Generation of multipotent central nervous system stem cells
(19) AUSTRALIAN PATENT OFFICE (54) (51)6 Title Generation of multipotent central nervous system stem cells International Patent Classification(s) C12Q 001/02 C12N 005/00 A23J 001/00 A61K 038/00 A01N 063/00 (21) Application No: 2002306865 (87) WIPO No: WO03/016507 (30) Priority Data (31) Number (32) Date 60/278,510 2001 .03.23 (43) Publication Date : 2003 .03.03 (43) Publication Journal Date : 2003 .05.29 (21) Application No: 2002306865 (87) WIPO No: WO03/016507 (30) Priority Data (31) Number (32) Date 60/278,510 2001 .03.23 (43) Publication Date : 2003 .03.03 (43) Publication Journal Date : 2003 .05.29 (33) (22) Application Date: 2002 .03.23 Country US (71) Applicant(s) REGENTS OF THE UNIVERSITY OF CALIFORNIA; Hoi Sang; SALJOOQUE, Farid ALILAIN, Warren, J.; ALILAIN, Warren, J.; U, (72) Inventor(s) Saljooque, Farid; U, Hoi Sang; Alilain, Warren J.; Alilain, Warren J. -1- Methods for generating various cellular phenotypes from central nervous system stem cells are disclosed. Cellular differentiation into phenotypes of organs and tissues within and outside of the central nervous system is induced by co-culture with target cell types or by soluble trophic factors and elements of the extracellular matrix. Established pluripotent CNS stem cell lines are also disclosed. CLAIMSWhat is claimed is: 1. A pluripotent mammalian central nervous system (CNS) stem cell line, comprising : stem cells isolated from fetal, neonatal or adult brain having the capacity of proliferating perpetually in an undifferentiated state as CNS stem cells and differentiating into Emctional cçlls ofthe ectodem mesoderm or endoderm tissue groups, wherein said capacity is manifest when said stem cells are grown in an environment selected from the group consisting of an environment comprising cells selected from one of said tissue groups, an environment comprising one or more stimulating factors produced by selected cells from one of said tissue groups, an environment comprising one or more stimulating factors from a non-cell source,STDC0080 and an environment comprising the absence of one or more stimulating factors.
2. A cell line according to claim 1, wherein the presence or absence of stimulating factors or signals from other mammalian cell types induces said stem cells to differentiate into neurons and glia.
3. A cell line according to claim 2, wherein the absence of beta FibroblastGrowth Factor in the growth medium induces said stem cells to differentiate into cells with glial properties.
4. A cell line according to claim 1, wherein stimulating factors or signals from adjacent endocrine cell types induces said stem cells to differentiate into endocrine cells.
5. A cell line according to claim 4, wherein the induced endocrine cells produce insulin.
6. A cell line according to claim 4, wherein the differentiated cells are insulin producing pancreatic beta cells.
7. A cell line according to claim 1, wherein said stem cells differentiate into endocrine cell types having the capability to produce one or more members of the group of pituitary factors consisting of growth hormone, prolactin, and pitl.
8. A cell line according to claim 7, wherein said differentiation is induced by factors or signals isolated from mammalian pituitary cells.
9. A cell line according to claim 7, wherein the differentiation is induced by contact with mammalian pituitary cells.
10. A cell line according to claim 7, wherein the endocrine cells are pituitary cells.
11. A cell line according to claim 1, wherein the stem cells differentiate into cardiac cell types through the exposure of said stem cells to horse serum andGDNF.
12. A cell line according to claim 11, wherein the cardiac cell types are pulsatile cardiac cells.
13. A cell line according to claim 12, wherein the pulsatile cardiac cells express one or more cardiac transcription factors.
14. A cell line according to claim 13, wherein the transcription factor is a member of me group consisting essentially of GATA-4, myosin, ortroponin IC.
15. A cell line according to claim 1, wherein said stem cells differentiate into glial cell types in the presence of other mammalian cell types.
16. A cell line according to claim 15, wherein said stem cells differentiate into glial cell types in the presence of mammalian Post Natal-5 days primary astrocytes culture.
17. A cell line according to claim 15, wherein said stem cells differentiate into glial cell types in the presence of mammalian glioma cultures.
18. A cell line according to claim 1, wherein said stem cells differentiate into glial cell types in the presence of isolated factors and or signals from other mammalian cell types.
19. A cell line according to claim 1, wherein said stem cells are capable of differentiating into neurons in the presence or absence of factors or signals from other mammalian cell types.
20. A cell line according to claim 20, wherein said stem cells respond to the presence of EGF and bFCiF by differentiating into neurons expressing microtubule associated protein 2 (Map-2) marker.
21. A cell line according to claim 20, wherein the cells respond to the presence of BDNF by differentiating into neurons expressing Map-2 marker.
22. A method for inducing trans-differentiation of pluripotent stem cells into other cell types, comprising : harvesting the pluripotent stem cells from tissues and/ororgans ; placing the harvested cells into cell culture ; culturing the cells under conditions suitable for maintainingpluripotency ; contacting the cultured pluripotent cells with differentiation inducing factors ; and determining differentiation into a particular cell type..
23The method according to claim 22, wherein the harvesting comprises teasing or trituration of fetal, neonatal or adult CNS tissue.
24. The method according to claim 22, wherein said harvested cells are placed on poly-1Ornithille coated culture plates.
25. The method according to claim 22, wherein the contacting is accomplished by differentiation-inducing factors.
26. The method according to claim 22, wherein the culturing conditions comprise maintaining inducing cells in standard media, harvesting the conditioned media, and exposing CNS stem cells to the conditioned media containing soluble stimulants secreted by the inducing cells.
27. The method according to claim 26, wherein the stimulants are isolated from the conditioned media.
28. The method according to claim 22, wherein the contacting is accomplished by co-culturing with organ-specific inducing cell types.
29. The method according to claim 22, wherein the determining is made by quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR).
30. The method according to claim 22, wherein the determination is made by immunocytochemical characterization of the expression of cell-specific markers.
31The method according to claim 22, wherein the cell-specific markers are members ofthe group consisting essentially of nestin, MAP-2 GFAP, Lhx-3, Pit-1 prolactin, Isl-1, insulin, GATA-4, myosin and troponin IC, and wherein the presence of nestin indicates stem cell properties, the presence of MAP-2 indicates differentiation ilto neuronal cells, the presence of GFAP indicates differentiation into glial cells, the presence of transcription factors Ll-3 and/or Pit-1 and/or the hormones hGH and Prl indicate differentiation into pituitary cells,STDC0197 the presence of GATA-4, myosin, and/or torponin IC indicate differentiation into pulsatile cardiac cells, and the presence of Isl-1 and/or insulin indicate differentiation into pancreatic cells.
32A method for treating a subject by populating and/or repopulating cells in depleted or defective organs and/or tissues with pluripotent CNS stem cells induced in vivo or in vitro to specifically differentiate into functional cell types of the affected organ or tissues, comprising : inducing trans-differentiation of pluripotent CNS stem cells into various other cell types by harvesting pluripotent stem cells from CNS tissue ; placing the harvested cells into cell culture, culturing the cells under conditions suitable for maintaining their pluripotency, contacting the cultured pluripotent cells in vitro or in vivo with differentiation-inducing factors ;STDC0253 determining presence of differentiation into a particular cell type by characterizing expression of cell-specific properties ; and introducing these differentiated cell types to populate and/or repopulate defective areas of said tissues angor organs.
33The method according to claim 32, wherein the differentiation-inducing factors are soluble.
34The method according to claim 32, wherein the source of differentiationinducing factors are cells in c-culture or the cells of said subjects vivo.
35The method according to claim 32, wherein the populating and/or repopulating is accomplished by a member ofthe group including grafting, gene therapy, factor delivery, tissue engineering and organ development.
36The method according to claim 32, wherein the differentiated CNS cells are used as a conduit for gene therapy or factor delivery to prevent or treat disease.
37A method for identifying functionality of certain genes, proteins and regulation in various organ and tissue cell types useful in gene discovery, drug discovery, elucidation of differentiation pathways, genetic markers, regulator factors and biological regulation, comprising : inducing trans-differentiation of pluripotent central nervous system stem cells into various other cell types by harvesting the pluripotent stem cells from tissues and organs, placing the harvested cells into cell culture, culturing the cells under conditions suitable for maintaining thei@ pluripotency, contacting the cultured pluripotent cells with differentiationinducing soluble factors or differentiated cells; determining the differentiation into a particular cell type by characterizing expression cell-specific properties ;STDC0201 and using these cell types to identify involvement of genes, efficacy of drugs, differentiation pathways, genetic markers and regulatory factors and biological regulation.
38The method according to claim? 37, wherein the differentiated CNS cells can be used to produce biological factors such as hormones and other vital proteins.
39. A method for isolating and identifying soluble differentiation-inducing factors capable of inducing differentiation of pluripotent central nervous system stem cells into various other cell types, comprising : placing differentiation-inducing cells into cell culture; culturing the cells under conditions suitable for maintaining their integrity; harvesting partially spent and conditioned culture medium; fractionating the conditioned medium ; contacting pluripotent stem cells with the fractions in cell culture; determining differentiation-inducing effectiveness of each fraction by characterizing expression of cell-specific properties acquired by the induced stem cells to identify the fraction comprising differentiation-inducing factor or factors; isolating the factor ; and identifying the molecular composition of the factor.
40. The method according to claim 39, wherein the isolated factors are produced in quantity to provide available resources for differentiating pluripotent cells from autologous, homologous, heterologous, or stem cell line sources.
41. The method according to claim 40, wherein the production is by chemical means.
42. The method according to claim 40, wherein the production is by genetic expression.
43. The method according to claim 42, wherein the expression is a natural occurrence in certain cell types.
44. The method according to claim 42, wherein the expression is induced by gene insertion.
45. The method according to claim 44, wherein the gene is inserted into pluripotent stem cells, which cells are capable of proliferation and expression of large amounts of said factors.
46. The method according to claim 44, wherein the gene is inserted into the gene pool of other organisms suitable for expression and recovery of large amounts of said factors.
47. The method according to claim 44, wherein the gene insertion is by methods known to those accomplished in the field.
48. The method according to claim 39, wherein the isolated factor is used to stimulate pluripotent stem cells into directed differentiation in the absence of inducing cell types. wherein the inducing cells are unavailable for co-culture, or are depleted or defective in a subject.
49. The method according to claim 48, wherein the stimulation is in vitro or in vivo.
50. The method according to claim 49, wherein the in vivo stimulation is accomplished by contacting a subject's cells with the isolated factor.
51. The method according to claim 50, wherein the contacting is by injection or infusion, or other means known to those in the field of administering drugs to subjects.
52. A pharmaceutical composition, comprising : an effective amount of a differention-inducing factor in a pharmaceutically acceptable carrier.