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WO2008044876A1 - Procédé de préparation d'un progéniteur lymphocytaire t à partir d'une cellule souche cd34 positive - Google Patents

Procédé de préparation d'un progéniteur lymphocytaire t à partir d'une cellule souche cd34 positive Download PDF

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WO2008044876A1
WO2008044876A1 PCT/KR2007/004951 KR2007004951W WO2008044876A1 WO 2008044876 A1 WO2008044876 A1 WO 2008044876A1 KR 2007004951 W KR2007004951 W KR 2007004951W WO 2008044876 A1 WO2008044876 A1 WO 2008044876A1
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Kyung Sun Kang
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Seoul National University Industry Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

  • the present invention relates to a method for preparing T cell progenitors from CD34 positive stem cells derived from the blood selected from the group consisting of human peripheral blood, bone marrow and umbilical cord blood, and more particularly, to a method for preparing T cell progenitors whose immunological properties are positive for CD4 but negative for CD2, the method comprising; isolating CD34 positive cells from human umbilical cord blood, and culturing and differentiating the CD34 positive cells in a cytokine-containing medium without adding thymic tissue.
  • T cells are a type of leucocyte that plays a central role in cell-mediated immunity.
  • T cells activate B cells producing antibodies, macrophages, and cytotoxic T cells.
  • T cells recognize the presence of antigens through an antigen receptor on the surface thereof, i.e., a T cell receptor (TCR).
  • TCR T cell receptor
  • Precursor T cells are made from bone marrow and they are transferred to thymus to develop therein. However, some T cells develop through extrathymic development.
  • T cells are developed in a three-step process, which is as follows; the first step is 'transfer and proliferation', in which T cell progenitors produced from bone marrow stem cells are selectctively transferred to the thymus to proliferate therein, the second step is the differentiation process in which the T cell progenitors enter the thymus through blood vessels in cortex of thymic lobules to differentiate into cells coexpressing CD3:TCR, CD4, and CD8 etc., during cortex-medulla migration, and the third step is the process of positive and negative selection based on recognition of self MHC through which many thymic lymphocytes (thymic cells, thymic T cell progenitors) die or is unable to proliferate, and only some of the cells survive to develop into T cells in peripheral blood.
  • the first step is 'transfer and proliferation', in which T cell progenitors produced from bone marrow stem cells are selectctively transferred to the thymus to proliferate therein
  • T cells are produced after colonization by bone marrow (BM) progenitors in the thymus, where the thymic microenvironment directs differentiation as well as positive and negative selection.
  • BM bone marrow
  • the progenitor cells develop their specific T cell markers, including T cell receptors (TCR), CD3, CD4 or CD8, and CD2.
  • T-lineage development is maintained through input of blood-derived progenitors (Maillard, I. et al., Blood, 107:3511, 2006). Notch signaling was regarded as essential during T-lineage development from multipotent hematopoietic progenitors (Radtke, F. et al., Nat. Immunol., 5:247, 2004). In the absence of Notch function, T-lineage development was arrested at an early stage (Radtke, F. et al., Immunity, 10:547, 1999). Ross et al. have evaluated the function of notch ligand delta like 1 on the development of T cell development in the culturing system with OP9 cells as feed layer (La Motte-Mohs, R.N. et al, Blood, 105:1431, 2005).
  • umbilical cord blood is the richest source of stem cells, recently, studies on that are actively being conducted. Since the umbilical cord blood was known as an enriched source of hematopoietic stem cells, there have been many attempts to clinically treat blood-related deseases through umbilical cord blood transplant, and many cord blood banks are already operating in Korea, in which umbilical cord blood is preserved by freezing to use for autotransplant treatment after a few years of preservation. Unlike bone marrow, umbilical cord blood can be obtained from umbilical cord discarded after the delivery through a simple method, and when considering its amount, relatively large number of hematopoietic cells and stem cells are contained therein.
  • CD 34+ cells regarded as the population containing hematopoietic stem cells, were the valuable fraction upon transplantation. However, it was reported that transplantation with purified CD34 + cells resulted in reduced diversity of the peripheral T cell repertoire (Bomberger, C. et al., Blood, 91:2588, 1998).
  • CD34 + cell- enriched blood transplantation has more priority than unselected blood transplantation due to the higher engraftment efficiency and provides prolonged disease-free survival rate
  • CD34 + progenitors might reduce the number of contaminating tumor cells while reserving hematopoietic engraftment, but transplantation with highly purified CD34 + cells result in reduced diversity of the peripheral T cell repertoire during the early post-transplant period compared with patients receiving unmanipulated or MoAb-depleted transplants.
  • the present inventors have isolated CD34 positive stem cells derevd from the blood selected from the group consisting of human pheripheral blood, bone marrow and umbilical cord blood, and cultured and differentiated them in a medium containing cytokine such as stem cell factor(SCF), thrombopoietin(TPO), flk-2/flt3 ligand(FL) and the like, and, as a result, found that it is possible to obtain T cell progenitors whose immunological properties are positive for CD4 but negative for CD2, thereby completing the present invention.
  • cytokine such as stem cell factor(SCF), thrombopoietin(TPO), flk-2/flt3 ligand(FL) and the like
  • the present invention provides a method for preparing T cell progenitors from CD34 positive stem cells, the method comprising the steps of (i) isolating CD34 positive cells selected from the group consisting of human pheripheral blood, bone marrow and umbilical cord blood; and (ii) obtaining T cell progenitors whose immunological properties are positive for CD4 but negative for CD2, by culturing and differentiating the isolated CD34 positive stem cells in a medium containing at least one cytokine selected from the group consisting of thrombopoietin(TPO), stem cell factor(SCF), and flk-2/flt3 ligand(FL), in which thymic tissue is not added.
  • FIG.l shows the changes of CD4 and CD34 expression in CD34 positive cells according to the present invention, using flow cytometry (FACS) (Panels A-D were the results right after isolating the CD34 enriched cells by MACs; while E-K show the result of the same population cells after 5 weeks of culturing; B,F,I was the fluoresce level in control, C, G and J shows the CD4 expression; and D,H and K shows the CD34 expression).
  • FACS flow cytometry
  • FIG.2 shows graphs; (2a) the rapid proliferation of the CD34 positive cells in the initial stage and (2b) entire growth curve from the start of seeding to the end of the 10th week.
  • FIG.3 is an image of the colony forming unit derived from long term cultured CD2 " /iow CD4 + ce ⁇ _ enr jched population.
  • FIG.4 shows the expression of several surface antigens of the long term cultured CD34 positive cells in STF medium (Panel A indicates two main populations, gated as Pl (red) and P2 (green); Panels B-E indicate the PE and FITC levels on the two populations in the control; Panels F-I indicate the expression of FITC-conjugate CD4 in the two populations; Panels J-M indicate the expression of PE-conjugated CD31; and Panels N-Q indicate PE conjugated CD44).
  • the present invention relates to a method for preparing T cell progenitors from CD34 positive stem cells, the method comprising the steps of (i) isolating CD34 positive cells derived from the blood selected from the group consisting of human peripheral blood, bone marrow and umbilical cord blood; and (ii) obtaining T cell progenitors whose immunological properties are positive for CD4 but negative for
  • CD2 by culturing and differentiating the isolated CD34 positive stem cells in a medium containing at least one cytokine selected from the group consisting of thrombopoietin(TPO), stem cell factor(SCF), and flk-2/flt3 ligand(FL), in which thymic tissue is not added.
  • TPO thrombopoietin
  • SCF stem cell factor
  • FL flk-2/flt3 ligand
  • T cell progenitors obtained according to the above method are positive for CD4 but negative for CD2.
  • said CD34 positive stem cells may be isolated from human umbilical cord blood, and said medium may be a STF medium containing stem cell factor(SCF), thrombopoietin(TPO), flk-2/flt3 ligand(FL) and FBS.
  • SCF stem cell factor
  • TPO thrombopoietin
  • FL flk-2/flt3 ligand
  • the CD34 positive cells used in the present invention may be obtained from human peripheral blood, bone marrow, or umbilical cord blood.
  • Peripheral blood has an advantage in that it could be easily obtained, while bone marrow has a problem in that patients suffer from severe pain post bone marrow harvest, even though the effectiveness thererof is excellent.
  • umbilical cord blood can be obtained from umbilical cords discarded after the delivery by a simple surgical operation, and when considering its amount, it contains relatively large nember of hematopoietic cells and stem cells. Therefore, a preferred embodiment of the present invention is to isolate CD34 positive cells from the blood derived from human umbilical cord blood.
  • CD34 antigen is a glyocophospoprotein having the molelcular weight of 16kDa, whose genes are located on the long arm of chromosome 1, and expressed in 1-2% of normal myelocytes, especially in parimary hematopoietic progenitor cells.
  • the veto cells are cells, which are typically positive for CD8 and express Fas ligand and class I MHC, and are called facilitating cells since they have the ability to increase survival rate of transplated fragments by selectively inducing cell death in T cell clones involved in immune rejection reactions in allogeneic stem cell transplantation. The activity of these veto cells are shown mostly in CD34 positive stem cells of bone marrow.
  • CD4 is a protein used in dividing T cells in peripheral blood into two groups, together with CD8, wherein about 2/3 of ⁇ / ⁇ T cells in pheripheral blood are
  • CD4+ and CD8- T cells and about 1/3 thereof are CD4- and CD8+ T cells.
  • CD4+ and CD8- T cells have a role as helper T cells, and MHC class II restricts antigen recognition by CD4+ and CD8- T cells.
  • CD4- and CD8+ T cells have cytotoxic T cells, and MHC class I restrics antigen recognition by CD4- and CD8+ T cells.
  • CD4 which is about 55kDa glycoprotein in cell membranes, is expressed in some T cells and on thymic cell surface.
  • CD4 genes are polymorphic protein belonging to the Ig superfamily(T4, Leu 3 in the human).
  • Human CD4 is a protein which is well known as a HIV virus receptor.
  • CD4 which is a cell adhesion molecule adhering to the polymorphic region of MHC class II, augments TCR recognition of peptide- MHC.
  • the cytoplasmic tail of CD4 is bound by the p561ck, a T cell- specific protein tyrosine kinase whose activation is essential for the signal transfer to T cells, thus playing an active role in signal transduction.
  • CD2 which is about 45-5OkDa membranous glycoprotein in cell membranes, is a protein expressed on approximately 90% of mature T cells, 50-70% of thymosytes, and NK cell surface, and variously referred to as Tl, LFA(lymphocyte function- associated antigen)-2, Leu5 and SRJBC receptor.
  • CD2 genes which also belong to the Ig superfamily, augments the adhesion between T cells, and plays a role in signal transduction.
  • the ligand for CD2 is a protein called LFA-3 or CD58.
  • the umbilical cord blood used in one embodiment of the present invention is blood taken from the umbilical vein which carry blood between the placenta and the unborn baby, and is taken before the placenta is removed at delivery.
  • UCB mononuclear cells (MNCs) were prepared from heparinized UCB taken from a parturient by Ficoll-Hypaque density centrifugation according to standard procedures (Tondreau, T. et al., Stem Cells, 23:1105, 2005).
  • umbilical cord blood taken before the placenta is removed at delivery was diluted with phosphate buffer solution(PBS), and overlapped on Ficoll- Hypaque having the same amount as the diluted umbilical cord blood.
  • PBS phosphate buffer solution
  • the f ⁇ coll-hypaque solution should be at room temperature before using, and it is preferable that the volume of the diluted umbilical cord blood should be less than 3- fold the volume of the Ficoll-Hypaque solution.
  • CD34 positive stem cells were isolated from umbilical cord blood mononuclear cell fraction using MACS magnetobead separation system (Miltenyi Biotecl Bergisch-Gladbach, Germany).
  • the medium used for culturing the isolated CD34 positive stem cells in the present invention is a medium for culturing animal cells, containing inorganic slats, amino aicds, vitamins, and/or cofactors, which is used for therapy with mammalian cell lines.
  • a medium which is conventionally used for culturing animal cells in the art for culture of animal cells, may be used.
  • Commercially available media for culturing animal cells include IMDM(Iscove's modified Dulbecco's medium) and the like.
  • the medium may contain at least one cytokine selected from the group consisting of thrombopoietin(TPO), stem cell factor(SCF), and flk-2/flt3 ligand(FL). Most preferably, the medium contains all of the stem cell factor(SCF), the thrombopoietin(TPO), and the flk-2/flt3 ligand(FL). In one embodiment of the present invention, the medium is supplemented with about 10% fetal bovine serume (FBS), stem cell factor(SCF), thrombopoietin(TPO), and flk-2/flt3 ligand(FL) to use.
  • FBS fetal bovine serume
  • the medium supplemented with cytokine-limited cocktail is STF medium (Stem cell factor, Thrembopoietin, Flk-2/Flt3 ligand), which is named after the initials of main ingredients.
  • STF medium Stem cell factor, Thrembopoietin, Flk-2/Flt3 ligand
  • the CD34 positive stem cells which are isolated from the umbilical cord blood mononuclear cell fraction, are cultured and differentiated under 5% CO 2 . Highly purified CD34+ fractions are cultured in STF medium in 25cm flask. At the second week, the cells are transferred to a medium in 75cm flask and 5ml of fresh medium is added therein. From the beginning of the third week, half of the medium is replaced with a fresh one every week.
  • thymic organ is not added unlike the conventional methods. In other words, T cell progenitors are directly obtained from the umbilical cord blood stem cells by culturing in the STF medium without adding thymic organ.
  • Methods for analyzing the obtained CD2 "/low CD4 + T cells include a FACS method using the flow cytometry with sorting ability ⁇ Int. Immunol., 10(3):275, 1998), a method using magnetic beads, a paning method using the antigens specifically recognizing stem cells ⁇ J. Immunol., 141(8):2797, 1998), and the like.
  • FACS method is used.
  • a method for obtaining multipotent stem cells from a large amount of culture broth there is a method in which the antigens specifically recognizing the molecules expressed on cellular surfaces(hereinafter, referred to surface antigens) are used alone or in combination as a column.
  • a waterdrop electric charge method As methods for sorting the flow cytometer, there are a waterdrop electric charge method, a cell-capture method and the like. Using the methods, it is possible to determine the amount of antigens expressed in cells by fluorescently labeling antibodies specifically recognizing the surface antigens in cells and converting the fluorescence intensity of antigen bonded with the fluorescence-labeled antibodies to an electric signal. Moreover, it is also possible to isolate cells expressing a pluarity of surface antigens by combining fluorescent materials to be used.
  • the fluorescent materials which can be used in the present invention, include FITC(fluorescein isothiocyanate), PE(phycoerythrin), APC(allo-phycocyanin), TR(TexasRed), Cy3, CyChrome, Redo 13 , Red670, TRI-Color, QuantumRed and the like
  • FACS methods using a flow cytometer include a method in which cell suspension is collected to isolate cells using centrifugation etc. and directly stain with antibodies, and a method of staining with antibodies after cells are cultured and proliferated in a sutable medium.
  • primary antibodies recognizing surface antigens and target cells are mixed and incubated on ice for 30 minutes ⁇ 1 hour.
  • isolation is carried out using a flow cytometer after washing.
  • primary antibodies are not labeled with fluorescent markers
  • cells reacted with primary antibodies were mixed with fluorescence-labeled secondary antibodies, which is adhesive to primary antibodies after washing, and incubated on ice for 30 minutes ⁇ 1 hour again. After washing, cells were stained with primary antibodies and secondary antibodies and analyzed by flow cytometry.
  • the cells can be applied to FACS analysis by being characterized by cell -surface CD antigen markers, for example, CD2 (T cell marker), CD4, CD20 (B cell marker), CD29 (mononuclear cell marker), CD31 (endothelial cell and stem cell marker), CD34, CD44(hematopoietic cell marker), CD51/61 (osteoclasT cell marker), CD90 (mononuclear stem cell marker), CDl 17 (stem cell marker) and CD 133 (hematopoietic stem marker).
  • CD2 T cell marker
  • CD4, CD20 B cell marker
  • CD29 monoonuclear cell marker
  • CD31 endothelial cell and stem cell marker
  • CD34 CD44(hematopoietic cell marker)
  • CD51/61 osteoclasT cell marker
  • CD90 monoonuclear stem cell marker
  • CDl 17 stem cell marker
  • CD 133 hematopoietic stem marker
  • T cell preginetors are prepared using CD34 positive stem cells derived human umbilical cord blood in the following examples, it will be obvious to those skilled in the art that CD34 positive stem cells derived from human peripheral blood or bone marrow, may also be used.
  • Example 1 Isolating and culturing CD34 positive cells from human umbilical cord blood (1) CD34 positive cell isolation from human umbilical cord blood
  • UCB mononuclear cells were prepared from heparinized UCB taken from a parturient by Ficoll-Hypaque density centrifugation according to standard procedures.
  • CD34 + cells were isolated from the UCB-MNC fraction, using the MACS magnetobead separation system (Miltenyi Biotec; Bergisch-Gladbach, Germany).
  • CD34 + cells (5 x 10 4 /ml) were cultured in 24- well plates with a medium that is composed of Iscove's modified Dulbecco's medium (IMDM), supplemented with a cytokine-limited cocktail and containing 5 ⁇ 50g/ml thrombopoietin (TPO), 10 ⁇ 100ng/ml stem cell factor (SCF), 10-100 ng/ml flk- 2/flt3 ligand (FL)(all recombinant human cytokines were purchased from PeproTech EC Ltd, London, UK).
  • IMDM Iscove's modified Dulbecco's medium
  • TPO thrombopoietin
  • SCF stem cell factor
  • FL 10-100 ng/ml flk- 2/flt3 ligand
  • the isolated CD34+ cells were cultured in a medium containing 5-20% FBS (hereinafter, referred to as STF medium) under 5% CO2. At this time, thymic organ was not added in the medium. At an interval of 7 days, a proper amount of cells were used for analyses. The remaining cells were subcultured in STF medium with replacing half the medium weekly after centrifuged down at 1500rpm, lOmin.
  • the selected CD34 + cells had a purity of
  • the highly purified CD34 + cell fraction was cultured in the STF medium in a 25cm flask. In the first week, the cell number was dramatically increased nearly 15 times
  • FIG. 2 At the second week, cells were transferred to a 75 cm flask with a medium, and another 5ml of new medium were added. From the third week, half the medium was changed weekly. Cells were expanded continuously with fresh medium replacement and maintained at a concentration of l x lO 6 /mL. Cells proliferated continuously at least for 10 weeks (FIG. 2). At the end of the 10th week, cells were expanded nearly 2.5> ⁇ 10 4 -fold, compared with the cells having seed concentration of 0.6x 10 5 /mL. Those result has shown that, while the cells shift from CD34 + population to CD2 "/low CD4 + cell-enriched population, they were expanded dramatically so that the number of cells even satisfy the requirements for adult transplantation.
  • CD 2 T cell marker
  • CD4, CD20 B cell marker
  • CD29 monoonuclear cell marker
  • CD31 endothelial cell and stem cell marker
  • CD34, CD44 hematopoietic cell marker CD51/61 (osteoclasT cell marker), CD90 (mononuclear stem cell marker), CDl 17 (stem cell marker), CD 133 (hematopoietic sem cell marker) were applied for Fluorescence Activated Cell Sorting (FACS) analysis.
  • FACS Fluorescence Activated Cell Sorting
  • Pl and P2 Two major populations were distinguished by FACS as Pl and P2 (FIG. 3).
  • Pl large fraction of the originally CD34 positive cells, which have been subjected to long term culture in STF medium, expressed CD29 (94%), CD31 (41%), CD4 (86.5%), CD44 (94.9%) and CD45 (95.6%).
  • P2 has the similar phonotype with some changes in the expression ratio as CD29 (59.6%), CD31 (63.3%), CD4 (63.3%), CD44 (76.8%), CD45 (52.7%).
  • the fraction expressed CD34 and CD133 lost in both Pl and P2 population (Table 1 ).
  • CD antigens especially CD44 , CD31 and CD45 which evolved to enable the development of T cells lineage, and the low/null expression of mature T cell marker CD2 (0% in Pl group, 0.87% in P2) might indicate the existence of different stage of lymphocyte progenitors, which have been distributed into the two population. This has been partly confirmed by the CFU assay(Colony formation unit assay).
  • Table 1 Expression of CD antigens respectively selected from CD2 /
  • Pl and P2 shows two main polulations, gated Pl (red) and P2(green) as shown in panel A ofFIG.4.
  • Colony formation unit assay Long term (7 to 8 weeks) cultured human UCB derived cells were sorted by FACS after stained by CD4 antibody (Becton-Dickinson). Sorted CD4 + cells were then plated in duplicate plates containing methylcellulose, supplemented with 100 ng/mL granulocyte-colony-stimulating factor (G-CSF, EC Ltd, London, UK ), 1 ng/mL granulocyte macrophage-colony-stimulating factor (GM-CSF, EC Ltd, London, UK), 5 ng/mL recombinant human 1L-3 (rh ⁇ L-3, EC Ltd, London, UK), and 20 ng/mL recombinant human stem cell factor (rhSCF). Colony- forming units (CFU- GM) with more than 100 cells were counted after 10 to 12 days.
  • G-CSF granulocyte-colony-stimulating factor
  • GM-CSF 1 ng/mL
  • the CD34 + cells purified in the STF medium according to the present invention has effects to solve a problem of a decrease in diversity of cell repertoire due to their differentiation capacity into T cell lineage, and to continuously expand the CD2 "/low CD4 + T cell progenitors for at least 10 weeks.
  • the method for preparing T cell progenitors from CD34 positive stem cells acoording to the present invention is useful for studies on T cell development as well as clinical transplantation and cellular treatment of AIDS, cancer, and autoimmune diseases, in which T cells play a main role, since it efficeintly controls differentiation of hematopoietic progenitors in a STF medium, and proliferates the T cell progenitors at large scale.

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Abstract

La présente invention concerne un procédé de préparation de progéniteurs lymphocytaires T à partir de cellules souches CD34 positives dérivées de sang choisi dans le groupe constitué par le sang périphérique humain, le sang de moelle osseuse et le sang de cordon ombilical, et, plus particulièrement, un procédé de préparation de progéniteurs lymphocytaires T dont les propriétés immunologiques sont positives pour CD4 mais négatives pour CD2. Ledit procédé consiste à isoler les cellules CD34 positives du sang de cordon ombilical humain et à cultiver et différencier les cellules CD34 positives dans un milieu contenant des cytokines sans ajout de tissu thymique. Le milieu STF selon la présente invention est utile pour des études sur le développement des lymphocytes T ainsi que pour la transplantation clinique et le traitement cellulaire du SIDA, du cancer et des maladies autoimmunes, dans lesquels les lymphocytes T jouent un rôle essentiel, ledit milieu permettant de réguler efficacement la différenciation de progéniteurs hématopoïétiques et d'assurer une prolifération des progéniteurs lymphocytaires T à large échelle.
PCT/KR2007/004951 2006-10-11 2007-10-10 Procédé de préparation d'un progéniteur lymphocytaire t à partir d'une cellule souche cd34 positive Ceased WO2008044876A1 (fr)

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KR1020060099049A KR100818215B1 (ko) 2006-10-11 2006-10-11 Ｃｄ３４양성 줄기세포로부터 ｔ 임파구 전구체를 제조하는 방법

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WO2011008277A3 (fr) * 2009-07-14 2011-05-26 Massachusetts Institute Of Technology Cellules progénitrices stromales hématopoïétiques et utilisations afférentes
WO2011068962A1 (fr) * 2009-12-03 2011-06-09 The University Of Utah Research Foundation Procédés de génération de lymphocytes t à partir de cellules souches hématopoïétiques
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CN109415699A (zh) * 2016-06-23 2019-03-01 国立大学法人京都大学 Cd4cd8双阳性t细胞的制备方法

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