1. Busulfan Study
Marie Olszewski (SH-ASCP) a clinical research specialist in the Stem Cell Transplant Lab is currently involved ( in conjunction with Dr Kletzel) in the study of Busulfan as an anticancer drug for Neuroblastoma. The in vitro model system utilizes neuroblastoma cell lines in the determination of apoptosis at different time points at various drug dose concentrations. In vitro testing allows for several methods of evaluating cell death ( Apoptosis, Cell cycle and Proliferation) allowing potential for more tumor cell lines and additional drug agents to be tested.
2. WT1 and Minimal Residual Disease
Earlier research of minimal residual disease (MRD) in leukemia and neuroblastoma, utilizing a novel technique at the molecular level is now clinically available as a tool to monitor for MRD in neuroblastoma. By utilizing the Wilms’ tumor (WT1) gene as a tumor marker in leukemia and Tyrosine hydroxylase (TH) gene in neuroblastoma, the capacity to quantitate MRD by RT-PCR offers another method of diagnosing to clinicians.
3. Chimerism studies
Wei Huang, a senior research tech in the Stem Cell transplant Lab continues her involvement in graft chimerism. Varying degrees of engrafment in an allogeneic transplant can be characterized at the cellular level including T cell, B cell, NK cell, myeloid and platelets chimerism. All research projects are under the direction of Dr Morris Kletzel.
Dr. Kletzel’s other ongoing research studies involve isolating mesenchymal stem cells from bone marrow and umbilical cord cells and attempting to differentiate them into nerve cells. The hope is that these cells can then be infused into patients to treat specific diseases, for instance new nerve cells could help replace cells that have been injured or removed in children who suffer from neurological disorders. These studies represent critical early steps toward developing and fine-tuning the techniques to successfully isolate these highly promising cells and ultimately test their therapeutic potential.
Xiaoxia Long, MD, an esteemed stem cell research scientist, works alongside Dr. Kletzel to further his mesenchymal stem cell research. Their early investigations have focused on bone marrow derived mesenchymal stem cells. Bone marrow derived stem cells have the capacity to develop into bone, cartilage, fat and connective tissue. They have been shown to possess an extraordinary degree of developmental ‘plasticity’ capable of becoming specialized cell types of different tissues such as skeletal and cardiac muscle cells and thus represent an enormously promising target for investigation.
In the Laboratory
In their studies using human bone marrow samples, Drs. Kletzel and Long attempted to differentiate mesenchymal stem cells using a combination of growth factors. Through the testing of unique chemical combinations, Drs. Kletzel and Long have been able to efficiently and effectively direct mesenchymal cells to express into mature neural markers, ultimately becoming robust, mature neural cells.
In the laboratory, two days after being cultured in a unique media, bone marrow derived mesenchymal stem cells started to present typical neural cell characteristics. On day 14, 70 percent of the cells presented with a typical neural phenotype. These findings represent a significantly higher percent response rate than has been achieved by other researchers conducting similar studies. The average differentiation is 25 percent. The differentiated cells were then tested to determine if specific cell surface markers were present, essentially to prove that they were neural cells, and then compared with the undifferentiated cells and human neural cells. Drs. Kletzel and Long’s results showed the cells developed early and had mature neural cell markers.
Overall, these results are very exciting because they further demonstrate the potential to generate nerve cells from mesenchymal stem cells. Bone marrow derived mesenchymal stem cells are promising because they appear to be resistant to rejection from a patient’s immune system, even though they are adult cells. Consequently, the use of nerve cells developed from mesenchymal stem cell for transplantation may help avoid major problems associated with organ rejection and the use of powerful, highly toxic immunosuppressant drugs.
As a testament to her early success, Dr. Long’s abstract was accepted for presentation at the 2003 International Society for Cellular Therapy annual meeting. And in July 2004, Dr. Long was invited to make a presentation of her research results at the 33rd Annual Scientific Meeting of the International Society for Experimental Hematology in New Orleans. These results strongly support the need for follow-up studies to determine an optimal media that will present the best microenvironment for the neural differentiation of mesenchymal stem cells.
Moving forward, Drs. Kletzel and Long are now in the process of developing the techniques necessary to isolate mesenchymal stem cells from umbilical cord blood. Umbilical cord blood stem cells are present in the blood of the umbilical cord and placenta after delivery. While umbilical cord stem cells are similar to bone marrow stem cells in that they can be used for the treatment of leukemia and other diseases of the blood, they are more immature than bone marrow cells. It is believed that these cells will cause less immune rejection, and therefore may offer greater potential for effective use in patients.
Once Dr. Long is able to successfully harvest and isolate the mesenchymal stem cells from the cord blood, she will utilize the same differentiation media used for the bone marrow stem cells. This is an essential next step since there has not yet been any widely published evidence of the successful isolation of stem cells within cord blood. Once she is successful, she will move on to test the new neural cells in a mouse model. The hope is that the injected cells will grow and function properly. If they do, we will be one step closer to attempting clinical trials. In essence, each study is another pivotal step towards validating Drs. Kletzel and Long’s initial findings. Ultimately, we hope to participate in clinical trials to bring better treatments to benefit children and adults suffering from diseases ranging from spinal cord injuries and massive brain damage to Alzheimer’s and Parkinson’s disease.
5. Helping the Body Heal Itself: Developing A Cancer Vaccine
Vaccine studies are a burgeoning area of cancer research. Unlike traditional vaccines, which generally aim to prevent the occurrence of disease, some experimental cancer vaccines are designed to treat or cure existing disease.
While the data is very preliminary, the findings of vaccine research have been quite promising. Early laboratory studies have shown that novel vaccines could slow the growth of melanoma, bladder and breast cancers implanted into genetically unrelated mice. No other vaccine has been able to induce such a broad immune response. In fact, some ongoing studies have shown complete and long lasting regression of lung cancer by stimulating the immune system to attack cancer cells.
As several researchers across the country continue working on developing vaccines for a wide range of cancer types, Dr. Kletzel and his team are focused on developing a vaccine to target the lethal neuroblastoma tumor – one of the deadliest tumors that affect the youngest and most vulnerable among us – children under the age of two. Dr. Kletzel’s first-hand experience caring for neuroblastoma patients in the clinic greatly compelled him to pursue this important goal.
IN THE LABORATORY
In this project, Dr. Kletzel is focused on developing a specialized system in which dendritic cells help regulate the immune system after stem cell transplantation to enhance an anti-tumor response in patients with high-risk neuroblastoma.
Beth Stephan, a researcher in the laboratory, is working closely with Dr. Kletzel to develop a neuroblastoma vaccine. To create the dendritic cell vaccine, she and Dr. Kletzel extract dendritic cells-which are unique antigen presenting cells- from either human bone marrow or peripheral blood and culture these specialized cells with specific cytokines. RNA is isolated from a neuroblastoma cell line to be used as the antigen-proteins that are found specifically in the tumor. Then these cells are non-virally transfected with the antigen and then combined with T cells to generate a tumor specific immune response.
Preliminary studies in the laboratory have shown that dendritic cells can be successfully transfected using a non viral method. Our current data shows a 30% transfection efficiency of the RNA into the cells using the non viral method of choice called lipofectin. This data has been presented as a poster presentation at the Tandem BMT Meeting in Orlando Florida in February. Our non viral method of transfecting immature dendritic cells with RNA from a neuroblastoma cell line was designed to avoid the potentially harmful use of viruses in the future event of human clinical trials. Our next step in the preliminary stage of development is to see how much of an immune response is generated by combining the transfected dendritic cells with T cells and measuring the level of cytoxicity of the tumor cells.
Once the preliminary stages of the project is complete and the highly specialized system is developed, Dr. Kletzel and his team will next turn to animal models to further develop the vaccine for neuroblastoma. Future goals involve injecting mice with the deadly disease and treating them with antigen specific transfected dendritic cells and monitor the immune response from the vaccine.
The hope then for the future is that the vaccine, a combination of dendritic cells and tumor-specific antigens, can be injected into our pediatric patients post stem cell transplant. There the newly programmed dendritic cells will work to show the body’s immune system how to recognize cancer cells as foreign cells. This will in turn illicit a full fledged attack by the body’s T-cells – the cells that fight disease – to cause the immune system to develop antibodies that will kill any circulating cancer cells. Our ultimate goal being a cure to preventing cancer relapses. These groundbreaking investigations could provide a revolutionary method for treating this aggressive form of cancer. Once the preliminary studies are complete, the same research will need to be conducted in animal models on the path to clinical trials and ever closer to a cure.
Dr. Kletzel and his team’s passion of working towards a goal to curing cancer is a long and hard battle that everyone is so dedicated on completing. The preliminary studies have taken two years to complete and have moved forward smoothly. They will continue working hard to complete each stage of the development of a neuroblastoma vaccine. Currently today researchers are working together to fight the deadly disease of cancer, but many more things still need to be learned about it and that is why ongoing efforts of research needs to continue.