WO1997032992B1 - Immortalized hematopoietic cell lines, cell system thereof with stromal cells, in vitro, ex vivo and in vivo uses, and in vitro generation of dendritic cells and macrophages - Google Patents
Immortalized hematopoietic cell lines, cell system thereof with stromal cells, in vitro, ex vivo and in vivo uses, and in vitro generation of dendritic cells and macrophagesInfo
- Publication number
- WO1997032992B1 WO1997032992B1 PCT/US1997/003186 US9703186W WO9732992B1 WO 1997032992 B1 WO1997032992 B1 WO 1997032992B1 US 9703186 W US9703186 W US 9703186W WO 9732992 B1 WO9732992 B1 WO 9732992B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- stem cells
- hematopoietic stem
- cell
- hematopoietic
- Prior art date
Links
Abstract
Extented life hematopoietic cell lines include stromal cell lines useful for the in vitro maintenance of undifferentiated pluripotent hematopoietic stem cells. Undifferentiated and differentiated immortalized stem cells are suitable for bone marrow transplantation, gene therapy and cell therapy applications, and as an in vitro model system for drug discovery and toxicological testing.
Claims
1. A transfection vector in the form of a complex, comprising a polypeptide comprising a basic peptide segment, a nuclear localization segment (NLS) peptide, and a neutral hinge peptide linking the basic peptide and the NLS; and at least one DNA segment selected from the group consisting of oncogenes, genes associated with cell cycle regulation, and segments thereof.
2. The vector of claim 1, comprising two oncogene segments.
3. The vector of claim 1, wherein the DNA segment is selected from the group consisting of SV40 DNA, SV40 large T-antigen gene, transcriptional factor E2F gene, adenovirus EIA, adenovirus E1B, polyoma large T and small t antigen genes, DHFR, myc, ras, papilloma virus E6/E7 genes, and fragments, mixtures, and combinations thereof.
4. The vector of claim 1, wherein the polypeptide and the DNA segment are non¬ covalently linked.
5. The vector of claim 1, wherein the polypeptide comprises the amino acid sequence PKKKRKVSGGGGGKKKKKKKKKKKK.
6. The vector of claim 1, further comprising a selectively targeted ligand, which is coupled to the polypeptide.
7. The vector of claim 6, further comprising an additional hinge peptide linking the targeted peptide ligand to the polypeptide.
8. The vector of claim 6, wherein the target polypeptide comprises an antibody, a fragment thereof, or an asialoglycoprotein.
9. An immortalized stromal cell, produced by transfection of a mononuclear hematopoietic cell with the vector of claim 1.
10. The cell of claim 9, being a bone-marrow or cord cell.
11. The cell of claim 9, being a human cell.
12. The cell of claim 9, being selected from the group consisting of fibroblasts, adipocytes, endothelial cells, osteoblasts, and macrophages.
13. A cell culture, comprising a plurality of the cells of claim 9.
14. An in vitro method of producing pluripotent, continuously growing, undifferentiated, transfected or transformed hematopoietic stem cells, comprising 30 allowing the cell culture of claim 13, to grow continuously in vitro; transfecting hematopoietic stem cells with a vector in the form of a complex, comprising a polypeptide comprising a basic peptide segment, a nuclear localization segment (NLS) peptide, and an intermediate hinge neutral peptide linking the basic peptide, NLS, and a DNA segment selected from the group consisting of oncogenes, genes associated with cell cycle regulation, and segments thereof, and combinations thereof with structural genes and segments thereof; and adding the transfected hematopoietic stem cells to the continuously growing stromal cells and culturing in the presence of a medium comprising a cytokine selected from the group consisting of IL-3, SCF, MlP-lα, IL-6, TNF-α, LIF, IL-2, GM-CSF, M-CSF, EPO, and mixtures thereof, under conditions effective for the hematopoietic stem cells to remain undifferentiated and pluripotent while growing and dividing.
15. The method of claim 14, wherein the hematopoietic stem cells comprise human bone marrow or cord cells.
16. The method of claim 14, wherein the stromal cells are layered on a solid substrate, and the transfected hematopoietic stem cells are layered over them for culturing.
17. An ex vivo method of expanding hematopoietic stem cells, comprising obtaining hematopoietic stem cells from a first subject; culturing the stem cells in the presence of the culture of claim 13, under conditions effective for the stem cells to remain pluripotent.
18. The method of claim 17, wherein the hematopoietic cells comprise human bone marrow or cord cells.
19. The method of claim 17, wherein the stromal cells are layered on a solid substrate, and the transfected hematopoietic stem cells are layered over them for culturing.
20. An in vitro method of producing dendritic cells, comprising obtaining continuously growing, undifferentiated hematopoietic stem cells by the method of claim 14; removing said undifferentiated hematopoietic stem cells from medium comprising said stromal cells and culturing said hematopoietic stem cells in medium comprising a cytokine selected from the group consisting of GM-CSF, TNF-α and mixtures thereof to produce dendritic cells.
21. A method of transplanting hematopoietic stem cells, comprising the method of claim 17; and transplanting the undifferentiated stem cells into a second subject with matching HLA cell type.
22. The method of claim 21, wherein the first and the second subjects are the same.
23. The method of claim 19, further comprising transfecting the stem cells with a vector, in the form of a complex, comprising a polypeptide formed of a basic peptide segment, a nuclear localization segment (NLS) peptide, and an intermediate hinge neutral peptide linking the basic peptide and the NLS; and oncogene segments, and optionally structural gene segments, prior to transplantation.
24. The method of claim 23, wherein the cells comprise a human bone marrow or cord cells.
25. The method of claim 23, wherein the pre-selected medium comprises an agent selected from the group consisting of GM-CSF, and TNF-α, and the cells produced comprise dendritic cells.
26. The method of claim 23, wherein the medium comprises an agent selected from the group consisting of M-CSF, and the cells produced comprise macrophages.
27. The method of claim 23, wherein the transfected hematopoietic stem cells comprise transfected CD34+ cells.
28. An immortalized undifferentiated hematopoietic cell produced by the method of claim
29. An undifferentiated transfected or transformed hematopoietic cell capable of continuously growing in vitro in the presence of an immortalized stromal cell.
30. A cell culture comprising a plurality of said hematopoietic cells of claim 29.
31. A continuously growing cell system, comprising the continuously growing undifferentiated transfected or transformed hematopoietic cell culture of claim 30, and immortalized stromal cells.
32. An immortalized differentiated hematopoietic cell produced by the method of claim
33. A cell culture comprising a plurality of the cells of claim 32.
34. A differentiated dendritic cell produced by the method of claim 25.
35. A cell culture comprising a plurality of dendritic cells of claim 34.
36. A differentiated macrophage produced by the method of claim 26.
37. A cell culture comprising a plurality of macrophages of claim 36. 32
38. An ex vivo method of gene therapy, comprising contacting in vitro mammalian hematopoietic stem cells with the vector of claim 1 , under conditions effective to effect transfection of the hematopoietic cells.
39. An in vivo cell therapy method comprising transplanting the hematopoietic stem cell culture of claim 33, into a subject afflicted by an immunodeficiency associated with defective or diminished activity of hematopoietic cells.
40. An in vitro method of testing the bone marrow toxicity of a drug, comprising adding a drug to be tested to the cell culture of claim 33, under conditions and for a period of time effective to induce toxicity; and detecting the presence of any toxicity signs in the cells by comparing to cells grown in the absence of the drug.
41. A method of testing the bone marrow activity of a drug, comprising adding a drug to be tested to the cell culture of claim 33, under conditions and for a period of time effective for the drug to interact with the cells to produce an effect on them; and detecting the presence of any effect of the drug on the cells by comparison with a control conducted in the absence of the drug.
42. An in vitro method of producing macrophage cells, comprising: obtaining continuously growing, undifferentiated hematopoietic stem cells by the method of claim 14; removing said hematopoietic stem cells from medium comprising said stromal cells and culturing said hematopoietic stem cells in medium, comprising the cytokine M-CSF, to produce macrophage cells.
43. An in vitro method of producing megakaryocytic cells, comprising: obtaining continuously growing, undifferentiated hematopoietic stem cells by the method of claim 14; removing said hematopoietic stem cells from medium comprising said stromal cells and culturing said hematopoietic stem cells in medium comprising a cytokine selected from the group consisting of IL-3, GM-CSF and mixtures thereof to produce megakaryocytic cells.
44. An in vitro method of producing erythroid cells, comprising: 33 obtaining continuously growing, undifferentiated hematopoietic stem cells by the method of claim 14; removing said undifferentiated hematopoietic stem cells from medium comprising said stromal cells and culturing said hematopoietic stem cells in medium comprising EPO and, optionally, a cytokine selected from the group consisting of IL-3, SCF, and mixtures thereof to produce erythroid cells.
45. The method of claim 19, wherein said genes associated with cell cycle regulation are selected from the group consisting of a dihydrofolate reductase gene (DHFR), a thymidine kinase gene, a thymidylate synthetase gene, a DRTF1/E2F transcription factor encoding DNA, or a E2F1 transcriptional factor gene.
46. The method of claim 45, wherein said genes associated with cell cycle regulation are selected from the group consisting of a dihydrofolate reductase gene (DHFR), or a E2F1 transcriptional factor gene.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9531851A JP2000508885A (en) | 1996-03-07 | 1997-03-07 | Immortalized hematopoietic cell lines, cell systems with these cell lines and stromal cells, use in vitro, ex vivo and in vivo, and in vitro production of dendritic cells and macrophages |
| EP97915851A EP0954594A2 (en) | 1996-03-07 | 1997-03-07 | Immortalized hematopoietic cell lines, cell system thereof with stromal cells, in vitro, ex vivo and in vivo uses, and in vitro generation of dendritic cells and macrophages |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/612,302 | 1996-03-07 | ||
| US08/612,302 US5811297A (en) | 1996-03-07 | 1996-03-07 | Immortalized hematopoietic cell lines, cell system thereof with stromal cells, in vitro, ex vivo and in vivo uses, & in vitro generation of dendritic cells and macrophages |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1997032992A1 WO1997032992A1 (en) | 1997-09-12 |
| WO1997032992B1 true WO1997032992B1 (en) | 1997-11-06 |
Family
ID=24452603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1997/003186 WO1997032992A1 (en) | 1996-03-07 | 1997-03-07 | Immortalized hematopoietic cell lines, cell system thereof with stromal cells, in vitro, ex vivo and in vivo uses, and in vitro generation of dendritic cells and macrophages |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US5811297A (en) |
| EP (1) | EP0954594A2 (en) |
| JP (1) | JP2000508885A (en) |
| CA (1) | CA2248555A1 (en) |
| WO (1) | WO1997032992A1 (en) |
Families Citing this family (30)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5670347A (en) * | 1994-05-11 | 1997-09-23 | Amba Biosciences Llc | Peptide-mediated gene transfer |
| US5795587A (en) | 1995-01-23 | 1998-08-18 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
| US6008202A (en) * | 1995-01-23 | 1999-12-28 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
| US20030069173A1 (en) * | 1998-03-16 | 2003-04-10 | Life Technologies, Inc. | Peptide-enhanced transfections |
| AUPO723097A0 (en) * | 1997-06-06 | 1997-07-03 | Australian National University, The | A method for culturing cells |
| EP1071747B1 (en) * | 1998-04-17 | 2004-08-18 | Societe Des Produits Nestle S.A. | Immortalised cell lines derived from normal human skin tissues |
| KR20010080722A (en) * | 1998-12-09 | 2001-08-22 | 추후제출 | Toxicity typing using embryoid bodies |
| AU3755800A (en) * | 1999-03-15 | 2000-10-04 | Introgen Therapeutics, Inc. | Dendritic cells transduced with a wild-type self gene elicit potent antitumor immune responses |
| DE60044125D1 (en) | 1999-04-14 | 2010-05-20 | Novartis Vaccines & Diagnostic | COMPOSITIONS AND METHODS FOR TRIGGING AN IMMUNE RESPONSE BASED ON ALPHAVIRUS VECTOR SYSTEMS |
| AU5178800A (en) * | 1999-06-01 | 2000-12-18 | Cornell Research Foundation Inc. | Activation of dendritic cells to enhance immunity |
| US20020045179A1 (en) * | 2000-06-14 | 2002-04-18 | Snodgrass H. Ralph | Toxicity typing using mesenchymal stem cells |
| ATE445158T1 (en) | 2000-06-14 | 2009-10-15 | Vistagen Inc | TOXICITY TYPING USING LIVER STEM CELLS |
| US20030202963A1 (en) * | 2000-10-12 | 2003-10-30 | Cornell Research Foundation, Inc. | Method of treating cancer |
| US20030027334A1 (en) * | 2000-12-13 | 2003-02-06 | Fitzpatrick David R. | Method for generating immortal dendritic cell lines |
| WO2002048167A1 (en) * | 2000-12-13 | 2002-06-20 | Immunex Corporation | Method for generating immortal dendritic cell lines |
| DE10139428A1 (en) * | 2001-08-17 | 2003-03-27 | Nemod Immuntherapie Ag | Preparation of dendritic cells, useful e.g. as antitumor or antimicrobial vaccines, by treating CD124- and CD116-positive cells with stimulatory molecules |
| JPWO2003038076A1 (en) * | 2001-10-31 | 2005-02-24 | 株式会社レノメディクス研究所 | Immortalized mesenchymal cells and their use |
| MXPA04004310A (en) * | 2001-11-09 | 2005-03-31 | Artecel Sciences Inc | Methods and compositions for the use of stromal cells to support embryonic and adult stem cells. |
| EP1463821A4 (en) * | 2001-12-21 | 2006-05-31 | Us Health | INCREASED GENTRANSFEREFFICIENCY BY PRE-INCUBATION WITH ENDOTHELIAL CELLS |
| CN101291687A (en) * | 2005-05-12 | 2008-10-22 | 因特罗根治疗公司 | P53 Vaccines for Cancer Therapy |
| WO2007026353A2 (en) | 2005-08-29 | 2007-03-08 | Technion Research & Development Foundation Ltd. | Media for culturing stem cells |
| EP2059586B1 (en) | 2006-08-02 | 2016-07-20 | Technion Research & Development Foundation Ltd. | Methods of expanding embryonic stem cells in a suspension culture |
| EP2154243B1 (en) * | 2007-05-17 | 2016-01-13 | ID Pharma Co., Ltd. | Method for production of dendritic cell |
| JP2010529987A (en) * | 2007-06-15 | 2010-09-02 | ガーネット バイオセラピューティクス インコーポレイテッド | Treatment of diseases and disorders using self-replicating colony-forming cells cultured and grown in vitro |
| US10894944B2 (en) * | 2009-04-10 | 2021-01-19 | Monash University | Cell culture media |
| EP2499236B1 (en) | 2009-11-12 | 2020-01-01 | Technion Research & Development Foundation Ltd. | Culture media, cell cultures and methods of culturing pluripotent stem cells in an undifferentiated state |
| CA2815426A1 (en) | 2010-10-25 | 2012-05-10 | The Children's Hospital Of Philadelphia | Compositions and methods for the generation of platelets and methods of use thereof |
| EP2983696B1 (en) * | 2013-04-09 | 2018-07-11 | INSERM - Institut National de la Santé et de la Recherche Médicale | Use of m-csf for preventing or treating myeloid cytopenia and related complications |
| WO2016168492A1 (en) | 2015-04-14 | 2016-10-20 | Hur Soojung Claire | Electrode array for vortex-assisted electroporation |
| US20180340186A1 (en) * | 2015-10-15 | 2018-11-29 | President And Fellows Of Harvard College | Cell immortalization via vortex electroporation gene delivery |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR950700403A (en) * | 1992-03-04 | 1995-01-16 | 죤 제이. 쉬바르츠 | CULTURING OF HEMATOPOIETIC STEM CELLS AND THEIR GENETIC ENGINEERING |
| EP0563485A1 (en) * | 1992-03-30 | 1993-10-06 | Schering-Plough | In vitro generation of human dendritic cells and uses thereof |
| WO1995009640A1 (en) * | 1993-10-07 | 1995-04-13 | Systemix, Inc. | Methods for analysis of human stem cells |
| US5599703A (en) * | 1993-10-28 | 1997-02-04 | The United States Of America As Represented By The Secretary Of The Navy | In vitro amplification/expansion of CD34+ stem and progenitor cells |
| US5599705A (en) * | 1993-11-16 | 1997-02-04 | Cameron; Robert B. | In vitro method for producing differentiated universally compatible mature human blood cells |
| US5670347A (en) * | 1994-05-11 | 1997-09-23 | Amba Biosciences Llc | Peptide-mediated gene transfer |
| US6103522A (en) * | 1994-07-20 | 2000-08-15 | Fred Hutchinson Cancer Research Center | Human marrow stromal cell lines which sustain hematopoiesis |
-
1996
- 1996-03-07 US US08/612,302 patent/US5811297A/en not_active Expired - Lifetime
-
1997
- 1997-03-07 CA CA002248555A patent/CA2248555A1/en not_active Abandoned
- 1997-03-07 JP JP9531851A patent/JP2000508885A/en active Pending
- 1997-03-07 EP EP97915851A patent/EP0954594A2/en not_active Withdrawn
- 1997-03-07 WO PCT/US1997/003186 patent/WO1997032992A1/en not_active Application Discontinuation
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