A pilot study of the role of CD133+ as a cell marker for human haematopoietic stem cells

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Therapeutic transplantation of haematopoietic stem cells (HSCs) is an increasing area of interest in medicine. In autologous stem cell transplantation, there is the ability to harvest stem cells soon after chemotherapeutic treatment, and then cryo-preserve and store such cells for stem cell transplantation at a later date. CD34 is the typical cell marker used to identify HSCs for transplantation. There exist certain limitations to CD34+ HSCs which include its expression on cells not capable engraftment, and also on acute leukaemic cells. CD133 is an alternative cell surface marker on HSCs which could prove useful in identifying cells for transplantation. Preliminary research suggests that CD133 is a more primitive marker, which is expressed by early progenitor cell line, and it has the additional advantage of not being expressed on acute leukaemic cells. The Launceston General Hospital is the site for stem cell harvest for the North and North-West of Tasmania. Annually, approximately 20-30 patients undergo stem cell harvest with the aim of later transplantation. This project is a pilot study to evaluate the value of CD133 in addition to the usual marker CD34+ as a cell marker for HSCs while using the current protocol for stem cell harvest. There is currently little data on the validity of CD133 as a HSC marker, and this project, as a part of Master degree at the School of Human Life Sciences at the University of Tasmania, aims to add to the current data and potentially identify the role of CD133 as HSC marker. We are aiming to study 30-50 patients with different haematological malignancies that require stem cell harvest with subsequent stem cell transplantation during a period of 24 months at the LGH, Tasmania. Current numbers of autologous stem cell harvest at a single centre; the LGH are varied between 20 and 30 episodes per year. Our current autologous stem cell harvest-protocol for patients with malignant haematological disorders is employing mobilisation chemotherapy with cyclophosphamide 5g/m2 and subsequent administration of granulocyte colony stimulating factor (G-CSF) subcutaneously daily at a dose of 10mcg/kg body weight (BW) until harvesting sufficient stem cells per transplant defined as at least 2x106 stem cells/Kg BW. We will start to measure both CD 34+ and CD 133+ daily using the flow cytometr (Becton-Dickenson) when the white cell count (WCC) recovers above 1.0/nL post chemotherapy and until the time of stem cell harvest at the Holman Clinic. Thereafter we will measure both markers according to manufacturer reccomendations (with commercially available CD34 and CD133 kits) in the harvest product for each patient. Correlation between the amount of harvested CD 34+/CD133+ cells and patient engraftment will be documented for each patient. Using commercially available kits, we are proposing to study the progenitors cells which express CD133 antigen by flow cytometry technique according to the manufacturer recommendations as a part of the usual daily measurement of CD34+ cells. There is no additional sample will be required from patients and hence this will not expose the participants to any extra burden. Also we are proposing to measure CD133+ in addition to CD34+ stem cells in the stem cell product garnered by the stem cell harvest. Using flow cytometry to study stem cell characteristics in the leukaphoresis product will provide useful information regarding engraftment kinetics. Platelets engraftment is defined as reaching platelet count>20/nL without substitution, while neutrophil engraftment is defined as reaching neutrophil count above>0.5/nL.

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