Group Members: Ken-ichi Miharada, Ulrika Blank, Göran Karlsson, Emma Larsson, Ronan Quére and Stefan Karlsson
• To develop novel approaches that can be applied in the clinic to expand hematopoietic stem cells ex vivo.
Hematopoietic stem cells (HSCs) are rare cells that are found during development in the aorta gonad mesonephros region of the embryo and later in development the HSC have moved to the fetal liver where they expand. Postnatally, HSC reside primarily in the bone marrow. Blood and marrow transplantation is used as a life saving therapeutic option for patients with malignant blood disorders and genetic disease. The optimal donor in allogeneic transplantation is a close relative with a near perfect MHC match to avoid rejection of the transplanted cells. This is however not always possible. If a suitable donor cannot be found, an alternative source of HSCs can be found in umbilical cord blood (CB). The HSCs found in the CB are usually well suited for transplantation due to their naïve immunological status, thereby better tolerated by the recipient. A caveat however is that the number of HSCs found in one cord blood CB sample is low and this may be inadequate for efficient engraftment of the donor cells in adult patients in need of transplantation. Therefore, there is a need to develop a safe and effective protocol to enable expansion of human cord blood HSCs since this will make available an increased repertoire of donors for most patients with serious blood disorders in need of transplantation.
During the last 10-15 years several studies have revealed a number of factors involved in the regulation of murine HSCs, for example cytokines, developmental fate determinants and intracellular signals from transcription factors, cell cycle regulators and transcriptional repressors. At present, several research groups, including our own, have shown that murine HSCs can be expanded in vitro (1-3,12) . By overexpressing the transcription factor HOXB4 from a retroviral vector, repopulating hematopoietic cells can be expanded up to 40 fold over a 10 day expansion period. Other studies have shown that the purified HOXB4 protein can be added directly to the cells in culture giving similar results (4,5) . Other vector systems, such as adenoviral vectors has also been utilized, revealing that the level of HOXB4 expressed in the cells is critical for the effect on the cells (6) . However, retroviral gene transfer introduce a risk of insertional mutagenesis, and the production of the HOXB4 protein is laborious and the half-life of the protein is short (˜2 hrs) requiring additions to the culture every 2-3 hrs (5). Therefore, the current focus of the research is on finding cytokines and developmental cues that can be used safely to expand murine and human HSC.
Symmetric divisions of stem cells shown in red lead to expansion of stem cells. in the figure above showing maintenance of stem cell numbers, asymmetric divisions generate one stem cell, shown in red and one differentiated cell, shown in yellow. The top figure shows maintenance of stem cell numbers (Figure: U Blank).
Studies with murine HSC are essential to define conditions for HSC expansion because murine HSC can be prospectively isolated and transplantation studies in syngeneic mice allow exact evaluation of repopulating stem cells, including serial transplantation to determine HSC self-renewal. The challenge is to move these studies to human cord blood cells. Several strategies have been pursued to increase the number of stem cells in cord blood grafts. Recently, double cord blood transplantation has been introduced. The initial results are encouraging (John Wagner, University of Minnesota). However, the long-term effects of this treatment where three immunologically distinct hematopoietic cells (two donors and the recipient) are competing remain to be evaluated. Expansion of cord blood stem- and progenitor cells in liquid culture media represents another approach to increase the number of cord blood stem cells and to promote hematopoietic engraftment. The development of reliable methods that can expand the number of HSC 2-10 fold would make most CB samples suitable for BMT therapy of adult patients. Recently, Notch-mediated expansion of human CB progenitors (but not HSC) in vitro was successfully used to prevent delayed myeloid reconstitution following transplantation. Successful development of HSC expansion in vitro would also facilitate the efficiency of HSC-based gene therapy of autologous HSC for patients with genetic disorders.
Approaches are now under investigation in our laboratory to expand human HSCs derived from CB using expansion promoting proteins of the Angiopoietin-like family. It has already been demonstrated that Angptl 2 and 3 can expand murine HSC in vitro and Angptl5 can expand SCID-repopulating cells ex vivo as well. We have identified Angptl4 as a factor that can increase the engraftment of SCID repopulating cells in NOD/SCID mice and in NSG mice. In order to find additional novel developmental cues that might affect self-renewal of HSC, we scanned data bases and publications that have detected candidate molecules that regulate “stemness” and/or self-renewal of stem cells. Gene expression profile analysis in undifferentiated and differentiated human ES cells detected a collection of genes that were upregulated in human ES cells and downregulated upon differentiation by withdrawal of LIF. We identified 4 soluble developmental cues and/or signaling modulators as candidate regulators for HSC. Murine LSKCD34- HSC were purified and treated with the candidate factors (500 ng/ml) in the presence of 100ng/ml of SCF and TPO to ask whether the factors had additive/synergistic effect on growth of hematopoietic colonies after serial replating. Increased growth of multipotential four-lineage colony forming CFU-GEMM progenitors was used as criteria to identify the most potent factor. These studies will be extended to human CB HSC.
In summary, we aim to develop a stem cell expansion protocol for human HSC using developmental fate determinants and cytokines since their use can easily be applied in clinical GMP laboratories. Our goal is to allow expansion of stem cells in CB and to generate enough numbers of repopulating HSC to transplant adult patients successfully. These efforts may open up a whole new venue for treatment of a number of hematological malignancies.
• Harvey F Lodish and Cheng Cheng Zhang, Whitehead Institute for Biomedical Research, Cambridge, MA, USA
• Tariq Enver, University College London, UK and Lund University
• STEMEXPAND, EU Small to Intermediate Project Consortium
• Jonas Larsson, Lund University
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Last modified: 2010-07-30
