Culture of fetal stem cells from umbilical cord blood

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Culture of fetal stem cells from umbilical cord blood


Author: Lam, Oi-man Winnie
Title: Culture of fetal stem cells from umbilical cord blood
Degree: M.Sc.
Year: 1999
Subject: Hematopoietic stem cells
Hong Kong Polytechnic University -- Dissertations
Department: Multi-disciplinary Studies
Dept. of Nursing and Health Sciences
Pages: xiii, 85 leaves : ill. ; 30 cm
Language: English
Abstract: Hematopoietic stem cells are pluripotent stem cells that have the ability to self-renew and to give rise to cells of multilineage. On the surface of these cells, there are expression of specific antigens, designated as CD or cluster of differentiation. On the surface of hematopoietic stem cells, there is expression of CD34 (presented with positive sign) surface antigen but absent of CD33, CD38 and CD45 surface antigens (presented with negative sign). As the cells mature, the level of CD34 detected on the cell surface decrease with an increase of lineage specific marker antigen: CD33 (myeloid lineage) or CD38 (lymphoid lineage). CD45 surface antigen is found on all common leukocytes. Adult bone marrow has been the traditional source of hematopoietic stem cells for transplantation. However, the chance of getting a Human Leukocyte Antigen (HLA)-matched bone marrow graft is low. The use of umbilical cord blood as a potential source has been evaluated. It was found that the number of stem cells present in the cord blood might not be sufficient for transplantation in an adult case. Ex vivo expansion is therefore required. The aim of this study was to analyze the growth supporting ability of 2 culture media for stem cell growth. Venous blood samples were taken from umbilical cord. The CD34+ cells were isolated from the mononucleated cells fraction by magnetic activating cell sorting device. The cells were grown in 2 different stroma-free liquid culture media each containing 2 growth factors: ligand for Flt3 (FL) and recombinant human thrombopoietin, for 28 days. The growth supporting ability of these media in stem cells were analyzed every 7 days in terms of cell morphology, cell count, and colony assay. The changes in cell populations were monitored by flow cytometry using flurochrome labeled anti-CD34, anti-CD33, anti-CD38 and anti-CD45. The results obtained from both media were analyzed by Paired t-test. It was found that there was an increase in total cell number by 254.6+-134.2 fold and 251.5+-162.6 fold for Medium 1 and Medium 2 respectively (p=O.599). After 28 days of culture, the number of CD34+ cells had increase 7 fold and 3.4 fold in Medium 1 and Medium 2 respectively (p=O.O8). However, the percentage of CD34+ (p=O. 161), CD34+/CD38- (p=O.25), and CD34+/CD33- (p=O.337) cells were decreased, but with an increase of CD33+ (p=O.67), CD38+ (p=O.126) and CD45+ (p=O.536) cells, indicating that the stem cells had undergone differentiation. The percentage of colony forming cells decreased dramatically during the culture period. However, the number of colony forming unit-granulocyte and monocyte (CFU-GM) colonies increased by more than 150 fold, indicating that the cultured stem cells had differentiated into the myeloid series. There was no significant difference between Medium 1 and Medium 2 in supporting stem cell growth, although there might be difference in the CD34+ and CD34+/CD38- cell expansion. Since both media support a net increase of the number of CD34+ cells, the possibility of cultured cells for transplantation was analyzed. It was found that the culturing conditions of Medium 1 in this study might be able to produce enough CD34+ cells for transplant with an acceptable level of CFU-GM cells after 21 days of culturing. Further investigations are needed to decrease the culture period so that better quality of cultured cells can be obtained.

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