The Network - Issue 1, Spring 2011
Under the Microscope
A Quick Look at What's Happening in the Stem Cell Labs
By: Mike Halpenny, Charge Laboratory Technologist (Ottawa) and Brenda Letcher, Charge Laboratory Technologist (Edmonton), Canadian Blood Services
Canadian Blood Services' stem cell program actively seeks to contribute to the science of transfusion medicine by improving the health care available for our primary clients-the patients. The following outlines some of the initiatives currently underway at our stem cell processing laboratories in Ottawa and Edmonton. The results and further details of many of these activities will be shared in upcoming issues of The Network.
A novel assay to measure hematopoietic progenitor cell (HPC) potency
(The Edmonton lab is working with Drs. Jelena Holovati, Locksley McGann and Leah Marquez)
To treat many malignant hematological disorders, such as leukemia and lymphoma, hematopoietic progenitor cell (HPC) transplantation is widely used. How well hematopoietic reconstitution goes after the transplant largely depends on two parameters of the cells involved: viability (how many cells survive thawing and injection) and proliferative potential (how many new cells a single cell can produce). The US company HemoGenix recently introduced a novel assay for evaluating both HPC viability and proliferative potential. The assay is reported to be quantitative, standardized, capable of low-to-high throughput, and is less labour-intensive and time-consuming than the current assays used. The Edmonton laboratory is evaluating this assay to determine if it is an improved method for measuring progenitor cell proliferative ability.
Hematopoietic Progenitor Cells (HPCs) are cells capable of reconstituting normal bone marrow function. They are transplanted into a patient after high-dose chemotherapy which destroys not only its target, the cancerous infiltrates, but also bystander healthy bone marrow cells.
A systematic quality assessment protocol for HPC products
(The Edmonton lab enlisted the assistance of University of Alberta statistics professor Dr. Peng Zhang)
HPCs are critical to a patient's recovery after high-dose chemotherapy. In most transplant centres, clinical decisions regarding infusion of HPCs are based on the product's viable CD34+ cell concentration (or cell dose). The greater the CD34+ cell concentration in the HPC product, the more rapidly the patient recovers. CD34+ testing is performed on HPC products soon after collection and therefore reflects excellent cell viability. However, in the autologous setting, cells are frozen pending re-infusion. Since cell loss during freezing and thawing is inevitable, pre-freeze cell counts do not necessarily represent the most meaningful number on which to base the decision to release the product for use.
CD34 is a cell membrane protein molecule present on Hematopoietic Progenitor Cells (including multipotent stem cells), but is not present on mature cells. It allows these cells to be distinguished from other cells and counted.
The Edmonton team looked at patient outcomes versus pre- and post-thaw cell counts. Since products with low cell concentrations prior to freezing could be significantly impacted by unpredictable low cell recovery post thaw, the need to establish minimum pre-freeze values was a priority.
Advanced statistical analysis was used to determine the minimum post thaw cell dose associated with the maximum recovery time in 95% of the patients. By including the uncertainty in post thaw cell recovery, the minimum post thaw cell dose was converted to establish its mathematical equivalent for fresh samples. The laboratory uses these values as guidelines for collection and recommendations for physicians, as well as for release criteria.
Evaluation of a cryopreservation process for mesenchymal stem cells
(The Ottawa lab is working in partnership with Dr. Harry Atkins - OHRI / Ottawa Hospital)
In addition to HPCs, human bone marrow also contains Mesenchymal stem/progenitor cells (MSCs). MSCs have the potential to regenerate many different cell types, including cardiac muscle, liver cells, neuronal and non-neuronal cell types of the brain, and endothelial (the thin layer of cells that line the interior surface of blood vessels) cells. This "regenerative capability" has suggested the possibility of using MSCs in novel cellular therapies to treat diseases such as diabetes, heart disease, Multiple Sclerosis and Parkinson's disease. The Ottawa laboratory is learning and working with MSCs to assist current research requirements and prepare for future clinical demand for this type of stem cell.
Mesenchymal stem cells (MSCs) are cells found in the bone marrow and other tissues of adults. They are capable of self-renewal and differentiating into heart muscle, neurons and bone.
Buffy-coat derived HPCs
(Edmonton laboratory in partnership with Dr. Jelena Holovati of the University of Alberta)
In recent years, the number of clinical applications for HPC transplantation has increased considerably. However, HPC shortage remains an unresolved issue in transplant medicine and research.
Recent changes in processing whole blood donations into transfusable component products may help to address HPC shortage. Canadian Blood Services uses the buffy-coat method for blood component production, which results in donated blood being separated into three layers: red blood cells at the bottom of the bag, buffy coat containing white blood cells and platelets in the middle layer, and a plasma layer at the top. While many of the donated buffy coats are used to make pooled platelet concentrates, some are not suitable for continued production. Ongoing research is investigating whether the buffy coat layer from donor peripheral blood remaining after production of blood component products is a potential source of viable HPCs with the potential for in vitro proliferation and the ability to differentiate to the progenitor colony forming units. By identifying the buffy-coat layer as an important potential source of HPCs from peripheral blood for transplantation and research, this study may result in a novel application of biomaterials that are currently being discarded.
Understanding freezing (cooling) rates
When long-term storage of HPCs is required, they are routinely frozen, then stored at 150 oC. Successful freezing and adequate recovery of these cells after thawing is highly dependent on their biological characteristics, the solution in which they are frozen, and the rate at which they are cooled. Protocols for freezing HPCs have been optimized based on these factors, but other routine and sometimes random variables exist. It is important to understand the effect of these variables on the products cooling rate and ultimately, its cell recovery post thaw. Since there is limited information available that specifically addresses the effects of these variables, the Edmonton team is conducting studies that measure the cooling rate of products under different conditions while using their standard controlled-rate freezing protocol. Conditions such as product volume, load size and positioning, product specific gravity and run interruptions/deviations, are being investigated. Uncontrolled freezing protocols are being investigated so that they may be validated for back up. Storage temperature deviations are being investigated to assist the laboratory in assessing these types of deviations should they occur.
Novel cellular therapy: Development of a mesenchymal stem cell culture system
(The Ottawa lab is working in partnership with Dr. Dave Allan - Ottawa Hospital)
The primary objective of this project is to develop a closed-culture system to grow and harvest MSCs isolated from both bone marrow and cord blood. Translational research, "from bench to bedside" or "mouse to man," has always been the focus for the Ottawa stem cell group. Although not directly involved in basic stem cell research, the lab has significant expertise in manufacturing products in a regulated environment and therefore can assist researchers making the transition from basic research to clinical human trials.
Hematopoetic progenitor cell sterility verification study
(The Ottawa lab is working in partnership with Dr. Sandra Ramirez-Arcos - Canadian Blood Services)
This project verified that the in house BacT/ALERT® 3D Microbial Detection System was able to reproducibly detect contamination with fungi and bacteria from small volumes of cryopreserved HPC-A (Apheresis) or HPC-M, (Marrow) collections, as well as from cryopreserved or fresh HPC-C collections (cord blood).
Characterizing non-hematopoietic progenitor cells from cord blood
(The Ottawa lab is working in partnership with Dr. David Allan - Ottawa Hospital)
The primary focus for this project was to develop a process for obtaining cord blood from hospitals for research purposes, and to gain experience in understanding the progenitor cell makeup of cord blood.
Mesenchymal stem cell constructs during cartilage tissue engineering
(The (Ottawa lab is working in partnership with Dr. Max Hincke - University of Ottawa)
MSC (mesenchymal stem cell) isolated and grown at the Ottawa lab were transferred to the University of Ottawa group for further research involving the development of cartilage from these MSC. As mentioned previously, mesenchymal stem cell work is important for the Ottawa group to gain expertise in this area, preparing for possible future clinical demand.
Stem cell evaluation in peripheral blood of post myocardial infarct patients
(The Ottawa lab is working in partnership with Dr. Chris Glover - Ottawa Heart Institute)
What happens inside the blood system when a person suffers a heart attack? Does the blood system contribute to "repairing" or "healing" the damaged heart? The Ottawa Heart Institute requested the Ottawa lab's assistance in determining the progenitor (stem cell) content in a person's blood stream following a heart attack. The ultimate goal for this research would be to develop a treatment method using stem cells to repair or fix the heart after an attack.
Clinical trial: "Targeting Multiple Sclerosis using immunoablative therapy and immunological reconstitution"
(Principle Investigators - Drs. Harry Atkins and Mark Freedman - Ottawa Hospital)
A six-year, multi-million dollar human clinical trial for multiple sclerosis patients with the Ottawa lab performing all the stem cell processing for this project. The lab must perform a specialized processing procedure that includes "purifying" the stem cell graft to remove damaging T-cells and other cells that may contribute to the progression of Multiple Sclerosis (MS). This purified graft, still containing the valuable stem cells, is transplanted back to the MS patient after treatment at the hospital.
Freezing without chemical cryoprotectants (i.e. DMSO)
(The Edmonton lab is working in partnership with Drs. Janet Elliott and Lisa Ross-Rodriguez - University of Alberta)
After the first report of freezing mouse bone marrow using dimethyl sulfoxide (DMSO) by Ashwood-Smith in 1961, techniques have been developed for freezing human HPCs using DMSO as a cryoprotectant with good cell recovery after thawing. Cryopreserved bone marrow, peripheral blood progenitor cells and cord blood continue to be transplanted using these techniques in a large number of patients around the world.
At the concentration of 10% normally used, the amount of DMSO infused into patients can be significant, particularly for large-volume transplants. As there have been reports of morbidity and mortality associated with infusion of DMSO, for susceptible patients, some laboratories may choose to remove the DMSO by washing the thawed product before transplantation. However, the loss of cells during the washing process may pose an even greater risk to the patient. Although not approved by the FDA, the use of DMSO continues because there is currently no effective alternative. The objective of this research is to develop an alternative approach to freezing progenitor cells.
The Ins and Outs of Hematopoietic Stem Cells: Studies to Improve Transplantation Outcomes. Part 1: Matrix-degrading enzymes: breaking the ties that bind
(Project headed by Dr. Anna Janowska, Scientist, Canadian Blood Services, Edmonton)
Traditionally, HPCs for use in transplantation were collected by multiple aspirations of bone marrow. However, this harvesting procedure has now been almost completely replaced by the collection of peripheral blood (PB). This was made possible by the early discovery that HPC can be coaxed out of the bone marrow and into circulation in response to stress, chemotherapy and other drugs in a process referred to as mobilization. Upon transplantation, the HPC find their way back in to the bone marrow in a process referred to as homing.
During its research into the "ins and outs" of so-called HPC homing and mobilization, Dr. Janowska's team found that an enzyme called membrane type-1 matrix metalloproteinase (MT1-MMP) enhances HPC mobilization. In subsequent issues, we will discuss this discovery further, as well as update the reader on other factors that promote mobilization and strategies that could augment the homing of HPC.