1, Sankaranarayanan S2, Baskar S3, Senthil KR3, Senthilnagarajan R3, Murugan P3, Abraham S3,4

(PASRM** 2008-005)

1. Kavery Medical Centre, Trichy, India. 2. Karpaga Vinayagar Dental College, Chengalpet, India. 3. Nichi-In Centre for Regenerative Medicine, Chennai, India. 4. Yamanashi University - Faculty of Medicine, Chuo, Japan

Background: Stem Cell Therapy (SCT) although in vogue since the early seventies for Hematological malignancies, is slowly evolving and extending into other fields. In Spinal Cord Injury its indications and uses are still getting refined.

Materials and methods: In our centre, after extensive review of literature drew up our own inclusion/ exclusion criteria which was approved by our ethics committee. we selected six patients with Dorsal Spinal Cord Injury for this study. Epidemiological and clinical data was collected and Videography of the patient’s initial presentation was done. Clinical assessment was carried out by a team of specialist including the Neurosurgeon, Neurologist, Urologist and Physiotherapist.100 ml of Bone marrow harvested from the posterior iliac crest was sent in cold preservation to NCRM, Chennai. Bone Marrow Mono Nuclear cells were isolated and CD34+ cells quantified using FACS. The isolated cells were injected through lumbar puncture. One of the six patients received two sittings of SCT, while all others have undergone only one sitting. The patients were followed on a monthly basis during which regular neurological reassessment was done and domiciliary physiotherapy was also given.

Result: One patient who was given two sittings of SCT made very good improvement and is now ambulant with the aid of an Orthosis. Two other patients who received one sitting of SCT had objective sensory and mild motor improvement; Three other patients had no improvement. There were no adverse reactions in any of them.

Conclusion: SCT is an evolving option for Spinal Cord Injury patients and although it is too early to comment, results as of date show a positive influence. The numbers of variables are many, but the results are promising and definitely warrant further clinical trials. 

Corresponding Author: Dr. Jos G. Jasper, MBBS., M.Ch (Neurosurgery) Consultant Neurosurgeon, Dept of Neurosciences, No.1, K.C.Road, Kavery Medical Centre, Tennur, Trichy - 620017. India. Tel: +91 431 4022555.
Email: : josjasper@rediffmail.com

(JSRM Code: 004010700008)

Cell therapeutics to treat diseases of the retina.

Natarajan S1, Asghar SA1, Baskar S2, Senthil KR2, Srinivasan V2, Murugan P2, Abraham S2,3

(PASRM** 2008-006)

1. Aditya Jyot Eye Hospital, Mumbai, India. 2. Nichi-In Centre for Regenerative Medicine, Chennai, India. 3. Yamanashi University - Faculty of Medicine, Chuo, Japan

Background: The adult Bone Marrow Stem Cells (BMSCs) have distinct advantages over the other types of stem cells. They are multipotent, can be stored for upto 10 years and considered to be one of the best sources of hematopoietic and mesenchymal stem cells in an adult body. Genetically inherited diseases such as Retinitis Pigmentosa and Degenerative diseases such as Age Related Macular Degeneration remain unsolved as no definitive treatment is available to repair the damages caused to the RPE and Photoreceptors as of now. In this scenario, the technique of Bone Marrow aspiration & isolation of Mono Nucleated Cells (MNCs) & intra-vitreal injection of a very small volume of MNCs in human retinal disorders has been standardized and is safe and feasible for human studies (Mohanty et al) and autotransplantation of RPEs from periphery to affected area are underpractice(Coffey et al). In this study we report our research work on different approaches to the above diseases using cell therapeutics

Study 1

Materials & methods: Ciliary Pigment Epithelium was harvested from donor eyes from Aditya Jyot Eye Hospital, Mumbai and was taken to and grown at NCRM lab. The cells were grown in the earlier reported methodology of Brenda et al (Science 2004).

Results: The CPE derived Retinal stem cells grew well in the lab. However, the practical difficulties of harvesting the same in patients limited our further steps in this study.

Study II:?

Materials & methods: Cadaver eye RPE cells were harvested and grown using polymer scaffolds after transporting them over 6 to 12 Hrs. The RPEs were grown on conventional methods and in polymer scaffolds and were subjected to RT-PCR.

Results: Human RPEs were able to grow without amniotic membrane and the same was proven by RT-PCR. This would make it possible for the peripheral RPEs taken from patients to be stored and later expanded and used for replacing the diseased cells of the central portion of the retina in future, without having to harvest the RPEs again.

Study III:

Materials & methods: Bone marrow mono nuclear cells were isolated and transported in cold containers (4-8oC) over a period of 6-12 Hrs and viability was evaluated.

Results: The bone marrow mononuclear cells were viable up to 12 Hrs in our methodology with a viability of more than 95% making it possible for cells isolated from Chennai centre to be taken to Mumbai or any other destination within a reach of 12 Hrs for application as reported in earlier studies.

Conclusion: The in-vitro expansion of RPEs without Human Amniotic Membrane is expected to open up a new possibility for treating the Retinal Degenerative Diseases. However an animal study is needed before clinical application. Intra vitreal application of Bone Marrow Mono Nuclear cells to treat RP and AMD as reported earlier are considered safe. We plan to undertake treatment and long term follow-up of more numbers of patients with RP and AMD.  

Corresponding Author: Dr.S.Natarajan, Chairman & Managing Director, Aditya Jyot Eye Hospital Pvt. Ltd., Plot No. 153, Road No. 9, Major Parameshwaran Road, Opposite S.I.W.S. College, Near Five Gardens, Wadala, Mumbai - 400 03. India. Tel: +91 22 24165533. Email: ajeh@vsnl.com

(JSRM Code: 004010700009)

Buccal Epithelium in treating Ocular Surface Disorders

Srinivas KR1, Sujatha M2, Mohan R2, Senthilnagarajan R3, Baskar S3, Senthil KR3, Abraham S3,4

(PASRM** 2008-007)

1. Darshan Eye Clinic, Chennai, India. 2. Rajan Eye Care Hospital, Chennai, India. 3. Nichi-In Centre for Regenerative medicine, Chennai, India. 4. Yamanashi University - Faculty of Medicine, Chuo, Japan

Background - Ocular surface disorders due to limbal stem cell deficiency are an important cause of ocular morbidity and visual loss. Although autologous limbal stem cell transplants have helped in the management of unilateral disease, allografts in those with bilateral disease often fail due to immunological reasons. The use of autologous buccal epithelium cultivated on amniotic membrane has been described as a useful approach in the management of this condition. It is the purpose of this study to explore the feasibility of using a novel thermo-gelatin polymer (TGP) as a substrate to culture these cells, and to characterize them using RNA extraction and RT-PCR.

Methods - Oral cheek mucosal biopsies were obtained from 5 adult patients undergoing Modified Osteo-Odonto Keratoprosthesis surgery. The specimens were transported to the laboratory in transport medium. The cells were released using enzymatic digestion and seeded in both convention culture medium and TGP. The resulting cellular growth was characterized using RNA extraction and RT-PCR.

Results - Cells could be cultured from 4 of the 5 specimens. In one specimen, contamination occurred and this was discarded. In the other specimens, the cheek epithelial cells could be cultured in both the conventional culture medium and TGP, with equal ease. RT-PCR revealed the presence of K3, a marker for epithelial cells, and GAPDH indicating the presence of some adipose tissue as well.

Conclusions - It is possible to culture autologous cheek mucosal epithelial cells using TGP, a synthetic scaffold, without the need for other biological substrates. Since the specimens are obtained from the oral cavity, stringent asepsis is required. Further studies are required for histopathological characterization of the cultured cells and to create a model for delivery onto the ocular surface of eyes with bilateral surface disease due to limbal stem cell deficiency.

Cover Image:

 

A: Buccal mucosal epithelial cells in culture on the 28th day in novel TGP synthtic scaffold

 

B: Histopathological examination and H&E staining of the cells grown in the method as shown above in Picture A
.

Corresponding Author: Dr Srinivas K Rao, DO, DNB, FRCSED, Director - Darshan Eye Clinic, T-80 (New 24) V- Main Road Anna Nagar, Chennai 600 040. India. Tel : +91 44 4350 0003. Email : srinikrao@gmail.com

(JSRM Code: 004010700010)

Envisaging an allogenic Corneal endothelial precursor/Stem Cell Bank (CESBANK)

Parikumar P1, Nelson J2, John S2, Baskar S3, Senthil KR3, Murugan P3, Senthilnagarajan R3, Srinivasan V3, Abraham S3,4, Amano S5

(PASRM** 2008-008)

1. The Light Eye Hospital, Dharmapuri, India. 2. Joseph Eye Hospital, Trichy, India. 3. Nichi-In Centre for Regenerative Medicine, Chennai, India. 4. Yamanashi University - Faculty of Medicine, Chuo, Japan. 5. Tokyo University-School of Medicine, Tokyo, Japan

Background: Bullous Keratopathy (BK) affects thousands of people in India every year. Though in early stages it is manageable medically, advanced disease warrants either total corneal transplantation or partial thickness transplantation for which a donor-cadaver cornea is necessary. Amano et al have reported the successful treatment of BK in animal models using in-vitro expanded human corneal endothelial precursors; though the rabbits had to be kept facing eye down to allow gravity assisted settling of the cells to the summit of the cornea where the damage had been created. For successful treatment using the above method, a human being has to lie prone with eyes immobilized for 24-36 Hrs. This is extremely discomforting and hence not practical. Corneal endothelium removed from the button and transported at varying temperature conditions for 48Hrs was successfully cultured in NCRM and this was reported earlier. We are working on a suitable scaffold to retain the cells in situ until their attachment to the damaged portion of the corneal endothelium enabling it to heal without the patient having to lie prone. With such capability, we envisage to make a corneal endothelial precursor/stem cell (CES) bank named as CESBANK to make in-vitro expanded CES available for patients with corneal diseases, most commonly Bullous Keratopathy (BK).

Materials & Methods for the project: The CESBANK will have a (i) Total ophthalmology diagnosis clinic, (ii) Operation theater and an out-patient clinic equipped for handling corneal procedures, (iii) An in-patient ward for post-CES transplantation patients, (iv) A cGMP cell screening, serology, immunology & molecular characterization lab, (v) Cell processing, expansion and cryopreservation lab, (vi) An eye bank, (vii) Documentation & networking facility (viii) Teaching accessories with lecture rooms, web & telecon capability (ix) A world class faculty for clinical & research projects. Earlier proven methodologies of corneal endothelial harvesting will be undertaken in CESBANK along with the collaborating hospitals. Studies comparing presently available scaffolds with novel nanomaterials for the application of CES into the affected portion of the eye will be undertaken. Long term cryopreservation and transportation to and fro from longer distances will be standardized to enlarge the network.

Expected Outcome: Ninety thousand patients are in the backlog every year, waiting for corneal transplantation in India, of which 30,000 may benefit from CESBANK. The CESBANK, as per the present plan, would be able to provide expanded CES for at least 14000 eyes to be treated every year when fully functional, provided it gets adequate number of donor eyes.

Conclusion:  The present project when implemented could make one donor eye be usable to more than 5 to 14 recipient eyes. By increasing the awareness for eye donation, more patients with corneal endothelial diseases awaiting donor cornea could be treated.

Corresponding Author: Dr. P. Parikumar, The Light Eye Hospital, 39D, By-pass Road, Dharmapuri Lodge Compound, Dharmapuri, Dharmapuri Dist, India. Tel: +91 4342 268368. Email: pparikumar@yahoo.com .

(JSRM Code: 004010700011)

The Gravity of Regenerative Medicine; Physics, Chemistry & Biology behind it

Dedeepiya V1, John S2, Abraham S1,3

(PASRM** 2008-009)

1. Nichi-In Centre for Regenerative Medicine, Chennai, India. 2. Joseph Eye Hospital, Trichy, India. 3. Yamanashi University - Faculty of Medicine, Chuo, Japan

The in-vitro expansion of cells of the organs/tissues and their re-implantation into the affected region/ tissue for treating cell/organ failure have been in practice for long, but in limited specialties. The in-vitro cell culture protocols use variety of biological reagents derived from animal sources and recombinant technologies. However, the optimal quantity of such biological components such as growth factors, cytokines etc.,needed for such cells to be grown in a non-physiological environment is still unknown. The use of such biological components have started to stir a controversy of late, due to the recognition of its potential hazards such as spread of prion diseases and contamination with non-human sialic acid proteins. Therefore synthetic reproducible biomaterials are gaining popularity in cell culture and tissue engineering.

The biomaterials made of several chemical components based on physical parameters are starting to change certain concepts about the niche of cell culture and that of stem cell expansion and differentiation to specific lineages. Engler et al have already proven that a simple change in the matrix elasticity alone could change the lineage of the cells. Spencer et al have reported that a change in bioelectricity could change the morphogenesis during development.

NCRM has been involved in cell culture and tissue engineering using approximately 240 different materials ranging from polymer hydrogel, gel with adherent inserts, nano composite materials, nano-coating technologies, nano-sheets and nano-films. These materials are used in cell culture in different hybrid combinations such as

  1. Floating 3D cell culture without adherent components in a homogenous hydrogel.
  2. Floating 3D cell culture with anchorage inserts.
  3. Flat surface- 2D adherent cell culture.
  4. Combined flat surface 2D cell culture (for differentiating cells) and floating 3D culture (for undifferentiated cells).

These combinations have started yielding several advantages in Corneal epithelial stem cells, Corneal endothelial precursors, Chondrocytes, Mouse Embryonic Stem Cells, Mouse Embryoid bodies.

Expansion of undifferentiated naive and Embryonic Stem (ES) cells has been made possible employing such hybrid techniques. With similar hybrid techniques we hope to make the undifferentiated expansion of fundamental hematopoietic stem cells possible. Nijnik et al have reported that the HSCs undergo damage with aging. If such in-vitro expansion technology without biological contamination could be made available in large scale, the day is not far off, when cryopreservation of one’s own hematopoietic stem cells harvested in youth can be cryopreserved and expanded and injected after a decade or two. If this becomes a reality, it would be the first step towards a physiological rejuvenation/infusion of youth and prolongation of the lifespan.

Physical parameters of such chemically constituted and reproducible hybrid scaffolds need to be studied in detail. We need to explore whether variability in these physical and chemical parameters would differentially influence the biological differentiation of cells. If this were to occur by simply manipulating either the physical or the chemical characteristics of these scaffolds, the cell of our choice can be obtained and used for treating varied human diseases affecting different cell types and organs.

The effect of all physical parameters on biological differentiation can be studied on earth, with an exception of the effect of gravity. If differential physical parameters allow varied biological differentiation, the day is not too far, when people will be sending space ships carrying their cells with automated systems to the outer space of lesser or zero gravity to get the cells of their choice expanded and the same be brought back after culture in outer space to be injected as a treatment modality.

Different planets with differing gravities could induce growth of different lineage of cells with different capabilities. This may herald the birth of a new scientific discipline called Astro-regenerative medicine and NCRM at that point would establish NGRM (Nichi-In Galaxy of Regenerative Medicine).  

Corresponding Author: Dr. V. Dedeepiya Devaprasad, Clinical Coordinator, Nichi-In Centre for Regenerative Medicine, C-16, Vijaya Health Centre Premises, 175, NSK Salai, Vadapalani, Chennai - 600026. India. Tel : +91 44 42321322. Email : dedeepiya_76@yahoo.co.in .

 


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