The Research Of Colony Culture And Differentiation Of Acute Myeloid Leukemia Stem/progenitor Cells In Vitro

Background & Objective : Study of AML stem/proginetor cells closely correlates with researches on Hematopoietic stem cells. The concept of HSC was first raised in 1961 and from then on, biology of stem cells research became an independent new subject that called for further exploration. In 1994, Lapidot isolated LSC of human AML through special molecule on cell surface. The result showed that only LSC performs with continuously self proliferation and stable malignant phenotype that proved the objective existance of CSC. It was reported that the phenotype of LSC is CD34⁺/ CD38^- or CD34⁺/HLA-DR^-.Nearly all of the subtypes of AML is related with LSC with the exception of APL.The major character of stem cell(SC) can be selfproliferation and committed differentiation. In 1980s, APL primitive cells was successfully induced into mature cells by Chinese scholars. Presently, analysis of LSC in vitro major relys on the detection into the ability of CFU-L. The proliferation differention and apoptosis of SC, progenitor cells (PC) and mature cells may be regulated by multi-channelled interactions of different cell factors. The research was designed to observe the ability of AML cells to induce myeloid stem/proginetor cells into mature granulocye cells with existance of IL-3, IL-6, SCF, GM-CSF and G-CSF.Methods: The research work comprised of three parts: Part 1, Two pre-experiments were performed as follows: Cells were cultured in the mixture of Agar, McCoy' 5A culture medium, FBS and nutrient layer cells of normal person peripheral blood white cells(radiated by ⁶⁰Co) and this mixture is short for Agar half-solid culture medium. The concrete experiment procedures were as follows: 1 .Comparing of colony growth ability of MNC from BM of AML patients with that collected from healthy donor. MNC that was sitmulated by IL-3, SCF and GM-CSF from BM MNC of AML patients as well as normal person were cultured into colony in CFU-L culture condition. The number of the colonies was calculated and compared on the 0d, 2^nd , 5^th, 7^th days. 2.The ability of colony formation test was also designed to compare the difference of fresh AML BM MNC from that kept in liquid nitrogen. Two groups of cells were cultured in the same condition of CFU-L to promote the formation of colonies. The number of the colonies was calcualted and compared on the 0d, 2^nd , 5^th, 7^th days. Part 2, hematopoietic stem/proginetor cells cultured into colonies: with the stimulation of IL-3, SCF,and GM-CSF, AML BM MNC was cultured into colonies in the Agar half-solid culture , being observed and detected so as to explicit whether the cells were CFU-L or not. Part 3, The differentiation of myeloid leukemia stem/proginetor cells in vitro: CFU-L cellls were separately inoculated into McCoy's 5A culture medium with and without FBS. Cell factors of IL-3, IL-6, SCF, GM-CSF and G-CSF were added to induce to mature granulocye cells. Cells collected on the 20^th day were picked out to identify the morphology and immuno-subtypes so as to confirm whether they are mature granulocye cells or not.Results: Part1, Observation under microscope: Very few BM MNC from healthy donor had the ability to form colonies, otherwise AML BM MNC begun to gather several colonies on the 2^nd day cultured and the number of the colony reached the top on the 7^th day. The colony number of the two groups was statistically different through k-square test. The result of fresh AML BM MNC cells and that kept in liquid nitrogen was identical. It appeared to gather colonies on 2^nd day and the number reached the top on the 7^th day. There was no significant variance between the two groups. Part 2, CFU-L is the colony of AML stem/proginetor cells. The morphology of which is smilar to small lymphoma cells. The colonies that we got was according with the cheracteriscis above-mentioned.The immuno-subtype of CFU was CD34+CD38- and it was tested that the granulocyte cell surface markers of CD13+CD15+CD33+ lowly expressed.The expression of CD 4+CD38- in the colony cells that we got was 16.94%±4.52%, and CD13⁺ 5.26%±1.92%,CD15⁺6.72%+1.03%,CD33⁺6.77%±3.58%. The result met the characteristics of CFU-L. Part 3,The in vitro average number of cells that we got in the differentiation experiment on 20^th day separately cultured with and without FBS was seperately 0.98×10⁶ and 90.67×10⁶. There was significant difference between two groups through couplet test. Cells that we got was out of special coloured grains that did not match the morphology of mature granulocye cells. The research of cell immunosuptypes were as follows: CD34⁺CD38^- 0.30±0.17%,CD13⁺10.73±3.05%,CD15⁺9.67±2.59%,CD33⁺12.35±3.05%. Conclusion: 1. BM MNC from healthy people is out of the ability to grow into colonies.that means we can exclude the possibility of polution. The ability to grow into colonies made no difference between fresh AML MNC and that kept in liquid nitrogen. This suggested that cells kept in liquid nitrogen could be the complementarity of our experiments. AML BM MNC that stimulated with IL-3, SCF and GM-CSF and cultured in special Agar half-solid culture medium could grow into myeloid CFU-L. Myeloid CFU-L (that is myeloid leukemia stem/proginetor cells ) fit the McCoy's 5A with FBS. Myeloid leukemia stem/proginetor cells could not be induced into mature granulocye cells with the stimulation of IL-3, IL-6, SCF, GM-CSF and G-CSF.