The present invention provides the use of sera separated from the clotted umbilical cord blood for growing human embryonic stem cells and adult cells such as Neural precursor cells, for therapeutic purposes in regenerative medicine. In particular, the present invention relates to transdifferentiation of CD+ 34 stem cells from Mononuclear cells derived from Umbilical Cord Blood to Neural precursors and such cells may be used in transplantation and repair of nerve damage, stroke, spinal injury, Parkinson's and Alzheimer's.
BACKGROUND OF THE INVENTION
Stem cell technology is an emerging field that may yield many promising therapies. Stem cells are special cells that have the ability to develop into many different types of tissue: bone, muscle, nerve, etc. In theory, they could be grown into replacements for almost any part of the human body. Stem cells are typically found in the embryo and umbilical cord of an organism, and in reservoirs within the human body. Researchers hope that stem cells will provide a solution to cure diseases caused by cell failure, and for repairing tissues that do not repair themselves. Heart damage, spinal cord injuries, Parkinson's disease, leukemia, and diabetes are among diseases named in relation to stem cell research. Hence, researchers are of the opinion, if these stem cells are controlled, they could cure a variety of debilitating diseases in the years to come. Stem cells are separated into 3 distinct categories viz. Totipotent, Pluripotent, and Multipotent. Stem cells are best described in relation to normal human development. Thus, a fertilized egg is totipotent. It produces an entire organism. After several cycles of cell division, these totipotent cells begin to specialize, becoming pluripotent. As the embryo begins to develop, these pluripotent cells become multipotent, specifically producing blood, skin, nerve, or other types of body cells. Multipotent stem cells are envisioned to potentially treat a variety of muscular-skeletal and neural disorders. While stem cells are extraordinarily important in early human development, multipotent stem cells are also found in children and adults. For example, one of the best understood stem cells are the blood stem cells. Blood stem cells reside in the bone marrow of every child and adult, and in fact, they can be found in very small numbers circulating in the blood stream. Blood stem cells perform the critical role of continually replenishing supply of blood cells—red blood cells, white blood cells, and platelets throughout the life span.
Stem cells are the building blocks of blood and immune systems. They form the white cells that fight infection, the red cells that carry oxygen and platelets that promote clotting. Stem cells are normally found in bone marrow where they continue to generate new blood cells throughout the live span of an individual. The presence of these stem cells in the bone marrow has made marrow transplantation an important therapeutic modality in the treatment of variety of malignant and non-malignant diseases. This is because of the realization that permanent clinical benefit from transfused blood cells can come from transplantation of multipotent haematopoietic stem cells. Besides bone marrow, Mobilized Peripheral Blood (MPB), and Umbilical Cord Blood (UCB) have also been used successfully for transplantation. In recent years although significant advances have been made in bone marrow transplantation (BMT), the basic problem of finding a suitable matching donor still remains. This is because a group of antigens expressed by the leukocytes called the human leukocyte antigens (HLA) need to match between the donor and the recipient. Further bone marrow harvesting is a painful and invasive procedure and many donors are unwilling to donate marrow. Therefore the search for alternate sources of stem cells has led to the development of stem cell transplant protocols from different tissues like liver (Kochupillai 1991), mobilized peripheral blood (Benboubker 1995), and cord blood (Mayani 1998). Of these, cord blood has significant advantages over the others. Increasingly, experts say cord blood transplants have distinct advantages over more traditional bone marrow transplants in stimulating the growth of healthy white blood cells. Stem cells can be collected from the bone marrow. However, the collection procedure is invasive, time-consuming, requires an anaesthetic and is painful for the donor. Also, cord blood is easily available, involves a non-invasive collection procedure and is better tolerated in transplants across the HLA barrier.
Like bone marrow, umbilical cord blood is rich in stem cells. Umbilical cord blood is the blood that remains in the placenta and umbilical cord following birth. Until recently the placenta and umbilical cord were discarded after delivery as medical waste, but now research has shown that cord blood is a rich source of blood (haematopoetic) stem cells, which can be collected, processed and frozen for potential future use. An experimental procedure to use umbilical cord blood instead of bone marrow to treat immune diseases is gaining attention from doctors and patients.
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, fetal stem cells, and adult stem cells. Recently, techniques have been developed for the in vitro culture of stem cells, providing unprecedented opportunities for studying and understanding human embryology. As a result, scientists can now carry out experiments aimed at determining the mechanisms underlying the conversion of a single, undifferentiated cell, the fertilized egg, into the different cells comprising the organs and tissues of the human body. Although it is impossible to predict the outcomes, scientists and the public will gain immense new knowledge in the biology of human development that will likely hold remarkable potential for therapies and cures.
Using cell replacement therapy, to cure diseases may prove to be one of the most significant advances in medicine. Unlike all current treatments that rely on surgical interventions or drugs that modulate cell activities, stem cells provide a replacement for dysfunctional or degenerating tissue.
Cells are regarded as stem cells if they retain the capacity to renew themselves as well as more specialized progeny stem cells can be obtained from early embryo, fetal tissues, adult blood, and umbilical cord blood. Identification of the full term umbilical cord blood (which is discarded at birth), as a source has made haematopoietic stem cells more accessible for study and clinical use. Cord blood stem cells are multipotent. These stem cells in addition to production of blood cells, have the ability to differentiate into cells of other tissue or organs. This ability has made cord blood stem cells more accessible for study and clinical use.
Cord blood stem cells express CD34 antigen. CD 34 antigen has been commonly used as a marker for the enrichment and isolation of candidate stem cells. Cord blood stem cells can be isolated on the basis of the presence of this marker. CD34 positive cells can be isolated from mononuclear cells of cord blood. Cord blood mononuclear cells constitute about 1-2% of CD34 positive cells. On exposure to a novel environment, cord blood stem cells are known to transdifferentiate to various cells like neural cells, liver cells, bone, cartilage etc.
Transdifferentiation is the ability of the adult stem cells from one tissue or organ, which can overcome their intrinsic restrictions upon exposure to novel environment perhaps via genomic reprogramming to cells of other organs either in vitro, or after transplantation in vivo.
Neural stem cell research is still in its early stages, is intriguing because scientists believe that the primitive cells can transform into virtually any cell type in the body and could be a source of tissue or organs to cure diseases such as repair of nerve damage, strokes, spinal injury, Parkinson's and Alzheimer's. For years, researchers studying stem cells have been intrigued by the possibility that these cells might be use to treat brain diseases. Recent studies have suggested neural stem cells transplanted into the brain can migrate throughout the brain and develop into other types of cells.
Up to the present, Stem Cells (Embryonic/Adult) are being cultured in animal serum such as Fetal Bovine Serum (FBS), or complex mixture of growth factors derived by mixing purified factors which are either isolated from FBS or Human Adult blood serum or a mixture of growth factors derived from recombinant methods.
However, these conventional culture media are associated with shortcomings and risks. Stem cells from adult/fetal as well as other sources are being widely used to regenerate tissues in patients after they have degenerated. For this purpose, these cells have to be grown in the tissue culture for varying periods of time using defined media, the principle constituent of which is animal serum such as Fetal Bovine Serum (FBS).
FBS is the most widely used serum in the culturing of cells, tissues and organs in vitro, in industry, medicine, and science. FBS has been shown to be essential for adhesion, proliferation and differentiation of the cells. However, animal serum such as FBS can be infected with several pathogens such as prions. Several known and unknown viruses may be present in the serum. Therefore cells/tissue cultured in the presence of FBS get infected and transmit these pathogens to the patient on transplantation. As stated FBS may have known and unknown pathogens, which may be transmitted to the human transplant subject if these cells are grown in FBS. The pathogens present in FBS are difficult to screen for likely causative agents of diseases in humans. Hence, using such cells in a human can be life threatening as there is every chance of a pathogen getting transmitted along with these cells. Human cells grown in FBS constitute a xenograft, if used for cells based therapies in humans.
Human adult blood serum also supports growth of several cells, however, it cannot substitute for FBS, since it does not provide growth factors, present in FBS. Hence, it is not used for culturing of stem cells in vitro.
Several investigators have tried to use a combination of complex mixture of growth factors, which are known to influence growth and differentiation of stem cells. However, the success is limited and it has been shown conclusively that 2% v/v of the tissue culture media should be made up of FBS for optimal growth of the cells.
There is a dire need to find an adequate substitute for conventional culture media for growing neural precursor cells. Looking to the need of the hour, the present inventors have resolved the above issue of concern and have come out with a solution, which will be of utmost importance in the field of regenerative medicine. The inventors of the present invention, have come out with a unique media for culturing cord blood stem cells which comprises of umbilical cord blood serum as a substitute for FBS and such cells may be used in transplantation and repair of nerve damage, strokes, spinal injury, Parkinson's and Alzheimer's. Cord blood being a natural substance, is found to be rich in growth factors. Taking this factor in mind, the inventors of the present invention have investigated a method of growing and differentiating cord blood stem cells into neural precursors. The present invention is advantageous over the prior art as it obviates the problems associated with the conventional culture media for growing stem cells for human use.
Use of umbilical cord blood stem cells in haematopoietic reconstitution has been around since 1970. However, no work has been done in using umbilical cord blood as a source for growing and differentiating stem cells in neural precursors. The inventors of the present invention have been successful in discovering this novel process for growing cord blood stem cells and differentiating into neural precursors.
OBJECTS OF THE INVENTION
It is an object of the present invention to grow human embryonic stem cells and adult stem cells such as cord blood stem cells for therapeutic purposes in regenerative medicine.
It is further object of the present invention to develop a method for growing and differentiating stem cells into neural precursors using a novel media consisting of cord blood serum and such cells may be used in transplantation and repair of nerve damage, stroke, spinal injury, Parkinson's and Alzheimer's.
It is still further object of the present invention to transdifferentiate CD34+stem cells from Mononuclear cells derived from Umbilical Cord blood to neural precursors.
SUMMARY OF THE INVENTION
In accordance with one aspect of this invention, there is provided the use umbilical cord blood sera for growing human embryonic stem cells and adult cells, like cord blood stem cells for therapeutic purposes in regenerative medicine.
To overcome the major obstacle described above, the inventors of the present invention have conducted research on human umbilical cord blood and have replaced conventionally used Fetal Bovine Serum (FBS) by cord blood serum, for culturing cord blood stem cells for neural transdifferentiation.
As stated, Human umbilical cord blood is a fetal product and a waste product during childbirth. During the gestation of the child in the mother's womb, the placenta and the blood present in the placenta nourish the developing fetus and are therefore rich in several growth promoting factors. The inventors of the present invention have taken advantage of this property of Human umbilical cord blood and have substituted it for FBS. The procedure by which Human umbilical cord blood is collected is given below. It is this cord blood that is used as a serum for growing cord blood stem cells of the present invention.
The method of the present invention may include the step of separating the pluripotent stem and progenitor cell population of mononuclear cells obtained from umbilical cord blood using a magnetic cell separator to separate out cells which contain a CD marker and then expanding these cells in a growth medium containing a differentiation agent such as retinoic acid and growth factors such as BDNF, GDNF, NGF, FGF or mixtures thereof. Preferably the a mixture of retinoic acid and at least one growth factor for example nerve growth factor is used as the differentiation agent. The retinoic acid may be 9-cis retinoic acid, all-trans retinoic acid or mixtures thereof. The separation and incubation steps maybe interchanged.
The umbilical cord blood sample from which the pluripotent stem/progenitor cells are obtained may be fresh umbilical cord blood, reconstituted cryopreserved umbilical cord blood or a fresh or reconstituted cryopreserved mononuclear cell fraction thereof.