Ex vivo expansion of haematopoietic stem cells in a co-culture bioreactor system with mesenchymal stromal cells

Matthew Cook (2011). Ex vivo expansion of haematopoietic stem cells in a co-culture bioreactor system with mesenchymal stromal cells PhD Thesis, School of Medicine, The University of Queensland.

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Author Matthew Cook
Thesis Title Ex vivo expansion of haematopoietic stem cells in a co-culture bioreactor system with mesenchymal stromal cells
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2011-05
Thesis type PhD Thesis
Supervisor Professor Kerry Atkinson
Associate Professor Gary Brooke
Dr Michael Doran
Associate Professor Jean-Pierre Levesque
Total pages 225
Total colour pages 25
Total black and white pages 200
Subjects 11 Medical and Health Sciences
Abstract/Summary Haematopoietic stem cell (HSC) transplantation is an established cell-based therapy for a number of haematological malignancies and immunodeficiency diseases. However, adequate supply of HSC from some donor tissues, namely umbilical cord blood (UCB), limits the efficacy of such transplants. The theory that this limitation could be overcome by expanding the HSC population prior to transplantation has motivated numerous laboratories to develop ex vivo expansion processes. The successful self-renewal of HSC in vitro is thought to require replication of the bone marrow (BM) stem cell niche. Pioneering work in this field used stromal cells as support cells in co-cultures with HSC to mimic the BM niche. The next few decades saw the evolution of chemically defined mediums which have so far failed to support in vitro HSC self-renewal, thus stimulating renaissance in co-culture. This study compared expansion of murine HSC co-cultured on murine mesenchymal stromal cells (MSC) and a population of murine osteoblasts (Ob) as support monolayers to establish an artificial environment for the controlled expansion of HSC. Murine populations of HSC and MSC were first purified and characterisation. HSC were isolated as lineagedim Sca1+ and c-Kit+ (LSK) by magnetic and fluorescent activated cell sorting (MACS and FACS respectively) from murine BM. These LSK cells showed functionally ability by being able to rescue lethally irradiated recipient mice. MSC were isolated from digested bone fragments and expanded by plastic adherence. They were further purified as Sca1+ and CD45- by FACS and characterised as CD44+, CD11b- and CD31-. They were definitively characterised by their functional ability to differentiate into Ob, chondrocytes and adipocytes. The LSK cells were then co-cultured on either MSC or MSC-derived Ob monolayers for seven days. The culture conditions were first optimised for haematopoietic cell expansion and it was found that co-cultures initiated with 1x103 LSK and grown in X-Vivo-15 media supplemented with 20 % fetal calf serum was optimal. Using these conditions, LSK demonstrated a proliferative capacity on both these stromal monolayers, with no additional haematopoietic growth factors required. Over 95 % of the generated haematopoietic cells were found to be adherent to either MSC or Ob after seven days of culture. The Ob monolayer displayed a significantly superior capacity for expanding both total nucleated haematopoietic cells as well as more primitive LSK, as defined by cell surface phenotyping, compared to co-culture with MSC. However, in both cases, a majority of the expanded cells were found to be committed to mature haematopoietic lineages. The quality and functional capacity of the cultured HSC was then tested in a murine competitive reconstitution assay. Upon transplantation, the cultured haematopoietic cells were capable of engrafting and repopulating lethally irradiated mice in both primary and secondary recipients at 12 weeks post-transplant. LSK co-cultured on MSC showed comparable reconstitution ability to that of freshly isolated HSC. Surprisingly, however, LSK-Ob co-cultures showed significantly poorer haematopoietic reconstitution compared to LSK-MSC co-cultures. Subsequently, recipients of LSK-Ob co-cultures displayed a significantly higher mortality rate compared to those receiving fresh LSK or LSK-MSC co-cultures. Investigations into this occurrence revealed that the likely cause of mortality was due to a delay in short-term reconstitution. Next, periodic or continuous media exchange was explored in an attempt to generate a more stable culture microenvironment for HSC culture. Furthermore, previous studies have reported that the mature cell progeny of HSC expansion cultures generate signalling networks that negatively influence the expansion of primitive HSC subsets. In this study media exchange was not shown to enhance LSK proliferation in this co-culture system when either bulk media exchanges were performed or when co-cultures were grown in customised continuous perfusion bioreactor devices. However, as this was preliminary work, future directions will be aimed at optimising this system for more robust HSC expansion. Finally, the classical two-dimensional (2D) co-culture system was translated to a three-dimensional (3D) system to more closely replicate spatial aspects of the HSC niche. Thus, LSK-MSC co-cultures were established in scaffold-free microaggregates, which we term micromarrows. Using this system, 3D LSK-MSC cultures generated a similar number of total haematopoietic cells when compared to 2D controls. However, a significantly higher proportion and fold increase of LSK was observed when the cultures were assessed by flow cytometry. Thus, the micromarrow platform represents a promising first step towards 3D co-culture system that enables HSC niche recapitulation and potentially true HSC self-renewal. Overall, this project has demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSC in a 2D system without the need for additional growth factors. We also demonstrate that despite apparently superior in vitro performance, co-injection of bulk cultures of Ob and LSK in vivo should be avoided. Finally, it is shown that scaffold-free 3D HSC-MSC co-culture may be a valuable method to further promote the self-renewal of HSC in ex vivo expansion systems.
Keyword Haematopoietic stem cells (HSC)
Haematopoietic Stem Cell Transplantation (HSCT)
Ex Vivo Expansion
Mesenchymal stem/stromal cells (MSC)
Additional Notes Colour pages – 41, 43, 63, 94, 97, 99, 103, 113-115, 120, 122, 125, 127, 134, 144, 148, 161-167, 183

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Created: Tue, 11 Oct 2011, 17:48:24 EST by Mr Matthew Cook on behalf of Library - Information Access Service