Hematopoietic stem cells (HSCs) present in our bone marrow generate all the cells of our blood system. Throughout our lifetime, they continuously generate our red blood cells, white blood cells and platelets. HSCs can be isolated and transplanted to save the lives of patients with some types of cancer or blood disorders. After transplantation, HSCs settle (engraft) in the recipient’s bone marrow, where they begin to proliferate and generate the cells of the hematopoietic system to replace the defective or malignant cells that were originally there.
There are three ways to collect HSCs for transplantation. The two most common ways are collecting from the bone marrow or whole blood of an adult donor. HSCs can also be collected from the cord blood that remains in the umbilical cord and placenta after birth. There are several advantages to using HSCs from cord blood, including availability (umbilical cord blood is usually discarded after birth) and flexibility: because the immune cells in cord blood are less mature, a patient who is unable to find a matching adult donor may be able to find a matching cord blood unit.
The number of cells in cord blood units can be increased before stem cell transplantation by growing them in culture medium exposed to bone cells.
Because cord blood does not contain many HSCs, engraftment can be delayed or unsuccessful. To improve engraftment, researchers are developing methods to increase the number of cord blood HSCs before they are transplanted into patients. Although these methods have improved overall engraftment outcomes, there are still issues with some aspects of cord blood HSC engraftment. The regeneration of platelets, which are crucial for blood clotting, has been particularly challenging.
Dr. Nicolas Pineault, a development scientist at Canadian Blood Services, has established a procedure for expanding cord blood HSCs which may help overcome this problem. His method uses osteoblasts, which are bone-generating cells. When grown in the laboratory, osteoblast cells release molecules into the liquid surrounding them (culture medium). This osteoblast-conditioned culture medium (OCM) can be collected and used to increase the numbers of cord blood HSCs. Dr. Pineault showed several years ago that OCM increases the proliferation rate of cord blood HSCs and improves their regeneration of platelets in mouse models. He has recently followed up on these findings by optimizing the process for producing OCM and investigating which factors in OCM are responsible for increasing the growth of cord blood HSCs.
What did the researchers do?
Dr. Pineault’s group used donated cord blood units obtained from Canadian Blood Services’ Cord Blood for Research Program to compare several OCM production methods and to clarify how OCM promotes cell growth. They produced the OCM by growing osteoblasts in culture medium and collecting the conditioned culture medium after one, two, three or six days. To determine what size of molecules were important in mediating the activity of OCM, they filtered the OCM to remove particles larger than 0.2 µm and compared the activity of filtered OCM with unfiltered OCM and with control conditions (culture medium not exposed to osteoblasts).
The researchers evaluated OCM’s ability to increase HSC proliferation by looking at changes in the subpopulations of cord blood cells that have specific combinations of molecules on their surface (for example, cells that have the cell surface markers CD34 and CD90 but do not have CD38 and CD45RA). They also used colony-forming cell assays as a test of biological function.
What did the researchers find?
OCM increased proliferation of cord blood cells, including several types of HSC subpopulations.
OCM produced with the shortest exposure to osteoblasts (one day) worked the best for enriching these HSC subpopulations.
Filtration of OCM greatly reduced or eliminated most of its growth promoting activity, suggesting that the most relevant particles in OCM are larger than 0.2 µm. However, the filtered OCM retained some activity, suggesting that smaller particles like growth factors also contribute to the observed effects.
OCM activity was not impaired by irradiation or by freezing.
How can you use this research?
This study has optimized the production methods for OCM and improved our understanding of how OCM promotes the growth of cord blood HSCs. By using filtration to separate the components of OCM, Dr. Pineault’s group has helped to narrow down which factors are most relevant for increasing HPC proliferation.
Regeneration of platelets after HSC engraftment has been a major obstacle with cord blood HSC transplantation. Dr. Pineault showed several years ago that OCM increases platelet regeneration in a mouse model; clinical trials are needed to determine whether OCM could help overcome this obstacle in HSC transplant patients. OCM may also be useful for basic scientific research because it is more simple compared with many other methods for expanding HSCs (such as co-culture systems), which means it could be used in complex cell model systems such as bioreactors. OCM activity was not altered by irradiation, suggesting that irradiation could be used to reduce potential biological safety concerns.
Although this work has revealed some of the mechanisms underlying OCM’s growth-promoting activity, the exact factors responsible for its effects are still unclear; more studies are needed to clarify these mechanisms. Identifying key factors that promote growth could lead to improved culture methods and therefore increased expansion of cord blood HSCs.
If these findings are confirmed in mouse models and later in clinical trials, they could one day lead to improved outcomes with cord blood transplantation and/or increase the proportion of cord blood donations that can be used for transplantation.
About the research team
The senior author, Dr. Nicolas Pineault, is a professor in the department of biochemistry, microbiology and immunology at the University of Ottawa and a development scientist at Canadian Blood Services. The first author, Dr. Ahmad Abu-Khader, was a post-doctoral fellow in the Pineault lab. Roya Pasha is a senior research assistant. Gwendoline Ward was a research technician and Gavin Boisjoli was an undergraduate honours student who was the recipient of a Canadian Blood Services summer internship award.
This research unit is derived from the following publication(s)
 Abu-Khader A, Pasha R, Ward GC, Boisjoli G, Pineault N. Characterization of the Growth Modulatory Activities of Osteoblast Conditioned Media on Cord Blood Progenitor Cells. Cytotechnology 2016; 68: 2257-69.
Acknowledgements: This research received financial support from Canadian Blood Services (Canadian Blood Services/Canadian Institutes of Health Research partnership operating grant program) and Health Canada. Canadian Blood Services is funded by the federal government (Health Canada) and provincial and territorial ministries of health. The views herein do not reflect the views of the federal, provincial, or territorial governments of Canada. Canadian Blood Services is grateful to the cord blood donors who made this research possible.
Keywords: cord blood, hematopoietic stem and progenitor cells, osteoblast, ex vivo expansion, conditioned medium