A Proposal of project On

Stem Cells Culture

Submitted towards the partial fulfillment of I Semester of M.B.A. & M.B.L. Degree course, for the subject

Research Methodology

Submitted to: Dr. V.S. Shastri Parinita Jhawar Faculty In Charge National Law University

Submitted by: M.B.A. - M.B.L. I Semester Roll No. 261 NATIONAL LAW UNIVERSITY, JODHPUR

TOPIC: STEM CELL CULTURE.

OBJECT: Development of Stem Cell Culture in India

INTRODUCTION: Research on stem cells is advancing knowledge about how an organism develops from a single cell and how healthy cells replace damaged cells in adult organisms. This promising area of science is also leading scientists to investigate the possibility of cell-based therapies to treat disease, which is often referred to as regenerative or reparative medicine. Stem cell research in India has achieved encouraging results. New players are entering with interesting findings and public-private partnership funding is on the rise, said Dr Geeta Jotwani, assistant director general, Indian Council of Medical Research (ICMR). Stem cells are the foundation cells for every organ and tissue in the body. They are like a blank microchip that can ultimately be programmed to perform particular tasks. Under proper conditions, stem cells begin to develop or differentiate into specialized cells that carry out a specific function, such as in the skin, muscle or liver. Additionally, stem cells can self renew, that is they can divide and give rise to more stem cells.1 The main characteristics of stem cells are: First, they are unspecialized cells that renew themselves for long periods through cell division. Second, under certain physiologic or experimental conditions, they can be induced to become cells with special functions such as the beating cells of the heart muscle or the insulin producing cells of the pancreas.2 There are many different types of stem cells. These include embryonic stem cells that exist only at the earliest stages of development, and various types of tissue-specific (sometimes referred to as adult or somatic) stem cells that exist in a number of different fetal and adult
1 2

ISSCR08 Published Brochure www.stemcell.com

tissues and organs. Recently, cells with properties similar to embryonic stem cells, known as induced pluripotent stem cells (iPS cells), have been engineered from specialized cells such as adult skin cells. 1 A stem cell transplant may be used so that you can have intensive high dose chemotherapy (and sometimes radiotherapy) to kill cancerous cells. The chemotherapy is higher than conventional chemotherapy and also kills the stem cells in the bone marrow that would normally make blood cells. A stem cell transplant is sometimes called a bone marrow transplant. However, stem cells can be obtained from blood as well as from the bone marrow. So, the term stem cell transplant is now used. A stem cell transplant is an option which is considered for various cancer conditions 3 Umbilical cord blood (UCB) is a valuable, yet limited resource. On average 50 - 100 ml of blood containing stem cells is obtained from the umbilical cord and the placenta after birth. When used in treatment the following applies: The greater the number of stem cells used, the better the prospects for healing. Currently, this is the reason the entire umbilical cord blood specimen is always used in transplantation.4 Stem cell research allows us to gain a fundamental understanding of how organisms develop and grow, and how tissues are maintained throughout adult life. Studying stem cells is helping researchers to study disease, test drugs and develop increasingly effective therapies. Scientists are pursuing stem cell research from many angles and investigating cells of various origins to address what we need to know. The study of stem cells has led to many new insights into how cancers develop and may be treated. Many cancers contain dysfunctional cells with the properties of stem cells that propagate the cancer. RESEARCH OBJECTIVE: a) To examine the unique features of Stem Cells. b) To know about the development of the Stem Cell Culture in India. c) To study the significance. d) Determining precisely how stem cells remain unspecialized and self renewing for many years.
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4

Cancer Management 9th Edition 2005. Edited by Richard Pazdur et al. www.corcell.com

e) Identifying the signals that cause stem cells to become specialized cells.

RESEARCH METHODOLOGY: The methodology of the study includes study of library references and latest literature on the various educational sites , and compilation of the secondary data and information obtained from various journals. The method of research would be deductive as conclusion would be drawn after the analysis and interpretation of data collected.

RESEARCH QUESTION: 1. What are the stem cells and why are they important? 2. What is the need of its culture? 3. Why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most adult stem cells cannot? 4. What are the factors in living organisms that normally regulate stem cell proliferation and self-renewal? 5. Can stem cells be used for the future treatment of Parkinsons disease? 6. What are the main risks in having stem cell transplant? 7. What is the potential for new medical treatments using stem cells?

HYPOTHESIS: Stem cells are one of the most fascinating areas of biology today. But like many expanding fields of scientific inquiry, research on stem cells raises scientific questions as rapidly as it generates new discoveries. Stem cell culture results in a social welfare.

LITERATURE REVIEWS

ABSTRACT 1: STEM CELL BASICS5 Stem cells have the remarkable potential to develop into many different cell types in the body. Serving as a sort of repair system for the body, they can theoretically divide without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell. a) Stem cells are unspecialized that is it does not have any tissue-specific structures that allow it to perform specialized functions. b) Stem cells are capable of dividing and renewing themselves for long periods. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells c) Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. There are two kinds of stem cells from animals and humans: 1. Embryonic stem cell . 2. Adult stem cells. Embryonic stem cells, as their name suggests, are derived from embryos. Specifically, embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitroin an in vitro fertilization clinicand then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman's body. As long as the embryonic stem cells in culture are grown under certain conditions, they can remain undifferentiated (unspecialized). But if cells are allowed to clump together to form embry id bodies, they begin to differentiate spontaneously. They can form muscle cells, nerve cells, and
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The official National Institute for Health Resource from Stem Cell Research

many other cell types. Although spontaneous differentiation is a good indication that a culture of embryonic stem cells is healthy, it is not an efficient way to produce cultures of specific cell types. An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found. Unlike embryonic stem cells, which are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is unknown. Adult stem cells have been identified in many organs and tissues.

ABSTRACT 2: STEM CELL RESEARCH AND APPLICATIONS MONITORING THE FRONTIERS OF BIOMEDICAL RESEARCH.6 In the face of extraordinary advances in the prevention, diagnosis, and treatment of human diseases, devastating illnesses such as heart disease, diabetes, cancer, and diseases of the nervous system, such as Parkinsons Disease and Alzheimers Disease, continue to deprive people of health, independence, and well-being. Research in human developmental biology has led to the discovery of human stem cells (precursor cells that can give rise to multiple tissue types), including embryonic stem (ES) cells, embryonic germ (EG) cells, and adult stem cells. Recently, techniques have been developed for the in vitro culture of stem cells. Derivation of ES cells from early human embryos, and EG and fetal stem cells from aborted, fetal tissues raise ethical, legal, religious, and policy questions.

ABSTRACT 3:

American Association for the Advancement of Science And Institute for Civil Society. 6

BLOOD AND MARROW STEM CELL TRANSPLANTATION7 Stem cell transplantation is a technique that can restore the marrow function of patients who have had severe injury to that site. Marrow injury can occur because of primary marrow failure, destruction of marrow by disease, or intensive chemical or radiation exposure. The source for the earliest transplants was the marrow of a healthy donor who had the same tissue type (HLA type) as the patient. Usually, the source was a brother or sister. Donor programs have been established to identify an unrelated donor who has a tissue type that matches that of a patient. This approach requires screening tens of thousands of unrelated individuals of similar ethnicity to the patient. The transplant is achieved by infusing a very small fraction of the marrow cells called stem cells. Stem cells not only reside in the marrow but a small number also circulate in the blood. They can be harvested from the blood by treating the donor with agents that cause a release of larger numbers of stem cells into the blood and collecting them by a process called hemapheresis. Stem cells also circulate in large numbers in fetal blood and can be recovered from placental and umbilical cord blood after childbirth. The harvesting, freezing, and storing of cord blood provide another source of stem cells for transplantation. Since blood and marrow are both good sources of stem cells for transplantation, the term stem cell transplantation has replaced bone marrow transplantation If the donor and recipient are identical twins, the transplant is called syngeneic, the medical term for genetically identical. With a syngeneic transplant there is no immune difference and no likelihood of a host versus graft (graft rejection) or a graft versus host reaction. If the donor and recipient are not identical twins, the transplant is called allogeneic. An allogeneic transplant means the donor is the same species and, in practice, nearly always a match in tissue type to the recipient. The term matched unrelated is applied to the donor who is not a family member, recruited by searching among a large pool of potential donors for the rare individual who is identical or very similar in HLA type to the recipient.The important technique of harvesting patients stem cells in marrow or blood, freezing the collection and returning it to patients after they have received intensive chemotherapy and/or radiation therapy for their underlying disease is referred to as autologous transplantation. This term is misleading since

The Lymphoma and Leukemia Society journal. 7

transplantation implies transferring tissue from one individual to another. This technique would better be referred to as autologous stem cell infusion

ABSTRACT 4: IN VITRO DEVELOPMENTAL TOXICITY TEST DETECTS INHIBITION OF STEM CELL DIFFERENTIATION BY SILICA NANOPARTICLES.8 While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 mug/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other two particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis. ABSTRACT 5:
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Toxicol Appl Pharmacol. 2009 Jul 23.(www.elsevier.com)

THERAPEUTIC STRATEGIES TARGETTING BRAIN TUMOR STEM CELLS.9 Progress in stem cell research reveals cancer stem cells to be present in a variety of malignant tumors. Since they exhibit resistance to anticancer drugs and radiotherapy, analysis of their properties has been rapidly carried forward as an important target for the treatment of intractable malignancies, including brain tumors. In fact, brain cancer stem cells (BCSCs) have been isolated from brain tumor tissue and brain tumor cell lines by using neural stem cell culture methods and isolation methods for side population (SP) cells, which have high drug-efflux capacity. Although the analysis of the properties of BCSCs is the most important to developing methods in treating BCSCs, the absence of BCSC purification methods should be remedied by taking it up as an important research task in the immediate future. Thus far, there are no effective treatment methods for BCSCs, and several treatment methods have been proposed based on the cell biology characteristics of BCSCs. In this article, I outline potential treatment methods damaging treatment-resistant BCSCs, including immunotherapy which is currently a topic of our research.

ABSTRACT 6:

AN

AUTOMATED

SYSTEM

FOR

DELIVERY

OF

AN

UNSTABLE

TRANSCRIPTION FACTOR TO HEMATOPOETIC STEM CELL CULTURE.10

An automated delivery system for cell culture applications would permit studying more complex culture strategies and simplify measures taken to expose cells to unstable molecules. We are interested in understanding how intracellular TAT-HOXB4 protein concentration affects hematopoietic stem cell (HSC) fate; however, current manual dosing strategies of this unstable protein are labor intensive and produce wide concentration ranges which may not promote
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Brain Nerve. 2009 Jul;61(7):799-803 (www.interscience.com)

10

Biotechnol Bioeng. 2009 Jun 1;103(2):402-12.(www.interscience.com) 9

optimal growth. In this study we describe a programmable automated delivery system that was designed to integrate into a clinically relevant, single-use, closed-system bioprocess and facilitate transcription factor delivery studies. The development of a reporter cell assay allowed for kinetic studies to determine the intracellular (1.4 +/- 0.2 h) and extracellular (3.7 +/- 1.8 h and 78 +/- 27 h at 37 degrees C and 4 degrees C, respectively) half-lives of TAT-HOXB4 activity. These kinetic parameters were incorporated into a mathematical model, which was used to predict the dynamic intracellular concentration of TAT-HOXB4 and optimize the delivery of the protein. The automated system was validated for primary cell culture using human peripheral blood patient samples. Significant expansion of human primitive progenitor cells was obtained upon addition of TAT-HOXB4 without user intervention. The delivery system is thus capable of being used as a clinically relevant tool for the exploration and optimization of temporally sensitive stem cell culture systems.

ABSTRACT 7:

PEEKING AT STEM CELLS IN THE BRAIN11.

For many years, researchers thought the adult brain did not make new neurons - - that we managed through adulthood with only those neurons that survived our youthful exuberance. Some years ago, however, that view was changed with the realization that the adult mammalian brain does indeed have an ongoing supply of fresh brain cells, or neurons, that come from neural stem cells. The new neurons were identified within the olfactory and hippocampal regions of the brain. The olfactory region processes the sense of smell. The hippocampal region remains a bit of a puzzle, although it seems to contribute to memory. Research reported by Manganas et al. now describes a non-invasive technology for visualizing new neurons in the human brain. The authors used nuclear magnetic resonance (NMR) spectroscopy to identify some compounds characteristic of neural stem cells that could be used as flags. The flags seem to represent a constellation of lipids-- unique to neural stem cells-- for which the specific function remains unknown. The authors then used magnetic resonance
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Pamela J. Hines, PhD*( http://www.isscr.org/public/briefings/peeking.html)

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spectroscopy (MRS) to follow these flags, and thus see the neural stem cells in the brains of living animals. Although much of this research was sorted out using mouse and rat brains, and cells in tissue culture, the applicability to the human brain was tested as well. MRS, tracking the neural stem cell-specific flags, confirmed the presence of stem cells in the hippocampus of the human brain, and hinted at a decline in neural stem cell activity with age. The non-invasive aspect of this imaging technology is a particular advantage for its application to understanding the normal aging of the human brain as well as our responses to disease and trauma to the brain.

ABSTRACT 8:

HUMAN

MESENCHYMAL

STEM

CELL

CULTURE

FOR

NEURAL

TRANSPLANTATION.12
Mesenchymal stem cells (MSCs) have the potential to play a role in autologous repair of central nervous system injury or disease, circumventing both the complications associated with immune rejection of allogenic cells, and many of the ethical concerns associated with embryonic stem cell use. Human bone marrow-derived MSCs can be extracted relatively simply from the marrow of adult patients and maintained and expanded in culture. More importantly, it has been previously demonstrated that MSCs have the capacity to differentiate into neurons and glia in vitro when grown under appropriate conditions. Multipotent MSCs have also been successfully used in transplantation studies in animal models of disease as diverse as demyelination, stroke, trauma and Parkinson's disease. MSCs therefore provide an attractive and practical source of stem cells for reparative therapy in patients, and in this paper we describe methods for the reproducible culture and neural differentiation of human MSCs generated from patient marrow.

12

Methods Mol Biol. 2009;549:103-18 (www.springer.com)

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ABSTRACT 9: STEM CELLS AND DIABETIES.13 The discovery of methods to isolate and grow human embryonic stem cells in 1998 renewed the hopes of the cure for type 1 diabetes, and perhaps type 2 diabetes as well. With a ready supply of cultured stem cells at hand, the theory is that a line of embryonic stem cells could be grown up as needed for anyone requiring a transplant. The cells could be engineered to avoid immune rejection. There are some evidence that differentiated cells derived from embryonic stem cells might be less likely to cause immune rejection. Recent studies in mice show that embryonic stem cells can be coaxed into differentiating into insulin-producing beta cells, and new reports indicate that this strategy may be possible using human embryonic cells as well. Researchers in Spain reported using mouse embryonic stem cells that were engineered to allow researchers to select for cells that were differentiating into insulin-producing cells. Manfred Ruediger of Cardion, Inc., in Erkrath, Germany, is using the approach developed by Soria and his colleagues to develop insulin-producing human cells derived from embryonic stem cells.

ABSTRACT 10: BONE MARROW STEM CELL TREATMENT SAVES LEGS.14 The transplant is achieved by infusing a very small fraction of the marrow cells called stem cells. The another application of bone marrow stem cell transplant is the treatment of Thrombo Angitis Obliterence(TAO). This ailment occurs mostly in the smokers and diabetic patient. Nicotin in the blood causes the muscles of the arteries to constrict. It also damages the vessels inner linings. The problem get compounded if the patients has diabeties.

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http://stemcells.nih.gov/staticresources/info/scireport/PDFs/chapter7.pdf
Article abstract : The Hindu ; August 6,2009 (15)

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The clinical trials is being conducted on the patients suffering from blocks in the artery of leg. The result of it to save volunteers leg from amputation by injecting stem cell concentrate to the limb affected by TMO. The stem cells were taken from the patients bone marrow. Thus, the injection of the stem cells help in creating new collaterals and these would provide the vital conduit for the blood flow to the parts of the leg below the block. The stem cells injected help in angiogenesis said Dr. Vijayaragavan, Head of the Department of Vascular Surgery, Sri Ramchandra University, Chennai. Despite of the success of the current trial Dr. insists that stem cells injection should be the last option.

PROPOSED LITERATURE: Books 1. Molecular Biology methods , Gene IX 2. Animal Tissue Culture, Primrose Websites 1. www.springerlink.com 2. www.isscr.co.in

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