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Friday Science Review: February 10, 2012

Automated Regulation of Inhibitory Feedback Signalling Leads to Rapid and Robust Expansion of Cord Blood-Derived Hematopoietic Stem Cells

University of Toronto ♦ Genomics Institute of the Novartis Research Foundation ♦ Heart and Stroke/Richard Lewar Centre of Excellence ♦ McEwen Centre for Regenerative Medicine

Published in Cell Stem Cell, February 3, 2012

The greatest current justification for the storage of cord blood stem cells, in the setting of both the private and public cord blood bank, is for hematopoietic stem cell transplantation (HSCT) for reconstitution of the bone marrow compartment; most often following intensive chemotherapy regimens that ablate the bone marrow completely.

Cord blood is a viable source of hematopoietic stem cells (HSCs), however a single cord blood unit in its original form only contains a small quantity of these cells. It has been found that the most critical factor in patient survival following HSCT is administering a threshold cell dose (roughly 30 million cells per kilogram patient) that must be met or surpassed in order to achieve successful engraftment and reconstitution of the bone marrow. As a result, the majority of clinical studies utilizing cord blood stem cells for HSCT have been reserved for the paediatric population. However, even in children, successful engraftment and recovery is by no means a given. Despite the widespread storage of cord blood units in public banks that can be HLA-matched to recipients, and the numerable benefits of this source of HSCs over others, cord blood has yet to become a viable solution for HSCT.

One treatment paradigm under current investigation in the clinic is double cord blood transplant, wherein two units are transplanted simultaneously in order to boost cell number. The shortcoming of this approach is the difficulty in acquiring two units that are HLA-matched to the recipient. Although cord blood is better tolerated by the host’s immune system following transplantation, relatively speaking, and typically exhibits lower levels of graft versus host disease than other sources of HSCs, finding two adequately safe units for a single patient is no trivial task. With a cord blood unit ringing in at $30,000 a hit (from a public bank) the economics of this approach are also prohibitive.

A second approach has been to expand cord blood units in the lab prior to transplantation. This process increases the number of HSCs in the unit by several-fold; the results depending on the protocol and hands at work. Preclinical studies show that expanded cord blood stem cells can reconstitute the bone marrow compartment in immune compromised mice. The medical community has begun preliminary studies in the clinic mixing expanded cord blood stem cells with unexpanded (mixed because there is currently not enough definitive evidence to suggest that expanded cord blood stem cells retain repopulating activity). The results of these studies so far have failed to show that the expanded product contributes significantly to engraftment and recovery. Expansion protocols have become iteratively better, however limitations on HSC number, and their ability to accurately home to the bone marrow compartment and engraft, have prevented expanded units from reaching their potential in the clinic.

Expansion protocols modulate molecular mechanisms that regulate stem cell fate and proliferation in order to maximize the number of HSCs produced. Two approaches have primarily been used. The first, cytokine-driven expansion, utilizes molecular messengers relevant to the bone marrow niche. These proteins interact with surface markers on HSCs, triggering pathways that help reinforce cell fate decision towards the HSC identity. Cytokines are amenable to expansion as cells can be grown in 3-dimensional space, often in bags, which allows for easier scale-up. This being said, it is arguable that the approach is flawed, as it fails to truly recapitulate signaling mechanisms in the bone marrow niche where cell-to-cell contact is critical for the maintenance of different HSC pools.

The second approach, stromal-driven expansion, expands HSCs in the presence of a second population of cells known as stromal cells. While this cell culture approach provides a microenvironment that more accurately reflects the niche an HSC would experience inside the body, it is exceedingly difficult to scale-up to produce clinically relevant cell numbers.

Both approaches struggle in producing large numbers of clinically relevant populations of HSCs. Emerging data supports the idea that not all HSCs in the bone marrow compartment are equivalent. Slight differences in their gene and protein expression stratify them into classes that behave differently in terms of their capability to home and engraft. Some HSCs exhibit the classical HSC markers but only retain repopulating capacity transiently. Other rarer HSC populations exhibit a unique and specialized capacity to form colonies over the long-term. These stem cells, known as long-term repopulating HSCs (LTR-HSCs), are the cells that expansion protocols must produce if they are to create a cord blood product that is useful to humans in the clinic. In addition, neither approach accounts for the production of HSC progeny that amass within the cell culture system. These differentiated cells produce high concentrations of inhibitory feedback molecules that prevent HSC proliferation.

A transformative development in this space is the advent of a cell culture platform that enables rapid expansion of hematopoietic stem cells from a cord blood unit at some of the highest levels ever achieved. Developed by Peter Zandstra at the University of Toronto, this closed-system approach utilizes a controlled fed-batch media dilution strategy to reduce concentrations of proteins that inhibit stem cell proliferation. Within 12 days, LTR-HSC populations can be scaled-up to 11 times their original number while retaining their capacity to self-renew and differentiate into cells of multiple lineages. At its core, the platform hinges on the concept that HSC self-renewal and differentiation are regulated tightly by secreted factors that either promote or inhibit stem cell proliferation.

Zandstra’s group took a computational approach based on the effects of feedback signaling to design the expansion protocol. Measurements of secreted factors were taken from previously established in vitro growth conditions to identify factors that had inhibitory effects on HSCs. Computational simulations were then performed to model the effects that inhibitory proteins would have on stem cell population dynamics. Simulations depicted an accumulation of predominantly inhibitory proteins within the system. Hence, the group rationalized that media exchange would be a key advance in stimulating stem cell expansion. Further investigation with the simulation predicted that a fed-batch process (continuous input of new media) would outperform both perfusion (continuous input of new media and output of old media) and frequent full or partial media exchange. The hypothesis generated was then tested utilizing an automated media delivery system, which confirmed that the fed-batch approach led to a significant increase in the absolute numbers of various HSC subpopulations over other media exchange processes.

A critical component of this study was evaluating the increase in expansion of LTR-HSCs. The only means in which to do this is to carry out transplantation studies in immune compromised mice. Repopulation of the bone marrow compartment was quantified by judging the extent of the contribution of human cells to hematopoietic reconstitution. All of the mice that experienced successful repopulation of the bone marrow exhibited multilineage differentiation, as indicated by the presence of human cells from the myeloid, lymphoid, and erythroid lineages, and the presence of T-cells. Importantly, human cells from repopulated mice could be transplanted to reconstitute the bone marrow of secondary mouse recipients, confirming the long-term engraftment potential of the HSCs at hand.

Limiting dilution analysis was used to quantify the expansion of LTR-HSCs. In fresh cord blood the frequency of LTR-HSCs was roughly 1 in 14,700. After 8 days of growth in the fed-batch system this expansion was increased by 7.6-fold to a frequency of 1 in 1,940. And finally, after 12 days the expansion had increased by 11-fold to a frequency of 1 in 1,334.

Zandstra has created a cell culture technology that rapidly and cost-effectively expands clinically relevant populations of blood stem cells that retain the ability to engraft and contribute to hematopoietic reconstitution over the long-term. A technology of this nature is truly enabling. Not only does it provide new potential to the hundreds of thousands of cord blood units currently stored in public stem cell banks, it ensures, at least in the eyes of HSCT with cord blood stem cells, that cord blood units stored in the future will be put to good medical use. On a high level, the technology is also a platform approach to the problem of scaling up any number of different stem cell types for cell therapies in the future.

An interesting innovation for the future of this technology would be the addition of a device that can measure the concentration of inhibitory proteins within the system as cell growth occurs. If this could be achieved in real-time, it is conceivable that media input could be regulated to create a dynamic stem cell expansion environment. This would be highly fitting for the expansion of stem cells from cord blood units, as the protocol would be tailored to every expanded unit; all of which are different in cellular composition and genetic make-up.

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