Ang, MF

Chan, JCK

Kwong, YL

Liang, RHS

Lie, AKW

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Title Ex vivo expansion of peripheral blood haematopoietic stemcells: preclinical studiesAuthor(s) Ang, MF; Lie, AKW; Chan, JCK; Liang, RHS; Kwong, YLCitation The 5th Medical Research Conference, Hong Kong, China,January 2000, v. 22 n. Supp 2, p. 11Issued Date 2000URL http://hdl.handle.net/10722/46846Rights Creative Commons: Attribution 3.0 Hong Kong LicenseG-H-1DONOR LYMPHOCYTE INFUSION (DLI) FOR LEUKEMIA RELAPSE: A revisitW.Y. Au. A.K.W. Lie, Y.L. Kwong, C.K. Lee, C.S. Chim, Y.H. Leung, R. Liang. Department of Medicine, QueenMary Hospital, Hong KongBackground: The curative effect of bone marrow transplantation (BMT) is partly due to the graft versus leukemiaeffect. This can be augmented by DLI at leukemia relapse and induce sustained remissions.Method: From 9/95-8/99, 18 cases (10 chronic myeloid leukemia (CML), 3 acute myeloid leukemia (AML), 4 acutelymphoblastic leukemia (ALL), 1 atypical CML) were treated. Their median age was 33 (range 18-49). Peripheralbuffy coat (median 2.3 x 108/kg lymphocytes, range 0.13-12.2) apheresed from the previous donor was infused in 2to 6 doses, with no graft versus host disease (GVHD) prophylaxis. For AML and ALL cases, chemotherapy wasadded for initial disease control.Results: The overall complete remission (CR) & partial remission (PR) rate was 13/18 (72%). 8/10 CML cases(80%) achieved CR (including 6 molecular remissions), and 1 achieved near CR (90%Ph-ve). DLI induced molecularCR in one AML case, while 2 cases (1AML, ALL) were maintained in PR with isolated extramedullary disease(EMD). Both CML-BT and aCML cases did not respond. CR was not achieved in 4 cases relapsing <6month postBMT. Notably 8/9 cases failing to achieve sustained CR have EMD components. The median time of follow up ofsurvivors was 22 mo, (range 1 to 48 mo.). The major side effects included self-limiting liver GVHD (n=5), severeskin GVHD (n=2), and herpes zoster virus/ cytomegalovirus reactivation (n=4).Conclusions: DLI is a valuable treatment option for patients relapsing after BMT. Factors predictive of a successfulresponse are: molecular relapse only, Ph+ve CML, relapse >1 year from BMT, no EMD, and preserved donorchimerism.G-H-2Ex Vivo Expansion of Peripheral Blood Haematopoietic Stem Cells: Preclinical Studies.M.F. Ang. A.K.W. Lie, J. Chan, R. Liang, Y.L. Kwong. Department of Medicine, Queen Mary Hospital, HongKong.Introduction. In autologous peripheral blood haematopoietic stem cell transplantation (aBMT), a period of severeneutropenia inevitably develops, owing to a deficiency of committed myeloid precursors that can give rise to matureneutrophils. Therefore, a combination of haematopoietic stem cells and more committed myeloid progenitor cellsmay overcome this problem, with the early haematopoietic recovery being mediated by more committed progenitors,and the long term haematopoiesis being maintained by repopulation with pluripotential stem cells. To test thishypothesis, we conducted preclinical studies to examine the expansion ex vivo of peripheral blood haematopoieticstem cells. Materials and methods. Circulating haematopoietic stem cells were collected with informed consentfrom patients undergoing stem cell mobilisation for aBMT. CD34 positive cells were isolated by positive selectionwith immunomagnetic beads coated with a monoclonal anti-CD34 antibody. The purity was verified by flowcytometry. Purified CD34 cells were plated at a concentration of 5,000 cells/ml/well, 10,000 cells/ml/well, and20,000 cells/ml/well, in a stroma free suspension culture, supplemented with the haematopoietic growth factorsinterleukin-3 (IL-3), IL-6, stem cell factor, granulocyte colony stimulating factor, stem cell factor, and megakaryocytesgrowth and development factor, for 12-14 days. Parameters measured included the percentage increase in cellnumbers, serial changes in surface antigen expression as defined by dual colour flow cytometric analysis, andclonogenic assays. Results. 4 cases were investigated. Cell number increased gradually until day 7, when dramaticincreases in the rate of cell growth were observed (a mean increase of 90.8 fold at day 10). It was also observed thatthe fold increase of cells in cultures initiated with 5,000 cells/ml was higher than cultures initiated with 20,000cells/ml. The number of granulocyte-macrohage colony-forming units (CFU-GM) paralleled nucleated cellproduction, peaking at day 10. Dual colour flow cytometry using antibodies to CD34, CD15, and CD11b evaluatedthe distribution and maturation stages of cells during culture. From days 9-14 there was emergence of a CD15+CD11b+ population, indicating the maturation of cultured cells toward more mature neutrophilic forms. This wasparalleled by a decrease of CD34+ cells, which dropped to only 1.6% of the starting number by day 14. Conclusion.At the end of a 14-day culture, cells produced were predominantly neutrophil precursors, and were developingnormally as assessed by immunophenotype and CFU-GM assay. The results suggest that ex vivo expansion ofhaematopoietic stem cells could generate sufficient number of progenitors as a supplement to stem cell transplantation.

Ex vivo expansion of peripheral blood haematopoietic stem cells: preclinical studies

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Ex vivo expansion of peripheral blood haematopoietic stem cells: preclinical studies

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